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Sample records for mutation affecting symbiosis

  1. The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis.

    PubMed

    Wells, Derek H; Long, Sharon R

    2002-03-01

    Sinorhizobium meliloti and host legumes enter into a nitrogen-fixing, symbiotic relationship triggered by an exchange of signals between bacteria and plant. S. meliloti produces Nod factor, which elicits the formation of nodules on plant roots, and succinoglycan, an exopolysaccharide that allows for bacterial invasion and colonization of the host. The biosynthesis of these molecules is well defined, but the specific regulation of these compounds is not completely understood. Bacteria control complex regulatory networks by the production of ppGpp, the effector molecule of the stringent response, which induces physiological change in response to adverse growth conditions and can also control bacterial development and virulence. Through detailed analysis of an S. meliloti mutant incapable of producing ppGpp, we show that the stringent response is required for nodule formation and regulates the production of succinoglycan. Although it remains unknown whether these phenotypes are connected, we have isolated suppressor strains that restore both defects and potentially identify key downstream regulatory genes. These results indicate that the S. meliloti stringent response has roles in both succinoglycan production and nodule formation and, more importantly, that control of bacterial physiology in response to the plant and surrounding environment is critical to the establishment of a successful symbiosis.

  2. Saltational symbiosis.

    PubMed

    Sapp, Jan

    2010-09-01

    Symbiosis has long been associated with saltational evolutionary change in contradistinction to gradual Darwinian evolution based on gene mutations and recombination between individuals of a species, as well as with super-organismal views of the individual in contrast to the classical one-genome: one organism conception. Though they have often been dismissed, and overshadowed by Darwinian theory, suggestions that symbiosis and lateral gene transfer are fundamental mechanisms of evolutionary innovation are borne out today by molecular phylogenetic research. It is time to treat these processes as central principles of evolution.

  3. The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.

    PubMed

    Cosme, Ana M; Becker, Anke; Santos, Mário R; Sharypova, Larissa A; Santos, Pedro M; Moreira, Leonilde M

    2008-07-01

    Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

  4. How Symbiosis Creates Diversity

    ERIC Educational Resources Information Center

    Lord, Joshua

    2010-01-01

    Diversity in habitats on Earth is astounding--whether on land or in the sea--and this is in part due to symbiosis. The lesson described in this article helps students understand how symbiosis affects different organisms through a fun and engaging game where they match hosts and symbionts based on their respective needs. This 45-minute lesson is…

  5. Designing symbiosis.

    PubMed

    Hosoda, Kazufumi; Yomo, Tetsuya

    2011-01-01

    Organisms rarely live as isolated species and usually show symbiosis in nature. As natural selection is not simple in symbiosis, the establishment and development of symbiosis is still unclear. Insight can be gained by not only retracing the history of well-developed natural symbiotic relationships, but also by observing the development of nascent symbiosis. By using synthetic symbiosis composed of two previously noninteracting populations, we can observe the establishment and its development. We have recently simulated the establishment of nascent symbiosis using two genetically engineered auxotrophic strains of Escherichia coli. One strain, 10 h after mixing with the partner strain, began to oversupply metabolites essential for the partner's growth, eventually leading to continual growth of both strains. Transcriptome analysis revealed that the oversupply was accompanied by global metabolic changes. This study demonstrated that an organism has the potential to adapt to the first encounter with another organism to establish symbiosis.

  6. Symbiosis between nitrogen-fixing bacteria and Medicago truncatula is not significantly affected by silver and silver sulfide nanomaterials.

    PubMed

    Judy, Jonathan D; Kirby, Jason K; McLaughlin, Mike J; McNear, David; Bertsch, Paul M

    2016-07-01

    Silver (Ag) engineered nanomaterials (ENMs) are being released into waste streams and are being discharged, largely as Ag2S aged-ENMs (a-ENMs), into agroecosystems receiving biosolids amendments. Recent research has demonstrated that biosolids containing an environmentally relevant mixture of ZnO, TiO2, and Ag ENMs and their transformation products, including Ag2S a-ENMs, disrupted the symbiosis between nitrogen-fixing bacteria and legumes. However, this study was unable to unequivocally determine which ENM or combination of ENMs and a-ENMs was responsible for the observed inhibition. Here, we examined further the effects of polyvinylpyrollidone (PVP) coated pristine Ag ENMs (PVP-Ag), Ag2S a-ENMs, and soluble Ag (as AgSO4) at 1, 10, and 100 mg Ag kg(-1) on the symbiosis between the legume Medicago truncatula and the nitrogen-fixing bacterium, Sinorhizobium melliloti in biosolids-amended soil. Nodulation frequency, nodule function, glutathione reductase production, and biomass were not significantly affected by any of the Ag treatments, even at 100 mg kg(-1), a concentration analogous to a worst-case scenario resulting from long-term, repeated biosolids amendments. Our results provide additional evidence that the disruption of the symbiosis between nitrogen-fixing bacteria and legumes in response to a mixture of ENMs in biosolids-amended soil reported previously may not be attributable to Ag ENMs or their transformation end-products. We anticipate these findings will provide clarity to regulators and industry regarding potential unintended consequences to terrestrial ecosystems resulting from of the use of Ag ENMs in consumer products.

  7. Arbuscular mycorrhizal symbiosis affects the grain proteome of Zea mays: a field study

    PubMed Central

    Bona, Elisa; Scarafoni, Alessio; Marsano, Francesco; Boatti, Lara; Copetta, Andrea; Massa, Nadia; Gamalero, Elisa; D’Agostino, Giovanni; Cesaro, Patrizia; Cavaletto, Maria; Berta, Graziella

    2016-01-01

    Maize is one of the most important crops worldwide and is strongly dependent on arbuscular mycorrhiza (AM) fungi, organisms that form a mutualistic association with land plants. In maize, AM symbiosis enhances spike dry weight, spike length, spike circumference, and the dry weight and dimensions of the grain. Notwithstanding its ubiquitous nature, the detailed relationship between AM fungal colonization and plant development is not completely understood. To facilitate a better understanding of the effects of AM fungi on plants, the work reported here assessed the effects of a consortium of AM fungi on the kernel proteome of maize, cultivated in open-field conditions. To our knowledge, this is the first report of the modulation of a plant seed proteome following AM fungal inoculation in the field. Here, it was found that AM fungi modify the maize seed proteome by up-regulating enzymes involved in energetic metabolism, embryo development, nucleotide metabolism, seed storage and stress responses. PMID:27216714

  8. Schoolyard Symbiosis.

    ERIC Educational Resources Information Center

    Allard, David W.

    1996-01-01

    Discusses different types of symbiosis--mutualism, commensalism, and parasitism--and examples of each type including lichens, legumes, mistletoe, and epiphytes. Describes how teachers can use these examples in the study of symbiosis which allows teachers to focus on many basic concepts in evolution, cell biology, ecology, and other fields of…

  9. Mycorrhizal symbiosis in leeks increases plant growth under low phosphorus and affects the levels of specific flavonoid glycosides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction- Mycorrhizae symbiosis is a universal phenomenon in nature that promotes plant growth and food quality in most plants, especially, under phosphorus deficiency and water stress. Objective- The objective of this study was to assess the effects of mycorrhizal symbiosis on changes in the le...

  10. Metal toxicity differently affects the Iris pseudacorus-arbuscular mycorrhiza fungi symbiosis in terrestrial and semi-aquatic habitats.

    PubMed

    Wężowicz, K; Turnau, K; Anielska, T; Zhebrak, I; Gołuszka, K; Błaszkowski, J; Rozpądek, P

    2015-12-01

    Phytoremediation offers an environmental friendly alternative to conventional cleanup techniques. In this study, mycorrhizal fungi isolated from the roots of Mentha longifolia grown in the basin of the Centuria River (S Poland) were used. Iris pseudacorus was grown in substratum from an industrial waste, enriched in Pb, Fe, Zn, and Cd in a terrestrial and water-logged habitat. Plant yield and photosynthetic performance was the highest in the aquatic environment; however, the presence of toxic metals (TM) negatively affected photosystem II (PSII) photochemistry as shown by the JIP test. Fungi colonization and Cd accumulation within plant tissues was decreased. In the terrestrial habitat, neither arbuscular mycorrhizal fungi (AMF) nor metal toxicity affected plant growth, although metal uptake, Cd in particular, as well as photosynthesis were affected. Inoculated plants accumulated significantly more Cd, and photosynthesis was downregulated. The results presented in this study clearly indicate that the I. pseudacorus-AMF symbiosis adapts itself to the presence of toxic metals in the environment, optimizing resource supply, energy fluxes, and possibly stress tolerance mechanisms. Plant/AMF consortia grown in terrestrial and water-logged habitats utilize different strategies to cope with metal toxicity. The use of AMF in improving the phytoremediation potential of I. pseudacorus needs, however, further research.

  11. Mutations affecting enzymatic activity in liver arginase

    SciTech Connect

    Vockley, J.G.; Tabor, D.E.; Goodman, B.K.

    1994-09-01

    The hydrolysis of arginine to ornithine and urea is catalyzed by arginase in the last step of the urea cycle. We examined a group of arginase deficient patients by PCR-SSCP analysis to characterize the molecular basis of this disorder. A heterogeneous population of nonsense mutations, microdeletions, and missense mutations has been identified in our cohort. Microdeletions which introduce premature stop codons downstream of the deletion and nonsense mutations result in no arginase activity. These mutations occur randomly along the gene. The majority of missense mutations identified appear to occur in regions of high cross-species homology. To test the effect of these missense mutations on arginase activity, site-directed mutagenesis was used to re-create the patient mutations for in vivo expression studies in a prokaryotic fusion-protein expression system. Of 4 different missense mutations identified in 6 individuals, only one was located outside of a conserved region. The three substitution mutations within the conserved regions had a significant effect on enzymatic activity (0-3.1 nmole/30min, normal is 1300-1400 nmoles/30min, as determined by in vitro arginase assay), while the fourth mutation, a T to S substitution, did not. In addition, site-directed mutagenesis was utilized to create mutations not in residues postulated to play a significant role in the enzymatic function or active site formation in manganese-binding proteins such as arginase. We have determined that the substitution of glycine for a histidine residue, located in a very highly conserved region of exon 3, and the substitution of a histidine and an aspartic acid residue within a similarly conserved region in exon 4, totally abolishes enzymatic activity. Mutations substituting glycine for an additional histidine and aspartic acid residue in exon 4 and two aspartic acid residues in exon 7 have also been created. We are currently in the process of characterizing these mutations.

  12. Factors affecting the nature of induced mutations

    SciTech Connect

    Russell, L.B.; Russell, W.L.; Rinchik, E.M.; Hunsicker, P.R.

    1989-01-01

    The recent considerable expansion of specific-locus-mutation data has made possible an examination of the effects of germ-cell stage on both quantity of mutation yield and nature of mutations. For chemicals mutagenic in poststem-cell stages, three patterns have been identified according to the stages in which they elicit maximum response: (1) early spermatozoa and late spermatids; (2) early spermatids; and (3) differentiating spermatogonia. The majority of chemicals tested fall into Pattern 1. Chemicals that are also mutagenic in stem-cell spermatogonia do not preferentially belong to any one of these three categories. For only one chemical (CHL) has an entire set of mutations been analyzed molecularly. However, the results of genetic and molecular analyses of genomic regions surrounding six of the specific-locus markers allow us to conclude that any mutation that causes lethality of homozygotes (in the case of d, prenatal lethality, specifically) must involve one or more loci in addition to the marked one. Such mutations have been classified as large lesions'' (LL), the remainder as other lesions'' (OL). Analysis of the data shows that, regardless of the nature of the chemical (Pattern-1, -2, or -3), (1) LLs constitute a very low proportion of the mutations induced in either stem-cell or differentiating spermatogonia, and (b) LLs constitute a high proportion of mutations induced in postmeiotic stages. Chemicals that are active in both pre- and postmeiotic stages produce LL or OL mutations depending on cell stage.

  13. Identification of mutations in Colombian patients affected with Fabry disease.

    PubMed

    Uribe, Alfredo; Mateus, Heidi Eliana; Prieto, Juan Carlos; Palacios, Maria Fernanda; Ospina, Sandra Yaneth; Pasqualim, Gabriela; da Silveira Matte, Ursula; Giugliani, Roberto

    2015-12-15

    Fabry Disease (FD) is an X-linked inborn error of glycosphingolipid catabolism, caused by a deficiency of the lisosomal α-galactosidase A (AGAL). The disorder leads to a vascular disease secondary to the involvement of kidney, heart and the central nervous system. The mutation analysis is a valuable tool for diagnosis and genetic counseling. Although more than 600 mutations have been identified, most mutations are private. Our objective was to describe the analysis of nine Colombian patients with Fabry disease by automated sequencing of the seven exons of the GLA gene. Two novel mutations were identified in two patients affected with the classical subtype of FD, in addition to other 6 mutations previously reported. The present study confirms the heterogeneity of mutations in Fabry disease and the importance of molecular analysis for genetic counseling, female heterozygotes detection as well as therapeutic decisions.

  14. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  15. Computer symbiosis: Emergence of symbiotic behavior through evolution

    SciTech Connect

    Ikegami, Takashi; Kaneko, Kunihiko

    1989-01-01

    Symbiosis is altruistic cooperation between distinct species. It is one of the most effective evolutionary processes, but its dynamics are not well understood as yet. A simple model of symbiosis is introduced, where we consider interactions between hosts and parasites and also mutations of hosts and parasites. It is found that a symbiotic state emerges for a suitable range of mutation rates. The symbiotic state is not static, but dynamically oscillates. Harmful parasites violating symbiosis appear periodically, but are rapidly extinguished by hosts and other parasites, and the symbiotic state is recovered. The emergence of ''Tit for Tat'' strategy to maintain symbiosis is discussed. 4 figs.

  16. Nickel tolerance of serpentine and non-serpentine Knautia arvensis plants as affected by arbuscular mycorrhizal symbiosis.

    PubMed

    Doubková, Pavla; Sudová, Radka

    2014-04-01

    Serpentine soils have naturally elevated concentrations of certain heavy metals, including nickel. This study addressed the role of plant origin (serpentine vs. non-serpentine) and symbiosis with arbuscular mycorrhizal fungi (AMF) in plant Ni tolerance. A semi-hydroponic experiment involving three levels of Ni and serpentine and non-serpentine AMF isolates and populations of a model plant species (Knautia arvensis) revealed considerable negative effects of elevated Ni availability on both plant and fungal performance. Plant growth response to Ni was independent of edaphic origin; however, higher Ni tolerance of serpentine plants was indicated by a smaller decline in the concentrations of photosynthetic pigments and restricted root-to-shoot Ni translocation. Serpentine plants also retained relatively more Mg in their roots, resulting in a higher shoot Ca/Mg ratio. AMF inoculation, especially with the non-serpentine isolate, further aggravated Ni toxicity to host plants. Therefore, AMF do not appear to be involved in Ni tolerance of serpentine K. arvensis plants.

  17. Plant hormones as signals in arbuscular mycorrhizal symbiosis.

    PubMed

    Miransari, Mohammad; Abrishamchi, A; Khoshbakht, K; Niknam, V

    2014-06-01

    Arbuscular mycorrhizal (AM) fungi are non-specific symbionts developing mutual and beneficial symbiosis with most terrestrial plants. Because of the obligatory nature of the symbiosis, the presence of the host plant during the onset and proceeding of symbiosis is necessary. However, AM fungal spores are able to germinate in the absence of the host plant. The fungi detect the presence of the host plant through some signal communications. Among the signal molecules, which can affect mycorrhizal symbiosis are plant hormones, which may positively or adversely affect the symbiosis. In this review article, some of the most recent findings regarding the signaling effects of plant hormones, on mycorrhizal fungal symbiosis are reviewed. This may be useful for the production of plants, which are more responsive to mycorrhizal symbiosis under stress.

  18. Mutations affecting xanthophore pigmentation in the zebrafish, Danio rerio.

    PubMed

    Odenthal, J; Rossnagel, K; Haffter, P; Kelsh, R N; Vogelsang, E; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kane, D A; Mullins, M C; Nüsslein-Volhard, C

    1996-12-01

    In a large-scale screen for mutants with defects in embryonic development we identified 17 genes (65 mutants) specifically required for the development of xanthophores. We provide evidence that these genes are required for three different aspects of xanthophore development. (1) Pigment cell formation and migration (pfeffer and salz); (2) pigment synthesis (edison, yobo, yocca and brie) and (3) pigment translocation (esrom, tilsit and tofu). The number of xanthophore cells that appear in the body is reduced in embryos with mutations in the two genes, salz and pfeffer. In heterozygous and homozygous salz and pfeffer adults, the melanophore stripes are interrupted, indicating that xanthophore cells have an important function in adult melanophore pattern formation. Most other genes affect only larval pigmentation. In embryos mutant for edison, yobo, yocca and brie, differences in pteridine synthesis can be observed under UV light and by thin-layer chromatography. Homozygous mutant females of yobo show a recessive maternal effect. Embryonic development is slowed down and embryos display head and tail truncations. Xanthophores in larvae mutant in the three genes esrom, tilsit and tofu appear less spread out. In addition, these mutants display a defect in retinotectal axon pathfinding. These mutations may affect xanthophore pigment distribution within the cells or xanthophore cell shape. Mutations in seven genes affecting xanthophore pigmentation remain unclassified.

  19. Mutations Affecting Expression of the rosy Locus in Drosophila melanogaster

    PubMed Central

    Lee, Chong Sung; Curtis, Daniel; McCarron, Margaret; Love, Carol; Gray, Mark; Bender, Welcome; Chovnick, Arthur

    1987-01-01

    The rosy locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Previous studies defined a "control element" near the 5' end of the gene, where variant sites affected the amount of rosy mRNA and protein produced. We have determined the DNA sequence of this region from both genomic and cDNA clones, and from the ry+10 underproducer strain. This variant strain had many sequence differences, so that the site of the regulatory change could not be fixed. A mutagenesis was also undertaken to isolate new regulatory mutations. We induced 376 new mutations with 1-ethyl-1-nitrosourea (ENU) and screened them to isolate those that reduced the amount of XDH protein produced, but did not change the properties of the enzyme. Genetic mapping was used to find mutations located near the 5' end of the gene. DNA from each of seven mutants was cloned and sequenced through the 5' region. Mutant base changes were identified in all seven; they appear to affect splicing and translation of the rosy mRNA. In a related study (T. P. Keith et al. 1987), the genomic and cDNA sequences are extended through the 3' end of the gene; the combined sequences define the processing pattern of the rosy transcript and predict the amino acid sequence of XDH. PMID:3036645

  20. Mutations affecting expression of the rosy locus in Drosophila melanogaster

    SciTech Connect

    Lee, C.S.; Curtis, D.; McCarron, M.; Love, C.; Gray, M.; Bender, W.; Chovnick, A.

    1987-05-01

    The rosy locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Previous studies defined a control element near the 5' end of the gene, where variant sites affected the amount of rosy mRNA and protein produced. The authors have determined the DNA sequence of this region from both genomic and cDNA clones, and from the ry/sup +10/ underproducer strain. This variant strain had many sequence differences, so that the site of the regulatory change could not be fixed. A mutagenesis was also undertaken to isolate new regulatory mutations. They induced 376 new mutations with 1-ethyl-1-nitrosourea (ENU) and screened them to isolate those that reduced the amount of XDH protein produced, but did not change the properties of the enzyme. Genetic mapping was used to find mutations located near the 5' end of the gene. DNA from each of seven mutants was cloned and sequenced through the 5' region. Mutant base changes were identified in all seven; they appear to affect splicing and translation of the rosy mRNA. In a related study, the genomic and cDNA sequences are extended through the 3' end of the gene; the combined sequences define the processing pattern of the rosy transcript and predict the amino acid sequence of XDH.

  1. The classical pink-eyed dilution mutation affects angiogenic responsiveness.

    PubMed

    Rogers, Michael S; Boyartchuk, Victor; Rohan, Richard M; Birsner, Amy E; Dietrich, William F; D'Amato, Robert J

    2012-01-01

    Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.

  2. Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

    PubMed Central

    Adams, A K; Holm, C

    1996-01-01

    To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. PMID:8756617

  3. Computer symbiosis-emergence of symbiotic behavior through evolution

    NASA Astrophysics Data System (ADS)

    Ikegami, Takashi; Kaneko, Kunihiko

    1990-06-01

    Symbiosis is cooperation between distinct species. It is one of the most effective evolutionary processes, but its dynamics are not well understood as yet. A simple model of symbiosis is introduced, in which we consider interactions between hosts and parasites and also mutations of hosts and parasites. The interactions and mutations form a dynamical system on the populations of hosts and parasites. It is found that a symbiotic state emerges for a suitable range of mutation rates. The symbiotic state is not static, but dynamically oscillates. Harmful parasites violating symbiosis appear periodically, but are rapidly extinguished by hosts and other parasites, and the symbiotic state is recovered. The relation between these phenomena and “TIT for TAT” strategy to maintain symbiosis is discussed.

  4. Nonredundant regulation of rice arbuscular mycorrhizal symbiosis by two members of the phosphate transporter1 gene family.

    PubMed

    Yang, Shu-Yi; Grønlund, Mette; Jakobsen, Iver; Grotemeyer, Marianne Suter; Rentsch, Doris; Miyao, Akio; Hirochika, Hirohiko; Kumar, Chellian Santhosh; Sundaresan, Venkatesan; Salamin, Nicolas; Catausan, Sheryl; Mattes, Nicolas; Heuer, Sigrid; Paszkowski, Uta

    2012-10-01

    Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.

  5. Mutations affecting the chemosensory neurons of Caenorhabditis elegans

    SciTech Connect

    Starich, T.A.; Herman, R.K.; Kari, C.K.

    1995-01-01

    We have identified and characterized 95 mutations that reduce or abolish dye filling of amphid and phasmid neurons and that have little effect on viability, fertility or movement. Twenty-seven mutations occurred spontaneously in strains with a high frequency of transposon insertion. Sixty-eight were isolated after treatment with EMS. All of the mutations result in defects in one or more chemosensory responses, such as chemotaxis to ammonium chloride or formation of dauer larvae under conditions of starvation and overcrowding. Seventy-five of the mutations are alleles of 12 previously defined genes, mutations which were previously shown to lead to defects in amphid ultrastructure. We have assigned 20 mutations to 13 new genes, called dyf-1 through dyf-13. We expect that the genes represented by dye-filling defective mutants are important for the differentiation of amphid and phasmid chemosensilla. 58 refs., 3 figs., 6 tabs.

  6. Mutations Affecting the Chemosensory Neurons of Caenorhabditis Elegans

    PubMed Central

    Starich, T. A.; Herman, R. K.; Kari, C. K.; Yeh, W. H.; Schackwitz, W. S.; Schuyler, M. W.; Collet, J.; Thomas, J. H.; Riddle, D. L.

    1995-01-01

    We have identified and characterized 95 mutations that reduce or abolish dye filling of amphid and phasmid neurons and that have little effect on viability, fertility or movement. Twenty-seven mutations occurred spontaneously in strains with a high frequency of transposon insertion. Sixty-eight were isolated after treatment with EMS. All of the mutations result in defects in one or more chemosensory responses, such as chemotaxis to ammonium chloride or formation of dauer larvae under conditions of starvation and overcrowding. Seventy-five of the mutations are alleles of 12 previously defined genes, mutations which were previously shown to lead to defects in amphid ultrastructure. We have assigned 20 mutations to 13 new genes, called dyf-1 through dyf-13. We expect that the genes represented by dye-filling defective mutants are important for the differentiation of amphid and phasmid chemosensilla. PMID:7705621

  7. Dominance of mutations affecting viability in Drosophila melanogaster.

    PubMed Central

    Fry, James D; Nuzhdin, Sergey V

    2003-01-01

    There have been several attempts to estimate the average dominance (ratio of heterozygous to homozygous effects) of spontaneous deleterious mutations in Drosophila melanogaster, but these have given inconsistent results. We investigated whether transposable element (TE) insertions have higher average dominance for egg-to-adult viability than do point mutations, a possibility suggested by the types of fitness-depressing effects that TEs are believed to have. If so, then variation in dominance estimates among strains and crosses would be expected as a consequence of variation in TE activity. As a first test, we estimated the average dominance of all mutations and of copia insertions in a set of lines that had accumulated spontaneous mutations for 33 generations. A traditional regression method gave a dominance estimate for all mutations of 0.17, whereas average dominance of copia insertions was 0.51; the difference between these two estimates approached significance (P = 0.08). As a second test, we reanalyzed Ohnishi 1974 data on dominance of spontaneous and EMS-induced mutations. Because a considerable fraction of spontaneous mutations are caused by TE insertions, whereas EMS induces mainly point mutations, we predicted that average dominance would decline with increasing EMS concentration. This pattern was observed, but again fell short of formal significance (P = 0.07). Taken together, however, the two results give modest support for the hypothesis that TE insertions have greater average dominance in their viability effects than do point mutations, possibly as a result of deleterious effects of expression of TE-encoded genes. PMID:12702680

  8. Survival through Symbiosis.

    ERIC Educational Resources Information Center

    Abdi, S. Wali

    1992-01-01

    Describes symbiosis and its significance in the day-to-day lives of plants and animals. Gives specific examples of mutualism, commensalism, and parasitism in the relationships among fungus and plant roots, animals and bacteria, birds and animals, fish, and predator and prey. (MDH)

  9. Symbiosis-mediated outbreaks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Symbiosis simply means "living together" and in its narrowest form can mean two species deriving mutual benefit from the association. Recent studies have made evident that insect associations with microorganisms can range the gamut from casual associations to obligate or context-dependent mutualisms...

  10. Kem Mutations Affect Nuclear Fusion in Saccharomyces Cerevisiae

    PubMed Central

    Kim, J.; Ljungdahl, P. O.; Fink, G. R.

    1990-01-01

    We have identified mutations in three genes of Saccharomyces cerevisiae, KEM1, KEM2 and KEM3, that enhance the nuclear fusion defect of kar1-1 yeast during conjugation. The KEM1 and KEM3 genes are located on the left arm of chromosome VII. Kem mutations reduce nuclear fusion whether the kem and the kar1-1 mutations are in the same or in different parents (i.e., in both kem kar1-1 X wild-type and kem X kar1-1 crosses). kem1 X kem1 crosses show a defect in nuclear fusion, but kem1 X wild-type crosses do not. Mutant kem1 strains are hypersensitive to benomyl, lose chromosomes at a rate 10-20-fold higher than KEM(+) strains, and lose viability upon nitrogen starvation. In addition, kem1/kem1 diploids are unable to sporulate. Cells containing a kem1 null allele grow very poorly, have an elongated rod-shape and are defective in spindle pole body duplication and/or separation. The KEM1 gene, which is expressed as a 5.5-kb mRNA transcript, contains a 4.6-kb open reading frame encoding a 175-kD protein. PMID:2076815

  11. Factors affecting mutational specificity induced by ionizing radiation and oxidizing radicals

    SciTech Connect

    Strauss, B.S.

    1992-01-01

    We propose to analyze the factors affecting the specificity of mutational change as induced by ionizing radiation and oxidizing radicals. We want to understand not only the rules that affect base substitution, but also the mechanism(s) by which additions and deletions are produced, since detections are a common consequence of radiation. We wish to carry out this analysis in an in vitro mutation system that permits us to analyze the role of base sequence, of polymerase and of mutagenic agent. Our system is designed to screen out most direct breaks as a cause of mutation and should indicate the changes resulting from base damage to the DNA.

  12. Parental Age Affects Somatic Mutation Rates in the Progeny of Flowering Plants1

    PubMed Central

    Singh, Amit Kumar; Bashir, Tufail; Sailer, Christian; Gurumoorthy, Viswanathan; Ramakrishnan, Anantha Maharasi; Dhanapal, Shanmuhapreya; Grossniklaus, Ueli; Baskar, Ramamurthy

    2015-01-01

    In humans, it is well known that the parental reproductive age has a strong influence on mutations transmitted to their progeny. Meiotic nondisjunction is known to increase in older mothers, and base substitutions tend to go up with paternal reproductive age. Hence, it is clear that the germinal mutation rates are a function of both maternal and paternal ages in humans. In contrast, it is unknown whether the parental reproductive age has an effect on somatic mutation rates in the progeny, because these are rare and difficult to detect. To address this question, we took advantage of the plant model system Arabidopsis (Arabidopsis thaliana), where mutation detector lines allow for an easy quantitation of somatic mutations, to test the effect of parental age on somatic mutation rates in the progeny. Although we found no significant effect of parental age on base substitutions, we found that frameshift mutations and transposition events increased in the progeny of older parents, an effect that is stronger through the maternal line. In contrast, intrachromosomal recombination events in the progeny decrease with the age of the parents in a parent-of-origin-dependent manner. Our results clearly show that parental reproductive age affects somatic mutation rates in the progeny and, thus, that some form of age-dependent information, which affects the frequency of double-strand breaks and possibly other processes involved in maintaining genome integrity, is transmitted through the gametes. PMID:25810093

  13. Factors affecting mutational specificity induced by ionizing radiation and oxidizing radicals. Progress report

    SciTech Connect

    Strauss, B.

    1992-07-01

    We propose to analyze the factors affecting the specificity of mutational change as induced by ionizing radiation and oxidizing radicals. We want to understand not only the rules the affect base substitution but also the mechanisms(s) by which additions and deletions are produced, since deletions are a common consequence of radiation. We wish to carry out this analysis in an in vitro mutation system that permits us to analyze the role of base sequence, of polymerase and of mutagenic agent. Our system is designed to screen out most direct breaks as a cause of mutation and should indicate the changes resulting from base damage to the DNA. Questions addressed include: 1. What types of base substitution mutations are induced by ionizing radiation and oxidizing radicals? 2. Are deletions and/or additions produced? 3. Is there a difference in type of mutation produced dependent on the polymerase used? Do mammalian polymerase plus their accessory factors result in different patterns of mutation. 4. What is the mechanism by which base damage is converted to mutation. Our proposal was based on utilization of an in vitro system in which mutations generated by the in vitro copying of a reporter gene sequence could be readily scored.

  14. Factors affecting mutational specificity induced by ionizing radiation and oxidizing radicals

    SciTech Connect

    Strauss, B.

    1992-01-01

    We propose to analyze the factors affecting the specificity of mutational change as induced by ionizing radiation and oxidizing radicals. We want to understand not only the rules the affect base substitution but also the mechanisms(s) by which additions and deletions are produced, since deletions are a common consequence of radiation. We wish to carry out this analysis in an in vitro mutation system that permits us to analyze the role of base sequence, of polymerase and of mutagenic agent. Our system is designed to screen out most direct breaks as a cause of mutation and should indicate the changes resulting from base damage to the DNA. Questions addressed include: 1. What types of base substitution mutations are induced by ionizing radiation and oxidizing radicals 2. Are deletions and/or additions produced 3. Is there a difference in type of mutation produced dependent on the polymerase used Do mammalian polymerase plus their accessory factors result in different patterns of mutation. 4. What is the mechanism by which base damage is converted to mutation. Our proposal was based on utilization of an in vitro system in which mutations generated by the in vitro copying of a reporter gene sequence could be readily scored.

  15. Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines.

    PubMed

    Chappuis-Flament, S; Pasini, A; De Vita, G; Ségouffin-Cariou, C; Fusco, A; Attié, T; Lenoir, G M; Santoro, M; Billaud, M

    1998-12-03

    The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.

  16. Tumor-specific mutations in low-frequency genes affect their functional properties.

    PubMed

    Erdem-Eraslan, Lale; Heijsman, Daphne; de Wit, Maurice; Kremer, Andreas; Sacchetti, Andrea; van der Spek, Peter J; Sillevis Smitt, Peter A E; French, Pim J

    2015-05-01

    Causal genetic changes in oligodendrogliomas (OD) with 1p/19q co-deletion include mutations in IDH1, IDH2, CIC, FUBP1, TERT promoter and NOTCH1. However, it is generally assumed that more somatic mutations are required for tumorigenesis. This study aimed to establish whether genes mutated at low frequency can be involved in OD initiation and/or progression. We performed whole-genome sequencing on three anaplastic ODs with 1p/19q co-deletion. To estimate mutation frequency, we performed targeted resequencing on an additional 39 ODs. Whole-genome sequencing identified a total of 55 coding mutations (range 8-32 mutations per tumor), including known abnormalities in IDH1, IDH2, CIC and FUBP1. We also identified mutations in genes, most of which were previously not implicated in ODs. Targeted resequencing on 39 additional ODs confirmed that these genes are mutated at low frequency. Most of the mutations identified were predicted to have a deleterious functional effect. Functional analysis on a subset of these genes (e.g. NTN4 and MAGEH1) showed that the mutation affects the subcellular localization of the protein (n = 2/12). In addition, HOG cells stably expressing mutant GDI1 or XPO7 showed altered cell proliferation compared to those expressing wildtype constructs. Similarly, HOG cells expressing mutant SASH3 or GDI1 showed altered migration. The significantly higher rate of predicted deleterious mutations, the changes in subcellular localization and the effects on proliferation and/or migration indicate that many of these genes functionally may contribute to gliomagenesis and/or progression. These low-frequency genes and their affected pathways may provide new treatment targets for this tumor type.

  17. Diaphanous gene mutation affects spiral cleavage and chirality in snails

    PubMed Central

    Kuroda, Reiko; Fujikura, Kohei; Abe, Masanori; Hosoiri, Yuji; Asakawa, Shuichi; Shimizu, Miho; Umeda, Shin; Ichikawa, Futaba; Takahashi, Hiromi

    2016-01-01

    L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus). PMID:27708420

  18. The callipyge mutation and other genes that affect muscle hypertrophy in sheep

    PubMed Central

    2005-01-01

    Genetic strategies to improve the profitability of sheep operations have generally focused on traits for reproduction. However, natural mutations exist in sheep that affect muscle growth and development, and the exploitation of these mutations in breeding strategies has the potential to significantly improve lamb-meat quality. The best-documented mutation for muscle development in sheep is callipyge (CLPG), which causes a postnatal muscle hypertrophy that is localized to the pelvic limbs and loin. Enhanced skeletal muscle growth is also observed in animals with the Carwell (or rib-eye muscling) mutation, and a double-muscling phenotype has been documented for animals of the Texel sheep breed. However, the actual mutations responsible for these muscular hypertrophy phenotypes in sheep have yet to be identified, and further characterization of the genetic basis for these phenotypes will provide insight into the biological control of muscle growth and body composition. PMID:15601596

  19. Activating Mutations Affecting the Dbl Homology Domain of SOS2 Cause Noonan Syndrome.

    PubMed

    Cordeddu, Viviana; Yin, Jiani C; Gunnarsson, Cecilia; Virtanen, Carl; Drunat, Séverine; Lepri, Francesca; De Luca, Alessandro; Rossi, Cesare; Ciolfi, Andrea; Pugh, Trevor J; Bruselles, Alessandro; Priest, James R; Pennacchio, Len A; Lu, Zhibin; Danesh, Arnavaz; Quevedo, Rene; Hamid, Alaa; Martinelli, Simone; Pantaleoni, Francesca; Gnazzo, Maria; Daniele, Paola; Lissewski, Christina; Bocchinfuso, Gianfranco; Stella, Lorenzo; Odent, Sylvie; Philip, Nicole; Faivre, Laurence; Vlckova, Marketa; Seemanova, Eva; Digilio, Cristina; Zenker, Martin; Zampino, Giuseppe; Verloes, Alain; Dallapiccola, Bruno; Roberts, Amy E; Cavé, Hélène; Gelb, Bruce D; Neel, Benjamin G; Tartaglia, Marco

    2015-11-01

    The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

  20. Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes.

    PubMed

    Gimenez, Luis E; Babilon, Stefanie; Wanka, Lizzy; Beck-Sickinger, Annette G; Gurevich, Vsevolod V

    2014-07-01

    Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor.

  1. Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes

    PubMed Central

    Gimenez, Luis E.; Babilon, Stefanie; Wanka, Lizzy; Beck-Sickinger, Annette G.; Gurevich, Vsevolod V.

    2014-01-01

    Based on the identification of residues that determine receptor selectivity in arrestins and the phylogenetic analysis of the arrestin (arr) family, we introduced fifteen mutations of receptor-discriminator residues in arr-3, which were identified previously using mutagenesis, in vitro binding, and BRET-based recruitment assay in intact cells. The effects of these mutations were tested using neuropeptide Y receptors Y1R and Y2R. NPY-elicited arr-3 recruitment to Y1R was not affected by these mutations, or even alanine substitution of all ten residues (arr-3-NCA), which prevented arr-3 binding to other receptors tested so far. However, NCA and two other mutations prevented agonist-independent arr-3 pre-docking to Y1R. In contrast, eight out of 15 mutations significantly reduced agonist-dependent arr-3 recruitment to Y2R. NCA eliminated arr-3 binding to active Y2R, whereas Tyr239Thr reduced it ~7-fold. Thus, manipulation of key residues on the receptor-binding surface generates arr-3 with high preference for Y1R over Y2R. Several mutations differentially affect arr-3 pre-docking and agonist-induced recruitment. Thus, arr-3 recruitment to the receptor involves several mechanistically distinct steps. Targeted mutagenesis can fine-tune arrestins directing them to specific receptors and particular activation states of the same receptor. PMID:24686081

  2. Uner Tan syndrome caused by a homozygous TUBB2B mutation affecting microtubule stability.

    PubMed

    Breuss, Martin W; Nguyen, Thai; Srivatsan, Anjana; Leca, Ines; Tian, Guoling; Fritz, Tanja; Hansen, Andi H; Musaev, Damir; McEvoy-Venneri, Jennifer; James, Kiely N; Rosti, Rasim O; Scott, Eric; Tan, Uner; Kolodner, Richard D; Cowan, Nicholas J; Keays, David A; Gleeson, Joseph G

    2016-12-23

    The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.

  3. Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions

    PubMed Central

    Erdeniz, Naz; Nguyen, Megan; Deschênes, Suzanne M.; Liskay, R. Michael

    2008-01-01

    Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLα, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X)2E(X)4E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMR-dependent suppression of homeologous recombination in yeast and responses to Sn1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLα is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions. PMID:17567544

  4. Symbiosis in eukaryotic evolution.

    PubMed

    López-García, Purificación; Eme, Laura; Moreira, David

    2017-02-28

    Fifty years ago, Lynn Margulis, inspiring in early twentieth-century ideas that put forward a symbiotic origin for some eukaryotic organelles, proposed a unified theory for the origin of the eukaryotic cell based on symbiosis as evolutionary mechanism. Margulis was profoundly aware of the importance of symbiosis in the natural microbial world and anticipated the evolutionary significance that integrated cooperative interactions might have as mechanism to increase cellular complexity. Today, we have started fully appreciating the vast extent of microbial diversity and the importance of syntrophic metabolic cooperation in natural ecosystems, especially in sediments and microbial mats. Also, not only the symbiogenetic origin of mitochondria and chloroplasts has been clearly demonstrated, but improvement in phylogenomic methods combined with recent discoveries of archaeal lineages more closely related to eukaryotes further support the symbiogenetic origin of the eukaryotic cell. Margulis left us in legacy the idea of 'eukaryogenesis by symbiogenesis'. Although this has been largely verified, when, where, and specifically how eukaryotic cells evolved are yet unclear. Here, we shortly review current knowledge about symbiotic interactions in the microbial world and their evolutionary impact, the status of eukaryogenetic models and the current challenges and perspectives ahead to reconstruct the evolutionary path to eukaryotes.

  5. Mutations of the Wiskott-Aldrich Syndrome Protein affect protein expression and dictate the clinical phenotypes.

    PubMed

    Ochs, Hans D

    2009-01-01

    Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and in rare instances congenital X-linked neutropenia (XLN). WASP is a regulator of actin polymerization in hematopoietic cells with well-defined functional domains that are involved in cell signaling and cell locomotion, immune synapse formation, and apoptosis. Mutations of WASP are located throughout the gene and either inhibit or disregulate normal WASP function. Analysis of a large patient population demonstrates a strong phenotype-genotype correlation. Classic WAS occurs when WASP is absent, XLT when mutated WASP is expressed and XLN when missense mutations occur in the Cdc42-binding site. However, because there are exceptions to this rule it is difficult to predict the long-term prognosis of a given affected boy solely based on the analysis of WASP expression.

  6. Suppressors of Mutations in the rII Gene of Bacteriophage T4 Affect Promoter Utilization

    PubMed Central

    Hall, Dwight H.; Snyder, Ronald D.

    1981-01-01

    Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in λ lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH2 reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L2 decreases the folate analog resistance and the inability of sip2 to grow on CTr5X. L2 itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L2 affects another regulatory gene related to the mot gene. PMID:7262547

  7. Determinant factors of industrial symbiosis: greening Pasir Gudang industrial park

    NASA Astrophysics Data System (ADS)

    Teh, B. T.; Ho, C. S.; Matsuoka, Y.; Chau, L. W.; Gomi, K.

    2014-02-01

    Green industry has been identified as an important element in attaining greater sustainability. It calls for harmonizing robust economic growth with environment protection. Industries, particularly in developing and transitional nations such as Malaysia, are in need of a reform. Many experts and international organizations suggest the concept of industrial symbiosis. Mainly, there are successful cases of industrial symbiosis practices around the world. However, there are numerous cases of failure too. As industrial symbiosis is an emerging new approach, with a short history of two decades, a lot of researches are generally focused on narrow context and technical details. There is a lack of concerted efforts to look into the drivers and barriers of industrial symbiosis across different cases. This paper aims to examine the factors influencing the development of industrial symbiosis from various countries to supports such networks to evolve in Pasir Gudang. The findings show institution, law and regulation, finance, awareness and capacity building, technology, research and development, information, collaboration, market, geography proximity, environmental issues and industry structure affect the formation of industrial symbiosis.

  8. Mutations Affecting RNA Polymerase I-Stimulated Exchange and Rdna Recombination in Yeast

    PubMed Central

    Lin, Y. H.; Keil, R. L.

    1991-01-01

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination. PMID:2016045

  9. Different inactivating mutations of the mineralocorticoid receptor in fourteen families affected by type I pseudohypoaldosteronism.

    PubMed

    Sartorato, Paola; Lapeyraque, Anne-Laure; Armanini, Decio; Kuhnle, Ursula; Khaldi, Yasmina; Salomon, Rémi; Abadie, Véronique; Di Battista, Eliana; Naselli, Arturo; Racine, Alain; Bosio, Maurizio; Caprio, Massimiliano; Poulet-Young, Véronique; Chabrolle, Jean-Pierre; Niaudet, Patrick; De Gennes, Christiane; Lecornec, Marie-Hélène; Poisson, Elodie; Fusco, Anna Maria; Loli, Paola; Lombès, Marc; Zennaro, Maria-Christina

    2003-06-01

    We have analyzed the human mineralocorticoid receptor (hMR) gene in 14 families with autosomal dominant or sporadic pseudohypoaldosteronism (PHA1), a rare form of mineralocorticoid resistance characterized by neonatal renal salt wasting and failure to thrive. Six heterozygous mutations were detected. Two frameshift mutations in exon 2 (insT1354, del8bp537) and one nonsense mutation in exon 4 (C2157A, Cys645stop) generate truncated proteins due to premature stop codons. Three missense mutations (G633R, Q776R, L979P) differently affect hMR function. The DNA binding domain mutant R633 exhibits reduced maximal transactivation, although its binding characteristics and ED(50) of transactivation are comparable with wild-type hMR. Ligand binding domain mutants R776 and P979 present reduced or absent aldosterone binding, respectively, which is associated with reduced or absent ligand-dependent transactivation capacity. Finally, P979 possesses a transdominant negative effect on wild-type hMR activity, whereas mutations G633R and Q776R probably result in haploinsufficiency in PHA1 patients. We conclude that hMR mutations are a common feature of autosomal dominant PHA1, being found in 70% of our familial cases. Their absence in some families underscores the importance of an extensive investigation of the hMR gene and the role of precise diagnostic procedures to allow for identification of other genes potentially involved in the disease.

  10. A novel mutation in TFL1 homolog affecting determinacy in cowpea (Vigna unguiculata).

    PubMed

    Dhanasekar, P; Reddy, K S

    2015-02-01

    Mutations in the widely conserved Arabidopsis Terminal Flower 1 (TFL1) gene and its homologs have been demonstrated to result in determinacy across genera, the knowledge of which is lacking in cowpea. Understanding the molecular events leading to determinacy of apical meristems could hasten development of cowpea varieties with suitable ideotypes. Isolation and characterization of a novel mutation in cowpea TFL1 homolog (VuTFL1) affecting determinacy is reported here for the first time. Cowpea TFL1 homolog was amplified using primers designed based on conserved sequences in related genera and sequence variation was analysed in three gamma ray-induced determinate mutants, their indeterminate parent "EC394763" and two indeterminate varieties. The analyses of sequence variation exposed a novel SNP distinguishing the determinate mutants from the indeterminate types. The non-synonymous point mutation in exon 4 at position 1,176 resulted from transversion of cytosine (C) to adenine (A) leading to an amino acid change (Pro-136 to His) in determinate mutants. The effect of the mutation on protein function and stability was predicted to be detrimental using different bioinformatics/computational tools. The functionally significant novel substitution mutation is hypothesized to affect determinacy in the cowpea mutants. Development of suitable regeneration protocols in this hitherto recalcitrant crop and subsequent complementation assay in mutants or over-expressing assay in parents could decisively conclude the role of the SNP in regulating determinacy in these cowpea mutants.

  11. Novel mutations affecting LRP5 splicing in patients with osteoporosis-pseudoglioma syndrome (OPPG)

    PubMed Central

    Laine, C M; Chung, B D; Susic, M; Prescott, T; Semler, O; Fiskerstrand, T; D'Eufemia, P; Castori, M; Pekkinen, M; Sochett, E; Cole, W G; Netzer, C; Mäkitie, O

    2011-01-01

    Osteoporosis-pseudoglioma sydrome (OPPG) is an autosomal recessive disorder with early-onset severe osteoporosis and blindness, caused by biallelic loss-of-function mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene. Heterozygous carriers exhibit a milder bone phenotype. Only a few splice mutations in LRP5 have been published. We present clinical and genetic data for four patients with novel LRP5 mutations, three of which affect splicing. Patients were evaluated clinically and by radiography and bone densitometry. Genetic screening of LRP5 was performed on the basis of the clinical diagnosis of OPPG. Splice aberrances were confirmed by cDNA sequencing or exon trapping. The effect of one splice mutation on LRP5 protein function was studied. A novel splice-site mutation c.1584+4A>T abolished the donor splice site of exon 7 and activated a cryptic splice site, which led to an in-frame insertion of 21 amino acids (p.E528_V529ins21). Functional studies revealed severely impaired signal transduction presumably caused by defective intracellular transport of the mutated receptor. Exon trapping was used on two samples to confirm that splice-site mutations c.4112-2A>G and c.1015+1G>T caused splicing-out of exons 20 and 5, respectively. One patient carried a homozygous deletion of exon 4 causing the loss of exons 4 and 5, as demonstrated by cDNA analysis. Our results broaden the spectrum of mutations in LRP5 and provide the first functional data on splice aberrations. PMID:21407258

  12. Identification and Functional Characterization of GAA Mutations in Colombian Patients Affected by Pompe Disease.

    PubMed

    Niño, Mónica Yasmín; Mateus, Heidi Eliana; Fonseca, Dora Janeth; Kroos, Marian A; Ospina, Sandra Yaneth; Mejía, Juan Fernando; Uribe, Jesús Alfredo; Reuser, Arnold J J; Laissue, Paul

    2013-01-01

    Pompe disease (PD) is a recessive metabolic disorder characterized by acid α-glucosidase (GAA) deficiency, which results in lysosomal accumulation of glycogen in all tissues, especially in skeletal muscles. PD clinical course is mainly determined by the nature of the GAA mutations. Although ~400 distinct GAA sequence variations have been described, the genotype-phenotype correlation is not always evident.In this study, we describe the first clinical and genetic analysis of Colombian PD patients performed in 11 affected individuals. GAA open reading frame sequencing revealed eight distinct mutations related to PD etiology including two novel missense mutations, c.1106 T > C (p.Leu369Pro) and c.2236 T > C (p.Trp746Arg). In vitro functional studies showed that the structural changes conferred by both mutations did not inhibit the synthesis of the 110 kD GAA precursor form but affected the processing and intracellular transport of GAA. In addition, analysis of previously described variants located at this position (p.Trp746Gly, p.Trp746Cys, p.Trp746Ser, p.Trp746X) revealed new insights in the molecular basis of PD. Notably, we found that p.Trp746Cys mutation, which was previously described as a polymorphism as well as a causal mutation, displayed a mild deleterious effect. Interestingly and by chance, our study argues in favor of a remarkable Afro-American and European ancestry of the Colombian population. Taken together, our report provides valuable information on the PD genotype-phenotype correlation, which is expected to facilitate and improve genetic counseling of affected individuals and their families.

  13. Expanding genomics of mycorrhizal symbiosis

    DOE PAGES

    Kuo, Alan; Kohler, Annegret; Martin, Francis M.; ...

    2014-11-04

    The mycorrhizal symbiosis between soil fungi and plant roots is a ubiquitous mutualism that plays key roles in plant nutrition, soil health, and carbon cycling. The symbiosis evolved repeatedly and independently as multiple morphotypes [e.g., arbuscular mycorrhizae (AM), ectomycorrhizal (ECM)] in multiple fungal clades (e.g., phyla Glomeromycota, Ascomycota, Basidiomycota). The accessibility and cultivability of many mycorrhizal partners make them ideal models for symbiosis studies. Alongside molecular, physiological, and ecological investigations, sequencing led to the first three mycorrhizal fungal genomes, representing two morphotypes and three phyla. The genome of the ECM basidiomycete Laccaria bicolor showed that the mycorrhizal lifestyle can evolvemore » through loss of plant cell wall-degrading enzymes (PCWDEs) and expansion of lineage-specific gene families such as short secreted protein (SSP) effectors. The genome of the ECM ascomycete Tuber melanosporum showed that the ECM type can evolve without expansion of families as in Laccaria, and thus a different set of symbiosis genes. The genome of the AM glomeromycete Rhizophagus irregularis showed that despite enormous phylogenetic distance and morphological difference from the other two fungi, symbiosis can involve similar solutions as symbiosis-induced SSPs and loss of PCWDEs. The three genomes provide a solid base for addressing fundamental questions about the nature and role of a vital mutualism.« less

  14. Expanding genomics of mycorrhizal symbiosis

    SciTech Connect

    Kuo, Alan; Kohler, Annegret; Martin, Francis M.; Grigoriev, Igor V.

    2014-11-04

    The mycorrhizal symbiosis between soil fungi and plant roots is a ubiquitous mutualism that plays key roles in plant nutrition, soil health, and carbon cycling. The symbiosis evolved repeatedly and independently as multiple morphotypes [e.g., arbuscular mycorrhizae (AM), ectomycorrhizal (ECM)] in multiple fungal clades (e.g., phyla Glomeromycota, Ascomycota, Basidiomycota). The accessibility and cultivability of many mycorrhizal partners make them ideal models for symbiosis studies. Alongside molecular, physiological, and ecological investigations, sequencing led to the first three mycorrhizal fungal genomes, representing two morphotypes and three phyla. The genome of the ECM basidiomycete Laccaria bicolor showed that the mycorrhizal lifestyle can evolve through loss of plant cell wall-degrading enzymes (PCWDEs) and expansion of lineage-specific gene families such as short secreted protein (SSP) effectors. The genome of the ECM ascomycete Tuber melanosporum showed that the ECM type can evolve without expansion of families as in Laccaria, and thus a different set of symbiosis genes. The genome of the AM glomeromycete Rhizophagus irregularis showed that despite enormous phylogenetic distance and morphological difference from the other two fungi, symbiosis can involve similar solutions as symbiosis-induced SSPs and loss of PCWDEs. The three genomes provide a solid base for addressing fundamental questions about the nature and role of a vital mutualism.

  15. Expanding genomics of mycorrhizal symbiosis

    PubMed Central

    Kuo, Alan; Kohler, Annegret; Martin, Francis M.; Grigoriev, Igor V.

    2014-01-01

    The mycorrhizal symbiosis between soil fungi and plant roots is a ubiquitous mutualism that plays key roles in plant nutrition, soil health, and carbon cycling. The symbiosis evolved repeatedly and independently as multiple morphotypes [e.g., arbuscular mycorrhizae (AM), ectomycorrhizal (ECM)] in multiple fungal clades (e.g., phyla Glomeromycota, Ascomycota, Basidiomycota). The accessibility and cultivability of many mycorrhizal partners make them ideal models for symbiosis studies. Alongside molecular, physiological, and ecological investigations, sequencing led to the first three mycorrhizal fungal genomes, representing two morphotypes and three phyla. The genome of the ECM basidiomycete Laccaria bicolor showed that the mycorrhizal lifestyle can evolve through loss of plant cell wall-degrading enzymes (PCWDEs) and expansion of lineage-specific gene families such as short secreted protein (SSP) effectors. The genome of the ECM ascomycete Tuber melanosporum showed that the ECM type can evolve without expansion of families as in Laccaria, and thus a different set of symbiosis genes. The genome of the AM glomeromycete Rhizophagus irregularis showed that despite enormous phylogenetic distance and morphological difference from the other two fungi, symbiosis can involve similar solutions as symbiosis-induced SSPs and loss of PCWDEs. The three genomes provide a solid base for addressing fundamental questions about the nature and role of a vital mutualism. PMID:25408690

  16. Mutations affecting mitotic recombination frequency in haploids and diploids of the filamentous fungus Aspergillus nidulans.

    PubMed

    Parag, Y; Parag, G

    1975-01-01

    A haploid strain of Asp. nidulans with a chromosome segment in duplicate (one in normal position on chromosome I, one translocated to chromosome II) shows mitotic recombination, mostly by conversion, in adE in a frequency slightly higher than in the equivalent diploid. A method has been devised, using this duplication, for the selection of rec and uvs mutations. Six rec mutations have been found which decrease recombination frequency in the haploid. One mutation selected as UV sensitive showed a hundred fold increase in recombination frequency in the haploid (pop mutation) and probably the same in diploids. The increased frequency is both in gene conversion and in crossing over, and the exchanges appear in clusters of two or more. pop is allelic to uvsB (Jansen, 1970) which had been found to affect mitotic but not meiotic recombination. It is suggested that mutations of this type interfere with the control mechanism which determines that high recombination is confirmed to the meiotic nuclei and avoided in somatic nuclei.

  17. Yield and yield components of hybrid corn (Zea mays L.) as affected by mycorrhizal symbiosis and zinc sulfate under drought stress.

    PubMed

    Sajedi, N A; Ardakani, M R; Rejali, F; Mohabbati, F; Miransari, Mohammad

    2010-12-01

    With respect to the significance of improving hybrid corn performance under stress, this experiment was conducted at the Islamic Azad University, Arak Branch, Iran. A complete randomized block design with three levels of irrigations (at 100%, 75% and 50% crop water requirement), two levels of arbuscular mycorrhizal (AM) fungi (Glumus intraradisis) (including control), and three levels of zinc (Zn) sulfate (0, 25 and 45 kg ha(-1)), was performed. Results of the 2-year experiments indicated that irrigation treatment significantly affected corn yield and its components at P = 1%. AM fungi and increasing Zn levels also resulted in similar effects on corn growth and production. Although AM fungi did not significantly affect corn growth at the non-stressed irrigation treatment, at moderate drought stress AM fungi significantly enhanced corn quality and yield relative to the control treatment. The combined effects of AM fungi and Zn sulfate at 45 kg ha(-1) application significantly affected corn growth and production. In addition, the tripartite treatments significantly enhanced corn yield at P = 1%. Effects of Zn and AM fungi on plant growth under drought stress is affected by the stress level.

  18. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

  19. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    SciTech Connect

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-08-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

  20. GENETIC MUTATIONS AFFECTING THE FIRST LINE ERADICATION THERAPY OF Helicobacter pylori-INFECTED EGYPTIAN PATIENTS

    PubMed Central

    RAMZY, Iman; ELGAREM, Hassan; HAMZA, Iman; GHAITH, Doaa; ELBAZ, Tamer; ELHOSARY, Waleed; MOSTAFA, Gehan; ELZAHRY, Mohammad A. Mohey Eldin

    2016-01-01

    SUMMARY Introduction: Several genetic mutations affect the first-line triple therapy for Helicobacter pylori. We aimed to study the most common genetic mutations affecting the metronidazole and clarithromycin therapy for H. pylori-infected Egyptian patients. Patients and Methods: In our study, we included 100 successive dyspeptic patients scheduled for diagnosis through upper gastroscopy at Cairo's University Hospital, Egypt. Gastric biopsies were tested for the presence of H. pylori by detection of the 16S rRNA gene. Positive biopsies were further studied for the presence of the rdxA gene deletion by Polymerase Chain Reaction (PCR), while clarithromycin resistance was investigated by the presence of nucleotide substitutions within H. pylori 23S rRNA V domain using MboII and BsaI to carry out a Restricted Fragment Length Polymorphism (RFLP) assay. Results: Among 70 H. pylori positive biopsies, the rdxA gene deletion was detected in 44/70 (62.9%) samples, while predominance of the A2142G mutations within the H. pylori 23S rRNA V domain was evidenced in 39/70 (55.7%) of the positive H. pylori cases. No statistically significant difference was found between the presence of gene mutations and different factors such as patients 'age, gender, geographic distribution, symptoms and endoscopic findings. Conclusion: Infection with mutated H. pylori strains is considerably high, a finding that imposes care in the use of the triple therapy to treat H. pylori in Egypt, since the guidelines recommend to abandon the standard triple therapy when the primary clarithromycin resistance rate is over 20%1. PMID:27982354

  1. Dynamics of a Recurrent Buchnera Mutation That Affects Thermal Tolerance of Pea Aphid Hosts

    PubMed Central

    Burke, Gaelen R.; McLaughlin, Heather J.; Simon, Jean-Christophe; Moran, Nancy A.

    2010-01-01

    Mutations in maternally transmitted symbionts can affect host fitness. In this study we investigate a mutation in an obligate bacterial symbiont (Buchnera), which has dramatic effects on the heat tolerance of pea aphid hosts (Acyrthosiphon pisum). The heat-sensitive allele arises through a single base deletion in a homopolymer within the promoter of ibpA, which encodes a universal small heat-shock protein. In laboratory cultures reared under cool conditions (20°), the rate of fixation (1.4 × 10−3 substitutions per Buchnera replication) is much higher than the previously estimated mutation rate for single base deletions in homopolymers in the Buchnera genome, implying a strong selective benefit. This mutation recurs in natural populations, but seldom reaches high frequencies, implying that it is only rarely favored by selection. Another potential source of physiological stress in pea aphids is infection by other microorganisms, including facultative bacterial symbionts, which occur in a majority of pea aphids in field populations. Frequency of the heat-sensitive Buchnera allele is negatively correlated with presence of facultative symbionts in both laboratory colonies and field populations, suggesting that these infections impose stress that is ameliorated by ibpA expression. This single base polymorphism in Buchnera has the potential to allow aphid populations to adapt quickly to prevailing conditions. PMID:20610410

  2. Mutations in the West Nile prM protein affect VLP and virion secretion in vitro.

    PubMed

    Calvert, Amanda E; Huang, Claire Y-H; Blair, Carol D; Roehrig, John T

    2012-11-10

    Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.

  3. Identification of quantitative trait loci affecting ectomycorrhizal symbiosis in an interspecific F1 poplar cross and differential expression of genes in ectomycorrhizas of the two parents: Populus deltoides and Populus trichocarpa

    SciTech Connect

    Labbe, Jessy L; Jorge, Veronique; Vion, Patrice; Marcais, Benoit; Bastien, Catherine; Tuskan, Gerald A; Martin, Francis; Le Tacon, F

    2011-01-01

    A Populus deltoides Populus trichocarpa F1 pedigree was analyzed for quantitative trait loci (QTLs) affecting ectomycorrhizal development and for microarray characterization of gene networks involved in this symbiosis. A 300 genotype progeny set was evaluated for its ability to form ectomycorrhiza with the basidiomycete Laccaria bicolor. The percentage of mycorrhizal root tips was determined on the root systems of all 300 progeny and their two parents. QTL analysis identified four significant QTLs, one on the P. deltoides and three on the P. trichocarpa genetic maps. These QTLs were aligned to the P. trichocarpa genome and each contained several megabases and encompass numerous genes. NimbleGen whole-genome microarray, using cDNA from RNA extracts of ectomycorrhizal root tips from the parental genotypes P. trichocarpa and P. deltoides, was used to narrow the candidate gene list. Among the 1,543 differentially expressed genes (p value 0.05; 5.0-fold change in transcript level) having different transcript levels in mycorrhiza of the two parents, 41 transcripts were located in the QTL intervals: 20 in Myc_d1, 14 in Myc_t1, and seven in Myc_t2, while no significant differences among transcripts were found in Myc_t3. Among these 41 transcripts, 25 were overrepresented in P. deltoides relative to P. trichocarpa; 16 were overrepresented in P. trichocarpa. The transcript showing the highest overrepresentation in P. trichocarpa mycorrhiza libraries compared to P. deltoides mycorrhiza codes for an ethylene-sensitive EREBP-4 protein which may repress defense mechanisms in P. trichocarpa while the highest overrepresented transcripts in P. deltoides code for proteins/genes typically associated with pathogen resistance.

  4. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

    PubMed Central

    Svensson, Lena; Howarth, Kimberley; McDowall, Alison; Patzak, Irene; Evans, Rachel; Ussar, Siegfried; Moser, Markus; Metin, Ayse; Fried, Mike; Tomlinson, Ian; Hogg, Nancy

    2009-01-01

    Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration. PMID:19234463

  5. Epilepsy due to PNPO mutations: genotype, environment and treatment affect presentation and outcome

    PubMed Central

    Mills, Philippa B.; Camuzeaux, Stephane S.M.; Footitt, Emma J.; Mills, Kevin A.; Gissen, Paul; Fisher, Laura; Das, Krishna B.; Varadkar, Sophia M.; Zuberi, Sameer; McWilliam, Robert; Stödberg, Tommy; Plecko, Barbara; Baumgartner, Matthias R.; Maier, Oliver; Calvert, Sophie; Riney, Kate; Wolf, Nicole I.; Livingston, John H.; Bala, Pronab; Morel, Chantal F.; Feillet, François; Raimondi, Francesco; Del Giudice, Ennio; Chong, W. Kling; Pitt, Matthew

    2014-01-01

    The first described patients with pyridox(am)ine 5’-phosphate oxidase deficiency all had neonatal onset seizures that did not respond to treatment with pyridoxine but responded to treatment with pyridoxal 5’-phosphate. Our data suggest, however, that the clinical spectrum of pyridox(am)ine 5’-phosphate oxidase deficiency is much broader than has been reported in the literature. Sequencing of the PNPO gene was undertaken for a cohort of 82 individuals who had shown a reduction in frequency and severity of seizures in response to pyridoxine or pyridoxal 5’-phosphate. Novel sequence changes were studied using a new cell-free expression system and a mass spectrometry-based assay for pyridoxamine phosphate oxidase. Three groups of patients with PNPO mutations that had reduced enzyme activity were identified: (i) patients with neonatal onset seizures responding to pyridoxal 5’-phosphate (n = 6); (ii) a patient with infantile spasms (onset 5 months) responsive to pyridoxal 5’-phosphate (n = 1); and (iii) patients with seizures starting under 3 months of age responding to pyridoxine (n = 8). Data suggest that certain genotypes (R225H/C and D33V) are more likely to result in seizures that to respond to treatment with pyridoxine. Other mutations seem to be associated with infertility, miscarriage and prematurity. However, the situation is clearly complex with the same combination of mutations being seen in patients who responded and did not respond to pyridoxine. It is possible that pyridoxine responsiveness in PNPO deficiency is affected by prematurity and age at the time of the therapeutic trial. Other additional factors that are likely to influence treatment response and outcome include riboflavin status and how well the foetus has been supplied with vitamin B6 by the mother. For some patients there was a worsening of symptoms on changing from pyridoxine to pyridoxal 5’-phosphate. Many of the mutations in PNPO affected residues involved in binding flavin

  6. ramR mutations affecting fluoroquinolone susceptibility in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198.

    PubMed

    Baucheron, Sylvie; Le Hello, Simon; Doublet, Benoît; Giraud, Etienne; Weill, François-Xavier; Cloeckaert, Axel

    2013-01-01

    A screening for non-target mutations affecting fluoroquinolone susceptibility was conducted in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198. Among a panel of representative isolates (n = 27), covering the epidemic, only three showed distinct mutations in ramR resulting in enhanced expression of genes encoding the AcrAB-TolC efflux system and low increase in ciprofloxacin MIC. No mutations were detected in other regulatory regions of this efflux system. Ciprofloxacin resistance in serovar Kentucky ST198 is thus currently mainly due to multiple target gene mutations.

  7. Recombination affects accumulation of damaging and disease-associated mutations in human populations.

    PubMed

    Hussin, Julie G; Hodgkinson, Alan; Idaghdour, Youssef; Grenier, Jean-Christophe; Goulet, Jean-Philippe; Gbeha, Elias; Hip-Ki, Elodie; Awadalla, Philip

    2015-04-01

    Many decades of theory have demonstrated that, in non-recombining systems, slightly deleterious mutations accumulate non-reversibly, potentially driving the extinction of many asexual species. Non-recombining chromosomes in sexual organisms are thought to have degenerated in a similar fashion; however, it is not clear the extent to which damaging mutations accumulate along chromosomes with highly variable rates of crossing over. Using high-coverage sequencing data from over 1,400 individuals in the 1000 Genomes and CARTaGENE projects, we show that recombination rate modulates the distribution of putatively deleterious variants across the entire human genome. Exons in regions of low recombination are significantly enriched for deleterious and disease-associated variants, a signature varying in strength across worldwide human populations with different demographic histories. Regions with low recombination rates are enriched for highly conserved genes with essential cellular functions and show an excess of mutations with demonstrated effects on health, a phenomenon likely affecting disease susceptibility in humans.

  8. Mutations affecting catabolite repression of the L-arabinose regulon in Escherichia coli B/r.

    PubMed

    Heffernan, L; Bass, R; Englesberg, E

    1976-06-01

    Expression of the L-arabinose regulon in Escherichia coli B/r requires, among other things, cyclic adenosine-3', 5'-monophosphate (cAMP) and the cAMP receptor protein (CRP). Mutants deficient in adenyl cyclase (cya-), the enzyme which synthesizes cAMP, or CRP (crp-) are unable to utilize a variety of carbohydrates, including L-arabinose. Ara+ revertants of a cya-crp- strain were isolated on 0.2% minimal L-arabinose plates, conditions which require the entire ara regulon to be activated in the absence of cAMP and CRP. Evidence from genetic and physiological studies is consistent with placing these mutations in the araC regulatory gene. Deletion mapping with one mutant localized the site within either araO or araC, and complementation tests indicated the mutants acted trans to confer the ability to utilize L-arabinose in a cya-crp- genetic background. Since genetic analysis supports the conclusion, that the mutant sites are in the araC regulatory gene, the mutants were designated araCi, indicating a mutation in the regulatory gene affecting the cAMP-CRP requirement. Physiological analysis of one mutant, araCi1, illustrates the trans-acting nature of the mutation. In a cya-crp- genetic background, araCi1 promoted synthesis of both isomerase, a product of the araBAD operon, and permease, a product of the araE operon. Isomerase and permease levels in araCi1 cya+ crp+ were hyperinducible, and the sensitivity of each to cAMP was altered. Two models are presented that show the possible mutational lesion in the araCi strains.

  9. Factors affecting mutational specificity induced by ionizing radiation and oxidizing radicals. Technical progress report, February 1, 1992--October 15, 1992

    SciTech Connect

    Strauss, B.S.

    1992-01-01

    We propose to analyze the factors affecting the specificity of mutational change as induced by ionizing radiation and oxidizing radicals. We want to understand not only the rules that affect base substitution, but also the mechanism(s) by which additions and deletions are produced, since detections are a common consequence of radiation. We wish to carry out this analysis in an in vitro mutation system that permits us to analyze the role of base sequence, of polymerase and of mutagenic agent. Our system is designed to screen out most direct breaks as a cause of mutation and should indicate the changes resulting from base damage to the DNA.

  10. Mutations in SGOL1 cause a novel cohesinopathy affecting heart and gut rhythm.

    PubMed

    Chetaille, Philippe; Preuss, Christoph; Burkhard, Silja; Côté, Jean-Marc; Houde, Christine; Castilloux, Julie; Piché, Jessica; Gosset, Natacha; Leclerc, Séverine; Wünnemann, Florian; Thibeault, Maryse; Gagnon, Carmen; Galli, Antonella; Tuck, Elizabeth; Hickson, Gilles R; El Amine, Nour; Boufaied, Ines; Lemyre, Emmanuelle; de Santa Barbara, Pascal; Faure, Sandrine; Jonzon, Anders; Cameron, Michel; Dietz, Harry C; Gallo-McFarlane, Elena; Benson, D Woodrow; Moreau, Claudia; Labuda, Damian; Zhan, Shing H; Shen, Yaoqing; Jomphe, Michèle; Jones, Steven J M; Bakkers, Jeroen; Andelfinger, Gregor

    2014-11-01

    The pacemaking activity of specialized tissues in the heart and gut results in lifelong rhythmic contractions. Here we describe a new syndrome characterized by Chronic Atrial and Intestinal Dysrhythmia, termed CAID syndrome, in 16 French Canadians and 1 Swede. We show that a single shared homozygous founder mutation in SGOL1, a component of the cohesin complex, causes CAID syndrome. Cultured dermal fibroblasts from affected individuals showed accelerated cell cycle progression, a higher rate of senescence and enhanced activation of TGF-β signaling. Karyotypes showed the typical railroad appearance of a centromeric cohesion defect. Tissues derived from affected individuals displayed pathological changes in both the enteric nervous system and smooth muscle. Morpholino-induced knockdown of sgol1 in zebrafish recapitulated the abnormalities seen in humans with CAID syndrome. Our findings identify CAID syndrome as a novel generalized dysrhythmia, suggesting a new role for SGOL1 and the cohesin complex in mediating the integrity of human cardiac and gut rhythm.

  11. Novel frameshifting mutations of the ZMPSTE24 gene in two siblings affected with restrictive dermopathy and review of the mutations described in the literature.

    PubMed

    Smigiel, Robert; Jakubiak, Aleksandra; Esteves-Vieira, Vera; Szela, Katarzyna; Halon, Agnieszka; Jurek, Tomasz; Lévy, Nicolas; De Sandre-Giovannoli, Annachiara

    2010-02-01

    Restrictive dermopathy (RD) is a rare, severe, lethal genodermatosis in which tautness of the skin causes fetal akinesia or hypokinesia deformation sequence. To date, about 60 cases of RD were described. The signs of the disease are very characteristic and include intrauterine growth retardation, thin, tightly adherent translucent skin, superficial vessels, typical facial dysmorphism as well as generalized joint contractures. The syndrome is caused in most cases by ZMPSTE24 autosomal recessive mutations, or, less frequently, by LMNA autosomal dominant mutations. We report on two brothers affected with RD, who died in the neonatal period. Molecular analyses were performed in the second child, for whom biological material was available, and both parents. Compound heterozygous frameshifting mutations were identified in exon 1 (c.50delA) and exon 5 (c.584_585delAT) of the ZMPSTE24 gene. The autosomal recessive inheritance was confirmed by the parents' genomic analysis. Besides, a review of the mutations causing RD is made.

  12. A Novel Forward Genetic Screen for Identifying Mutations Affecting Larval Neuronal Dendrite Development in Drosophila melanogaster

    PubMed Central

    Medina, Paul Mark B.; Swick, Lance L.; Andersen, Ryan; Blalock, Zachary; Brenman, Jay E.

    2006-01-01

    Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actin∷GFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of >4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actin∷GFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment. PMID:16415365

  13. Factors affecting germline mutations in a hypervariable microsatellite: a comparative analysis of six species of swallows (Aves: Hirundinidae).

    PubMed

    Anmarkrud, Jarl A; Kleven, Oddmund; Augustin, Jakob; Bentz, Kristofer H; Blomqvist, Donald; Fernie, Kim J; Magrath, Michael J L; Pärn, Henrik; Quinn, James S; Robertson, Raleigh J; Szép, Tibor; Tarof, Scott; Wagner, Richard H; Lifjeld, Jan T

    2011-03-15

    Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows (Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.

  14. SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome.

    PubMed

    Perez, Yonatan; Shorer, Zamir; Liani-Leibson, Keren; Chabosseau, Pauline; Kadir, Rotem; Volodarsky, Michael; Halperin, Daniel; Barber-Zucker, Shiran; Shalev, Hanna; Schreiber, Ruth; Gradstein, Libe; Gurevich, Evgenia; Zarivach, Raz; Rutter, Guy A; Landau, Daniel; Birk, Ohad S

    2017-02-09

    A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through

  15. Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles.

    PubMed

    Javot, Hélène; Pumplin, Nathan; Harrison, Maria J

    2007-03-01

    In response to the colonization by arbuscular mycorrhizal (AM) fungi, plants reprioritize their phosphate (Pi)-uptake strategies to take advantage of nutrient transfer via the fungus. The mechanisms underlying Pi transport are beginning to be understood, and recently, details of the regulation of plant and fungal Pi transporters in the AM symbiosis have been revealed. This review summarizes recent advances in this area and explores current data and hypotheses of how the plant Pi status affects the symbiosis. Finally, suggestions of an interrelationship of Pi and nitrogen (N) in the AM symbiosis are discussed.

  16. Rare Mutations of CACNB2 Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

    PubMed Central

    Breitenkamp, Alexandra F. S.; Matthes, Jan; Nass, Robert Daniel; Sinzig, Judith; Lehmkuhl, Gerd; Nürnberg, Peter; Herzig, Stefan

    2014-01-01

    Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism. PMID:24752249

  17. Transgenerational effects of plant sex and arbuscular mycorrhizal symbiosis.

    PubMed

    Varga, Sandra; Vega-Frutis, Rocío; Kytöviita, Minna-Maarit

    2013-08-01

    In gynodioecious plants, females are predicted to produce more and/or better offspring than hermaphrodites in order to be maintained in the same population. In the field, the roots of both sexes are usually colonized by arbuscular mycorrhizal (AM) fungi. Transgenerational effects of mycorrhizal symbiosis are largely unknown, although theoretically expected. We examined the maternal and paternal effects of AM fungal symbiosis and host sex on seed production and posterior seedling performance in Geranium sylvaticum, a gynodioecious plant. We hand-pollinated cloned females and hermaphrodites in symbiosis with AM fungi or in nonmycorrhizal conditions and measured seed number and mass, and seedling survival and growth in a glasshouse experiment. Females produced more seeds than hermaphrodites, but the seeds did not germinate, survive or grow better. Mycorrhizal plants were larger, but did not produce more seeds than nonmycorrhizal plants. Transgenerational parental effects of AM fungi were verified in seedling performance. This is the first study to show transgenerational mycorrhiza-mediated parental effects in a gynodioecious species. Mycorrhizal symbiosis affects plant fitness mainly through female functions with enduring effects on the next generation.

  18. TRNA mutations that affect decoding fidelity deregulate development and the proteostasis network in zebrafish.

    PubMed

    Reverendo, Marisa; Soares, Ana R; Pereira, Patrícia M; Carreto, Laura; Ferreira, Violeta; Gatti, Evelina; Pierre, Philippe; Moura, Gabriela R; Santos, Manuel A

    2014-01-01

    Mutations in genes that encode tRNAs, aminoacyl-tRNA syntheases, tRNA modifying enzymes and other tRNA interacting partners are associated with neuropathies, cancer, type-II diabetes and hearing loss, but how these mutations cause disease is unclear. We have hypothesized that levels of tRNA decoding error (mistranslation) that do not fully impair embryonic development can accelerate cell degeneration through proteome instability and saturation of the proteostasis network. To test this hypothesis we have induced mistranslation in zebrafish embryos using mutant tRNAs that misincorporate Serine (Ser) at various non-cognate codon sites. Embryo viability was affected and malformations were observed, but a significant proportion of embryos survived by activating the unfolded protein response (UPR), the ubiquitin proteasome pathway (UPP) and downregulating protein biosynthesis. Accumulation of reactive oxygen species (ROS), mitochondrial and nuclear DNA damage and disruption of the mitochondrial network, were also observed, suggesting that mistranslation had a strong negative impact on protein synthesis rate, ER and mitochondrial homeostasis. We postulate that mistranslation promotes gradual cellular degeneration and disease through protein aggregation, mitochondrial dysfunction and genome instability.

  19. Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells.

    PubMed

    Söllner, Christian; Rauch, Gerd-Jörg; Siemens, Jan; Geisler, Robert; Schuster, Stephan C; Müller, Ulrich; Nicolson, Teresa

    2004-04-29

    Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.

  20. P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster

    SciTech Connect

    Kania, A.; Salzberg, A.; Bhat, M.

    1995-04-01

    The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS. 52 refs., 5 figs., 5 tabs.

  1. Glutamine synthetase-constitutive mutation affecting the glnALG upstream promoter of Escherichia coli.

    PubMed

    León, P; Romero, D; Garciarrubio, A; Bastarrachea, F; Covarrubias, A A

    1985-12-01

    The spontaneous gln-76 mutation of Escherichia coli (Osorio et al., Mol. Gen. Genet. 194:114-123, 1984) was previously shown to be responsible for the cis-dominant constitutive expression of the glnA gene in the absence of a glnG-glnF activator system. Nucleotide sequence analysis has now revealed that gln-76 is a single transversion T.A to A.T, an up-promoter mutation affecting the -10 region of glnAp1, the upstream promoter of the glnALG control region. Both, wild-type and gln-76 DNA control regions were cloned into the promoter-probe plasmid pKO1. Galactokinase activity determinations of cells carrying the fused plasmids showed 10-fold more effective expression mediated by gln-76 than by the glnA wild-type control region. Primer extension experiments with RNA from strains carrying the gln-76 control region indicated that the transcription initiation sites were the same in both the gln-76 mutant and the wild type.

  2. Glutamine synthetase-constitutive mutation affecting the glnALG upstream promoter of Escherichia coli.

    PubMed Central

    León, P; Romero, D; Garciarrubio, A; Bastarrachea, F; Covarrubias, A A

    1985-01-01

    The spontaneous gln-76 mutation of Escherichia coli (Osorio et al., Mol. Gen. Genet. 194:114-123, 1984) was previously shown to be responsible for the cis-dominant constitutive expression of the glnA gene in the absence of a glnG-glnF activator system. Nucleotide sequence analysis has now revealed that gln-76 is a single transversion T.A to A.T, an up-promoter mutation affecting the -10 region of glnAp1, the upstream promoter of the glnALG control region. Both, wild-type and gln-76 DNA control regions were cloned into the promoter-probe plasmid pKO1. Galactokinase activity determinations of cells carrying the fused plasmids showed 10-fold more effective expression mediated by gln-76 than by the glnA wild-type control region. Primer extension experiments with RNA from strains carrying the gln-76 control region indicated that the transcription initiation sites were the same in both the gln-76 mutant and the wild type. Images PMID:2866175

  3. Social defeat interacts with Disc1 mutations in the mouse to affect behavior.

    PubMed

    Haque, F Nipa; Lipina, Tatiana V; Roder, John C; Wong, Albert H C

    2012-08-01

    DISC1 (Disrupted-in-schizophrenia 1) is a strong candidate susceptibility gene for psychiatric disease that was originally discovered in a family with a chromosomal translocation severing this gene. Although the family members with the translocation had an identical genetic mutation, their clinical diagnosis and presentation varied significantly. Gene-environment interactions have been proposed as a mechanism underlying the complex heritability and variable phenotype of psychiatric disorders such as major depressive disorder and schizophrenia. We hypothesized that gene-environment interactions would affect behavior in a mutant Disc1 mouse model. We examined the effect of chronic social defeat (CSD) as an environmental stressor in two lines of mice carrying different Disc1 point mutations, on behaviors relevant to psychiatric illness: locomotion in a novel open field (OF), pre-pulse inhibition (PPI) of the acoustic startle response, latent inhibition (LI), elevated plus maze (EPM), forced swim test (FST), sucrose consumption (SC), and the social interaction task for sociability and social novelty (SSN). We found that Disc1-L100P +/- and wild-type mice have similar anxiety responses to CSD, while Q31L +/- mice had a very different response. We also found evidence of significant gene-environment interactions in the OF, EPM and SSN.

  4. Mutation in fucose synthesis gene of Klebsiella pneumoniae affects capsule composition and virulence in mice.

    PubMed

    Pan, Po-Chang; Chen, Hui-Wen; Wu, Po-Kuan; Wu, Yu-Yang; Lin, Chun-Hung; Wu, June H

    2011-02-01

    The emerging pathogenicity of Klebsiella pneumoniae (KP) is evident by the increasing number of clinical cases of liver abscess (LA) due to KP infection. A unique property of KP is its thick mucoid capsule. The bacterial capsule has been found to contain fucose in KP strains causing LA but not in those causing urinary tract infections. The products of the gmd and wcaG genes are responsible for converting mannose to fucose in KP. A KP strain, KpL1, which is known to have a high death rate in infected mice, was mutated by inserting an apramycin-resistance gene into the gmd. The mutant expressed genes upstream and downstream of gmd, but not gmd itself, as determined by reverse transcriptase polymerase chain reaction. The DNA mapping confirmed the disruption of the gmd gene. This mutant decreased its ability to kill infected mice and showed decreased virulence in infected HepG2 cells. Compared with wild-type KpL1, the gmd mutant lost fucose in capsular polysaccharides, increased biofilm formation and interacted more readily with macrophages. The mutant displayed morphological changes with long filament forms and less uniform sizes. The mutation also converted the serotype from K1 of wild-type to K2 and weak K3. The results indicate that disruption of the fucose synthesis gene affected the pathophysiology of this bacterium and may be related to the virulence of this KpL1 strain.

  5. Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

    PubMed Central

    Alaa el Din, Ferdos; Patri, Sylvie; Thoreau, Vincent; Rodriguez-Ballesteros, Montserrat; Hamade, Eva; Bailly, Sabine; Gilbert-Dussardier, Brigitte; Abou Merhi, Raghida; Kitzis, Alain

    2015-01-01

    Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. PMID:26176610

  6. Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds

    SciTech Connect

    Doerk, T.; Wulbrand, U.; Tuemmler, B. )

    1993-03-01

    Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.

  7. Error-prone and error-restrictive mutations affecting ribosomal protein S12.

    PubMed

    Agarwal, Deepali; Gregory, Steven T; O'Connor, Michael

    2011-07-01

    Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.

  8. A Promoter Region Mutation Affecting Replication of the Tetrahymena Ribosomal DNA Minichromosome

    PubMed Central

    Gallagher, Renata C.; Blackburn, Elizabeth H.

    1998-01-01

    In the ciliated protozoan Tetrahymena thermophila the ribosomal DNA (rDNA) minichromosome replicates partially under cell cycle control and is also subject to a copy number control mechanism. The relationship between rDNA replication and rRNA gene transcription was investigated by the analysis of replication, transcription, and DNA-protein interactions in a mutant rDNA, the rmm3 rDNA. The rmm3 (for rDNA maturation or maintenance mutant 3) rDNA contains a single-base deletion in the rRNA promoter region, in a phylogenetically conserved sequence element that is repeated in the replication origin region of the rDNA minichromosome. The multicopy rmm3 rDNA minichromosome has a maintenance defect in the presence of a competing rDNA allele in heterozygous cells. No difference in the level of rRNA transcription was found between wild-type and rmm3 strains. However, rmm3 rDNA replicating intermediates exhibited an enhanced pause in the region of the replication origin, roughly 750 bp upstream from the rmm3 mutation. In footprinting of isolated nuclei, the rmm3 rDNA lacked the wild-type dimethyl sulfate (DMS) footprint in the promoter region adjacent to the base change. In addition, a DMS footprint in the origin region was lost in the rmm3 rDNA minichromosome. This is the first reported correlation in this system between an rDNA minichromosome maintenance defect and an altered footprint in the origin region. Our results suggest that a promoter region mutation can affect replication without detectably affecting transcription. We propose a model in which interactions between promoter and origin region complexes facilitate replication and maintenance of the Tetrahymena rDNA minichromosome. PMID:9566921

  9. A family with a dystrophin gene mutation specifically affecting dystrophin expression in the heart

    SciTech Connect

    Muntoni, F.; Davies, K.; Dubowitz, V.

    1994-09-01

    We recently described a family with X-linked dilated cardiomyopathy where a large deletion in the muscle promoter region of the dystrophin gene was associated with a severe dilated cardiomyopathy in absence of clinical skeletal muscle involvement. The deletion removed the entire muscle promoter region, the first muscle exon and part of intron 1. The brain and Purkinje cell promoters were not affected by the deletion. Despite the lack of both the muscle promoter and the first muscle exon, dystrophin was detected immunocytochemically in relative high levels in the skeletal muscle of the affected males. We have now found that both the brain and Purkinje cell promoters were transcribed at high levels in the skeletal muscle of these individuals. This phenomenon, that does not occur in normal skeletal muscle, indicates that these two isoforms, physiologically expressed mainly in the central nervous system, can be transcribed and be functionally active in skeletal muscle under specific circumstances. Contrary to what is observed in skeletal muscle, dystrophin was not detected in the heart of one affected male using immunocytochemistry and an entire panel of anti-dystrophin antibodies. This was most likely the cause for the pronounced cardiac fibrosis observed and eventually responsible for the severe cardiac involvement invariably seen in seven affected males. In conclusion, the mutation of the muscle promoter, first muscle exon and part of intron 1 specifically affected expression of dystrophin in the heart. We believe that this deletion removes sequences involved in regulation of dystrophin expression in the heart and are at the moment characterizing other families with X-linked cardiomyopathy secondary to a dystrophinopathy.

  10. An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control.

    PubMed

    Inuzuka, M; Wada, Y

    1988-02-08

    Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

  11. Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1.

    PubMed

    Pilz, D T; Kuc, J; Matsumoto, N; Bodurtha, J; Bernadi, B; Tassinari, C A; Dobyns, W B; Ledbetter, D H

    1999-09-01

    Subcortical band heterotopia (SBH) are bilateral and symmetric ribbons of gray matter found in the central white matter between the cortex and the ventricular surface, which comprises the less severe end of the lissencephaly (agyria-pachygyria-band) spectrum of malformations. Mutations in DCX (also known as XLIS ) have previously been described in females with SBH. We have now identified mutations in either the DCX or LIS1 gene in three of 11 boys studied, demonstrating for the first time that mutations of either DCX or LIS1 can cause SBH or mixed pachygyria-SBH (PCH-SBH) in males. All three changes detected are missense mutations, predicted to be of germline origin. They include a missense mutation in exon 4 of DCX in a boy with PCH-SBH (R78H), a different missense mutation in exon 4 of DCX in a boy with mild SBH and in his mildly affected mother (R89G) and a missense mutation in exon 6 of LIS1 in a boy with SBH (S169P). The missense mutations probably account for the less severe brain malformations, although other patients with missense mutations in the same exons have had diffuse lissencephaly. Therefore, it appears likely that the effect of the specific amino acid change on the protein determines the severity of the phenotype, with some mutations enabling residual protein function and allowing normal migration in a larger proportion of neurons. However, we expect that somatic mosaic mutations of both LIS1 and DCX will also prove to be an important mechanism in causing SBH in males.

  12. Balancing protein stability and activity in cancer: a new approach for identifying driver mutations affecting CBL ubiquitin ligase activation

    PubMed Central

    Li, Minghui; Kales, Stephen C.; Ma, Ke; Shoemaker, Benjamin A.; Crespo-Barreto, Juan; Cangelosi, Andrew L.; Lipkowitz, Stanley; Panchenko, Anna R.

    2015-01-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  13. Interaction between mutations in the suppressor of Hairy wing and modifier of mdg4 genes of Drosophila melanogaster affecting the phenotype of gypsy-induced mutations

    SciTech Connect

    Georgiev, P.; Kozycina, M.

    1996-02-01

    The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region. Mutations in a second gene, modifier of mdg4, also affect the gypsy-induced phenotype. Two major effects of the mod(mdg4){sup lu1} mutation can be distinguished: the interference with insulation by the su(Hw)-binding region and direct inhibition of gene expression that is not dependent on the su(Hw)-binding region position. The mod(mdg4){sup lu1} mutation partially suppresses ct{sup 6}, sc{sup D1} and Hw{sup 1} mutations, possibly by interfering with the insulation effect of the su(Hw)-binding region. An example of the second effect of mod(mdg4){sup lu1} is a complete inactivation of yellow expression in combination with the y{sup 2} allele. Phenotypic analyses of flies with combinations of mdg(mdg4){sup lu1} and different su(Hw) mutations, or with constructions carrying deletions of the acidic domains of the su(Hw) protein, suggest that the carboxy-terminal acidic domain is important for direct inhibition of yellow transcription in bristles, while the amino-terminal acidic domain is more essential for insulation. 31 refs., 1 fig., 6 tabs.

  14. Functional Assessment of Human Coding Mutations Affecting Skin Pigmentation Using Zebrafish

    PubMed Central

    Tsetskhladze, Zurab R.; Canfield, Victor A.; Ang, Khai C.; Wentzel, Steven M.; Reid, Katherine P.; Berg, Arthur S.; Johnson, Stephen L.; Kawakami, Koichi; Cheng, Keith C.

    2012-01-01

    A major challenge in personalized medicine is the lack of a standard way to define the functional significance of the numerous nonsynonymous, single nucleotide coding variants that are present in each human individual. To begin to address this problem, we have used pigmentation as a model polygenic trait, three common human polymorphisms thought to influence pigmentation, and the zebrafish as a model system. The approach is based on the rescue of embryonic zebrafish mutant phenotypes by “humanized” zebrafish orthologous mRNA. Two hypomorphic polymorphisms, L374F in SLC45A2, and A111T in SLC24A5, have been linked to lighter skin color in Europeans. The phenotypic effect of a second coding polymorphism in SLC45A2, E272K, is unclear. None of these polymorphisms had been tested in the context of a model organism. We have confirmed that zebrafish albino fish are mutant in slc45a2; wild-type slc45a2 mRNA rescued the albino mutant phenotype. Introduction of the L374F polymorphism into albino or the A111T polymorphism into slc24a5 (golden) abolished mRNA rescue of the respective mutant phenotypes, consistent with their known contributions to European skin color. In contrast, the E272K polymorphism had no effect on phenotypic rescue. The experimental conclusion that E272K is unlikely to affect pigmentation is consistent with a lack of correlation between this polymorphism and quantitatively measured skin color in 59 East Asian humans. A survey of mutations causing human oculocutaneous albinism yielded 257 missense mutations, 82% of which are theoretically testable in zebrafish. The developed approach may be extended to other model systems and may potentially contribute to our understanding the functional relationships between DNA sequence variation, human biology, and disease. PMID:23071798

  15. Ionic leakage underlies a gain-of-function effect of dominant disease mutations affecting diverse P-type ATPases.

    PubMed

    Kaneko, Maki; Desai, Bela S; Cook, Boaz

    2014-02-01

    Type II P-type ATPases (PAIIs) constitute a family of conserved proteins that actively generate ionic gradients across membranes. Mutations in genes encoding PAIIs can cause heritable dominant diseases, with suggested etiology of haploinsufficiency. Using a Drosophila melanogaster genetic screen, we identified a dominant mutation altering the PAII member sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA). This mutation conferred temperature-sensitive uncoordination in a gain-of-function manner. We established that this gain-of-function phenotype is linked to dominant disease-causing mutations affecting various human PAIIs. We further found that heterologous expression of mutant PAIIs elicited ion leakage that was exacerbated at elevated temperatures. Therefore, these dominant mutations result in ionic leakage and render PAIIs susceptible to deleterious effects from elevated temperatures. Accordingly, it was recently reported that missense mutations affecting the Na(+)/K(+) ATPase can elicit ionic leakage. We propose that ionic leakage is a pervasive gain-of-function mechanism that can underlie a variety of dominant PAII-related diseases.

  16. Mutations Affecting the SAND Domain of DEAF1 Cause Intellectual Disability with Severe Speech Impairment and Behavioral Problems

    PubMed Central

    Vulto-van Silfhout, Anneke T.; Rajamanickam, Shivakumar; Jensik, Philip J.; Vergult, Sarah; de Rocker, Nina; Newhall, Kathryn J.; Raghavan, Ramya; Reardon, Sara N.; Jarrett, Kelsey; McIntyre, Tara; Bulinski, Joseph; Ownby, Stacy L.; Huggenvik, Jodi I.; McKnight, G. Stanley; Rose, Gregory M.; Cai, Xiang; Willaert, Andy; Zweier, Christiane; Endele, Sabine; de Ligt, Joep; van Bon, Bregje W.M.; Lugtenberg, Dorien; de Vries, Petra F.; Veltman, Joris A.; van Bokhoven, Hans; Brunner, Han G.; Rauch, Anita; de Brouwer, Arjan P.M.; Carvill, Gemma L.; Hoischen, Alexander; Mefford, Heather C.; Eichler, Evan E.; Vissers, Lisenka E.L.M.; Menten, Björn; Collard, Michael W.; de Vries, Bert B.A.

    2014-01-01

    Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1. PMID:24726472

  17. Mutations affecting the SAND domain of DEAF1 cause intellectual disability with severe speech impairment and behavioral problems.

    PubMed

    Vulto-van Silfhout, Anneke T; Rajamanickam, Shivakumar; Jensik, Philip J; Vergult, Sarah; de Rocker, Nina; Newhall, Kathryn J; Raghavan, Ramya; Reardon, Sara N; Jarrett, Kelsey; McIntyre, Tara; Bulinski, Joseph; Ownby, Stacy L; Huggenvik, Jodi I; McKnight, G Stanley; Rose, Gregory M; Cai, Xiang; Willaert, Andy; Zweier, Christiane; Endele, Sabine; de Ligt, Joep; van Bon, Bregje W M; Lugtenberg, Dorien; de Vries, Petra F; Veltman, Joris A; van Bokhoven, Hans; Brunner, Han G; Rauch, Anita; de Brouwer, Arjan P M; Carvill, Gemma L; Hoischen, Alexander; Mefford, Heather C; Eichler, Evan E; Vissers, Lisenka E L M; Menten, Björn; Collard, Michael W; de Vries, Bert B A

    2014-05-01

    Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.

  18. Psychological Distress, Anxiety, and Depression of Cancer-Affected BRCA1/2 Mutation Carriers: a Systematic Review.

    PubMed

    Ringwald, Johanna; Wochnowski, Christina; Bosse, Kristin; Giel, Katrin Elisabeth; Schäffeler, Norbert; Zipfel, Stephan; Teufel, Martin

    2016-10-01

    Understanding the intermediate- and long-term psychological consequences of genetic testing for cancer patients has led to encouraging research, but a clear consensus of the psychosocial impact and clinical routine for cancer-affected BRCA1 and BRCA2 mutation carriers is still missing. We performed a systematic review of intermediate- and long-term studies investigating the psychological impact like psychological distress, anxiety, and depression in cancer-affected BRCA mutation carriers compared to unaffected mutation carriers. This review included the screening of 1243 studies. Eight intermediate- and long-term studies focusing on distress, anxiety, and depression symptoms among cancer-affected mutation carriers at least six months after the disclosure of genetic testing results were included. Studies reported a great variety of designs, methods, and patient outcomes. We found evidence indicating that cancer-affected mutation carriers experienced a negative effect in relation to psychological well-being in terms of an increase in symptoms of distress, anxiety, and depression in the first months after test disclosure. In the intermediate- and long-term, no significant clinical relevant symptoms occurred. However, none of the included studies used specific measurements, which can clearly identify psychological burdens of cancer-affected mutation carriers. We concluded that current well-implemented distress screening instruments are not sufficient for precisely identifying the psychological burden of genetic testing. Therefore, future studies should implement coping strategies, specific personality structures, the impact of genetic testing, supportive care needs and disease management behaviour to clearly screen for the possible intermediate- and long-term psychological impact of a positive test disclosure.

  19. Cell Biology of Cnidarian-Dinoflagellate Symbiosis

    PubMed Central

    Allemand, Denis; Weis, Virginia M.

    2012-01-01

    Summary: The symbiosis between cnidarians (e.g., corals or sea anemones) and intracellular dinoflagellate algae of the genus Symbiodinium is of immense ecological importance. In particular, this symbiosis promotes the growth and survival of reef corals in nutrient-poor tropical waters; indeed, coral reefs could not exist without this symbiosis. However, our fundamental understanding of the cnidarian-dinoflagellate symbiosis and of its links to coral calcification remains poor. Here we review what we currently know about the cell biology of cnidarian-dinoflagellate symbiosis. In doing so, we aim to refocus attention on fundamental cellular aspects that have been somewhat neglected since the early to mid-1980s, when a more ecological approach began to dominate. We review the four major processes that we believe underlie the various phases of establishment and persistence in the cnidarian/coral-dinoflagellate symbiosis: (i) recognition and phagocytosis, (ii) regulation of host-symbiont biomass, (iii) metabolic exchange and nutrient trafficking, and (iv) calcification. Where appropriate, we draw upon examples from a range of cnidarian-alga symbioses, including the symbiosis between green Hydra and its intracellular chlorophyte symbiont, which has considerable potential to inform our understanding of the cnidarian-dinoflagellate symbiosis. Ultimately, we provide a comprehensive overview of the history of the field, its current status, and where it should be going in the future. PMID:22688813

  20. A novel reef coral symbiosis

    NASA Astrophysics Data System (ADS)

    Pantos, O.; Bythell, J. C.

    2010-09-01

    Reef building corals form close associations with unicellular microalgae, fungi, bacteria and archaea, some of which are symbiotic and which together form the coral holobiont. Associations with multicellular eukaryotes such as polychaete worms, bivalves and sponges are not generally considered to be symbiotic as the host responds to their presence by forming physical barriers with an active growth edge in the exoskeleton isolating the invader and, at a subcellular level, activating innate immune responses such as melanin deposition. This study describes a novel symbiosis between a newly described hydrozoan ( Zanclea margaritae sp. nov.) and the reef building coral Acropora muricata (= A. formosa), with the hydrozoan hydrorhiza ramifying throughout the coral tissues with no evidence of isolation or activation of the immune systems of the host. The hydrorhiza lacks a perisarc, which is typical of symbiotic species of this and related genera, including species that associate with other cnidarians such as octocorals. The symbiosis was observed at all sites investigated from two distant locations on the Great Barrier Reef, Australia, and appears to be host species specific, being found only in A. muricata and in none of 30 other species investigated at these sites. Not all colonies of A. muricata host the hydrozoans and both the prevalence within the coral population (mean = 66%) and density of emergent hydrozoan hydranths on the surface of the coral (mean = 4.3 cm-2, but up to 52 cm-2) vary between sites. The form of the symbiosis in terms of the mutualism-parasitism continuum is not known, although the hydrozoan possesses large stenotele nematocysts, which may be important for defence from predators and protozoan pathogens. This finding expands the known A. muricata holobiont and the association must be taken into account in future when determining the corals’ abilities to defend against predators and withstand stress.

  1. Cancer-associated noncoding mutations affect RNA G-quadruplex-mediated regulation of gene expression.

    PubMed

    Zeraati, Mahdi; Moye, Aaron L; Wong, Jason W H; Perera, Dilmi; Cowley, Mark J; Christ, Daniel U; Bryan, Tracy M; Dinger, Marcel E

    2017-04-06

    Cancer is a multifactorial disease driven by a combination of genetic and environmental factors. Many cancer driver mutations have been characterised in protein-coding regions of the genome. However, mutations in noncoding regions associated with cancer have been less investigated. G-quadruplex (G4) nucleic acids are four-stranded secondary structures formed in guanine-rich sequences and prevalent in the regulatory regions. In this study, we used published whole cancer genome sequence data to find mutations in cancer patients that overlap potential RNA G4-forming sequences in 5' UTRs. Using RNAfold, we assessed the effect of these mutations on the thermodynamic stability of predicted RNA G4s in the context of full-length 5' UTRs. Of the 217 identified mutations, we found that 33 are predicted to destabilise and 21 predicted to stabilise potential RNA G4s. We experimentally validated the effect of destabilising mutations in the 5' UTRs of BCL2 and CXCL14 and one stabilising mutation in the 5' UTR of TAOK2. These mutations resulted in an increase or a decrease in translation of these mRNAs, respectively. These findings suggest that mutations that modulate the G4 stability in the noncoding regions could act as cancer driver mutations, which present an opportunity for early cancer diagnosis using individual sequencing information.

  2. Mutation-induced protein interaction kinetics changes affect apoptotic network dynamic properties and facilitate oncogenesis.

    PubMed

    Zhao, Linjie; Sun, Tanlin; Pei, Jianfeng; Ouyang, Qi

    2015-07-28

    It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant-wild-type and 16 matched SNP--wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation.

  3. Mutation-induced protein interaction kinetics changes affect apoptotic network dynamic properties and facilitate oncogenesis

    PubMed Central

    Zhao, Linjie; Sun, Tanlin; Pei, Jianfeng; Ouyang, Qi

    2015-01-01

    It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant–wild-type and 16 matched SNP—wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation. PMID:26170328

  4. Oakleaf: an S locus-linked mutation of Primula vulgaris that affects leaf and flower development.

    PubMed

    Cocker, Jonathan M; Webster, Margaret A; Li, Jinhong; Wright, Jonathan; Kaithakottil, Gemy; Swarbreck, David; Gilmartin, Philip M

    2015-10-01

    In Primula vulgaris outcrossing is promoted through reciprocal herkogamy with insect-mediated cross-pollination between pin and thrum form flowers. Development of heteromorphic flowers is coordinated by genes at the S locus. To underpin construction of a genetic map facilitating isolation of these S locus genes, we have characterised Oakleaf, a novel S locus-linked mutant phenotype. We combine phenotypic observation of flower and leaf development, with classical genetic analysis and next-generation sequencing to address the molecular basis of Oakleaf. Oakleaf is a dominant mutation that affects both leaf and flower development; plants produce distinctive lobed leaves, with occasional ectopic meristems on the veins. This phenotype is reminiscent of overexpression of Class I KNOX-homeodomain transcription factors. We describe the structure and expression of all eight P. vulgaris PvKNOX genes in both wild-type and Oakleaf plants, and present comparative transcriptome analysis of leaves and flowers from Oakleaf and wild-type plants. Oakleaf provides a new phenotypic marker for genetic analysis of the Primula S locus. We show that none of the Class I PvKNOX genes are strongly upregulated in Oakleaf leaves and flowers, and identify cohorts of 507 upregulated and 314 downregulated genes in the Oakleaf mutant.

  5. Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations.

    PubMed Central

    Léveillard, T; Andera, L; Bissonnette, N; Schaeffer, L; Bracco, L; Egly, J M; Wasylyk, B

    1996-01-01

    The p53 tumour suppressor is mutated in the majority of human tumours. p53's proposed role as the guardian of the genome is reflected in its multiple effects on transcription genome stability, cell growth and survival. We show that p53 interacts both physically and functionally with the TFIIH complex. There are multiple protein-protein contacts, involving two regions of p53 and three subunits of TFIIH, ERCC2 (XPD), ERCC3 (XPB) and p62. p53 and its C-terminus (amino acids 320-393) inhibit both of the TFIIH helicases and in vitro transcription in the absence of TFIIH. Transcription inhibition is overcome by TFIIH. The N-terminal region of p53 (1-320), lacking the C-terminus, is inactive on its own, yet apparently affects the activity of the C-terminus in the native protein. Interestingly, mutant p53s that are frequently found in tumours are less efficient inhibitors of the helicases and transcription. We hypothesize that the interactions provide an immediate and direct link for p53 to the multiple functions of TFIIH in transcription, DNA repair and possibly the cell cycle. Images PMID:8612585

  6. Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila.

    PubMed

    Martín, F; Charro, M J; Alcorta, E

    2001-06-01

    The rutabaga and dunce genes, encode two enzymes of the cyclic adenosine monophosphate transduction pathway in Drosophila, adenylyl cyclase and cyclic adenosine monophosphate phosphodiesterase, respectively. Two main second messenger systems, depending on inositol 1,4,5-triphosphate and cyclic adenosine monophosphate, have been associated with olfaction in vertebrates as well as invertebrates. A relationship between the cyclic adenosine monophosphate signaling pathway and olfactory reception in Drosophila is suggested by the presence of cyclic nucleotide gated channels and cyclic-nucleotide modulated K+ channels in the antennae, the main olfactory organs. In this report, molecular, electrophysiological and behavioral data support the role of cyclic adenosine monophosphate in olfactory function for this species. Expression of both genes in the antennae has been shown by messenger ribonucleic acid analysis. Changes in the electroantennogram kinetics have been observed specifically on the slope of the initial rising phase, as predicted for processes that affect cyclic adenosine monophosphate concentration. Olfactory behavior changes due to both mutations were coherent with a functional meaning of the reported electrophysiological phenotype in olfactory perception. Sensitivity level increases or decreases for the mutants compared to the control line depending on the odorant. These results are compatible with some olfactory coding at the reception level by differential activation of a dual transduction system involving the inositol 1,4,5-triphosphate and cyclic adenosine monophosphate cascades.

  7. Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis

    PubMed Central

    Samuelov, Liat; Bertolini, Marta; Weissglas-Volkov, Daphna; Eskin-Schwartz, Marina; Malchin, Natalia; Bochner, Ron; Fainberg, Gilad; Goldberg, Ilan; Sugawara, Koji; Tsuruta, Daisuke; Morasso, Maria; Shalev, Stavit; Gallo, Richard L.; Shomron, Noam; Paus, Ralf; Sprecher, Eli

    2016-01-01

    Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway. PMID:27736875

  8. Detection of three nonsense mutations and one missense mutation in the interleukin-2 receptor [gamma] chain gene in SCIDX1 that differently affect the mRNA processing

    SciTech Connect

    Markiewicz, S.; Fischer, A.; Saint Basile, G. de ); Subtil, A.; Dautry-Varsat, A. )

    1994-05-01

    The interleukin-2 receptor [gamma] (IL-2R[gamma]) chain gene encodes a 64-kDa protein that not only composes the high-affinity form of the IL-2 binding receptor in association with the 2R [alpha] and [beta] chains, but also participates in at least the IL-4 and IL-7 receptor complexes. Mutations in this gene have recently been shown to cause X-linked severe combined immunodeficiency (SCIDX1). This disease of the immune system results from an early block of T lymphocyte and natural killer (NK) cell differentiation, which leads to a severe cellular and humoral immune defect that is lethal unless treated by bone marrow transplantation. Analysis of the IL-2R[gamma] gene in SCIDX1 patients has revealed the presence of heterogeneous mutations principally located in the extracellular domain of the molecule. We report here three intraexonic mutations and one deletion in the IL-2R[gamma] gene in four SCIDX1 patients. These mutations appear to differentially affect RNA processing, either by decreasing IL-2R[gamma] mRNA level or by the skipping of a constitutive exon. 16 refs., 1 fig.

  9. Mutations affecting export and activity of cytolysin A from Escherichia coli.

    PubMed

    Ludwig, Albrecht; Völkerink, Guido; von Rhein, Christine; Bauer, Susanne; Maier, Elke; Bergmann, Birgit; Goebel, Werner; Benz, Roland

    2010-08-01

    Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

  10. Symbiosis through exploitation and the merger of lineages in evolution

    PubMed Central

    Law, R.; Dieckmann, U.

    1998-01-01

    A model for the coevolution of two species in facultative symbiosis is used to investigate conditions under which species merge to form a single reproductive unit. Two traits evolve in each species, the first affecting loss of resources from an individual to its partner, and the second affecting vertical transmission of the symbiosis from one generation to the next. Initial conditions are set so that the symbiosis involves exploitation of one partner by the other and vertical transmission is very rare. It is shown that, even in the face of continuing exploitation, a stable symbiotic unit can evolve with maximum vertical transmission of the partners. Such evolution requires that eventually deaths should exceed births for both species in the free-living state, a condition which can be met if the victim, in the course of developing its defences, builds up sufficiently large costs in the free-living state. This result expands the set of initial conditions from which separate lineages can be expected to merge into symbiotic units.

  11. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas.

    PubMed

    Palles, Claire; Cazier, Jean-Baptiste; Howarth, Kimberley M; Domingo, Enric; Jones, Angela M; Broderick, Peter; Kemp, Zoe; Spain, Sarah L; Guarino, Estrella; Guarino Almeida, Estrella; Salguero, Israel; Sherborne, Amy; Chubb, Daniel; Carvajal-Carmona, Luis G; Ma, Yusanne; Kaur, Kulvinder; Dobbins, Sara; Barclay, Ella; Gorman, Maggie; Martin, Lynn; Kovac, Michal B; Humphray, Sean; Lucassen, Anneke; Holmes, Christopher C; Bentley, David; Donnelly, Peter; Taylor, Jenny; Petridis, Christos; Roylance, Rebecca; Sawyer, Elinor J; Kerr, David J; Clark, Susan; Grimes, Jonathan; Kearsey, Stephen E; Thomas, Huw J W; McVean, Gilean; Houlston, Richard S; Tomlinson, Ian

    2013-02-01

    Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

  12. High prevalence of mutations affecting the splicing process in a Spanish cohort with autosomal dominant retinitis pigmentosa

    PubMed Central

    Ezquerra-Inchausti, Maitane; Barandika, Olatz; Anasagasti, Ander; Irigoyen, Cristina; López de Munain, Adolfo; Ruiz-Ederra, Javier

    2017-01-01

    Retinitis pigmentosa is the most frequent group of inherited retinal dystrophies. It is highly heterogeneous, with more than 80 disease-causing genes 27 of which are known to cause autosomal dominant RP (adRP), having been identified. In this study a total of 29 index cases were ascertained based on a family tree compatible with adRP. A custom panel of 31 adRP genes was analysed by targeted next-generation sequencing using the Ion PGM platform in combination with Sanger sequencing. This allowed us to detect putative disease-causing mutations in 14 out of the 29 (48.28%) families analysed. Remarkably, around 38% of all adRP cases analysed showed mutations affecting the splicing process, mainly due to mutations in genes coding for spliceosome factors (SNRNP200 and PRPF8) but also due to splice-site mutations in RHO. Twelve of the 14 mutations found had been reported previously and two were novel mutations found in PRPF8 in two unrelated patients. In conclusion, our results will lead to more accurate genetic counselling and will contribute to a better characterisation of the disease. In addition, they may have a therapeutic impact in the future given the large number of studies currently underway based on targeted RNA splicing for therapeutic purposes. PMID:28045043

  13. High prevalence of mutations affecting the splicing process in a Spanish cohort with autosomal dominant retinitis pigmentosa.

    PubMed

    Ezquerra-Inchausti, Maitane; Barandika, Olatz; Anasagasti, Ander; Irigoyen, Cristina; López de Munain, Adolfo; Ruiz-Ederra, Javier

    2017-01-03

    Retinitis pigmentosa is the most frequent group of inherited retinal dystrophies. It is highly heterogeneous, with more than 80 disease-causing genes 27 of which are known to cause autosomal dominant RP (adRP), having been identified. In this study a total of 29 index cases were ascertained based on a family tree compatible with adRP. A custom panel of 31 adRP genes was analysed by targeted next-generation sequencing using the Ion PGM platform in combination with Sanger sequencing. This allowed us to detect putative disease-causing mutations in 14 out of the 29 (48.28%) families analysed. Remarkably, around 38% of all adRP cases analysed showed mutations affecting the splicing process, mainly due to mutations in genes coding for spliceosome factors (SNRNP200 and PRPF8) but also due to splice-site mutations in RHO. Twelve of the 14 mutations found had been reported previously and two were novel mutations found in PRPF8 in two unrelated patients. In conclusion, our results will lead to more accurate genetic counselling and will contribute to a better characterisation of the disease. In addition, they may have a therapeutic impact in the future given the large number of studies currently underway based on targeted RNA splicing for therapeutic purposes.

  14. Genetic and biochemical characterization of mutations affecting the ability of the yeast Pachysolen tannophilus to metabolize D-xylose

    SciTech Connect

    James, A.P.; Zahab, D.M.; Mahmourides, G.; Maleszka, R.; Schneider, H. )

    1989-11-01

    Induced mutants, selected for their defective growth on D-xylose while retaining the ability to grow normally on D-glucose, were studied in Pachysolen tannophilus, a yeast capable of converting D-xylose to ethanol. Fourteen of the mutations were found to occur at nine distinct loci, and data indicated that many more loci remain to be detected. Most of the mutations were pleiotropic in character, and the expression of some of them was much affected by nutritional conditions and by genetic background. Mutations at several loci resulted in poor growth on at least one compound that was either an intermediate of the tricarboxylic acid cycle, succinate or {alpha}-ketoglutarate, or on compounds metabolizable via this cycle, ethanol or glycerol. An initial biochemical characterization of the mutants was undertaken. Analysis for xylose reductase, xylitol dehydrogenase, and xylulose kinase activity showed that one or more of these activities was affected in 12 of 13 mutants. However, drastic reduction in activity of a single enzyme was confined to that of xylitol dehydrogenase by mutations at three different loci and to that of D-xylose reductase by mutation at another locus. Growth of these latter four mutants was normal on all carbon sources tested that were not five-carbon sugars.

  15. A Mutator Affecting the Region of the Iso-1-Cytochrome c Gene in Yeast

    PubMed Central

    Liebman, Susan W.; Singh, Arjun; Sherman, Fred

    1979-01-01

    The mutator gene DEL1 in the yeast Saccharomyces cerevisiae causes a high rate of formation of multisite mutations that encompass the following three adjacent genes: CYC1, which determines the structure of iso-1-cytochrome c; RAD7, which controls UV sensitivity; and OSM1, which controls osomotic sensitivity. The simplest hypothesis is that these multisite mutations are deletions, although it has not been excluded that they may involve other types of gross chromosomal aberrations. In contrast, normal strains do not produce such multisite mutations even after mutagenic treatments.—The multisite mutations arise at a rate of approximately 10-5 to 10-6 per cell per division in DEL1 strains, which is much higher than rates observed for mutation of genes in normal strains. For example, normal strains produce all types of cyc1 mutants at a low rate of approximately 10-8 to 10-9. No evidence for multisite mutations was obtained upon analysis of numerous spontaneous ade1, ade2, met2 and met15 mutants isolated in a DEL1 strain. DEL1 segregates as a single Mendelian gene closely linked to the CYC1 locus. DEL1 appears to be both cis- and trans-dominant. The location of the DEL1 gene and the lack of effect on other genes suggest that the mutator acts only on a region adjacent to itself. PMID:231539

  16. Heavy metal stress in alders: Tolerance and vulnerability of the actinorhizal symbiosis.

    PubMed

    Bélanger, Pier-Anne; Bellenger, Jean-Philippe; Roy, Sébastien

    2015-11-01

    Alders have already demonstrated their potential for the revegetation of both mining and industrial sites. These actinorhizal trees and shrubs and the actinobacteria Frankia associate in a nitrogen-fixing symbiosis which could however be negatively affected by the presence of heavy metals, and accumulate them. In our hydroponic assay with black alders, quantification of the roots and shoots metal concentrations showed that, in the absence of stress, symbiosis increases Mo and Ni root content and simultaneously decreases Mo shoot content. Interestingly, the Mo shoot content also decreases in the presence of Ni, Cu, Pb, Zn and Cd for symbiotic alders. In symbiotic alders, Pb shoot translocation was promoted in presence of Pb. On the other hand, Cd exclusion in symbiotic root tissues was observed with Pb and Cd. In the presence of symbiosis, only Cd and Pb showed translocation into aerial tissues when present in the nutrient solution. Moreover, the translocation of Ni to shoot was prevented by symbiosis in the presence of Cd, Ni and Pb. The hydroponic experiment demonstrated that alders benefit from the symbiosis, producing more biomass (total, root and shoot) than non nodulated alders in control condition, and in the presence of metals (Cu, Ni, Zn, Pb and Cd). Heavy metals did not reduce the nodule numbers (SNN), but the presence of Zn or Cd did reduce nodule allocation. Our study suggests that the Frankia-alder symbiosis is a promising (and a compatible) plant-microorganism association for the revegetation of contaminated sites, with minimal risk of metal dispersion.

  17. Identification of a single ancestral CYP1B1 mutation in Slovak Gypsies (Roms) affected with primary congenital glaucoma

    PubMed Central

    Plasilova, M.; Stoilov, I.; Sarfarazi, M.; Kadasi, L.; Ferakova, E.; Ferak, V.

    1999-01-01

    Primary congenital glaucoma (PCG) is an autosomal recessive eye disease that occurs at an unusually high frequency in the ethnic isolate of Roms (Gypsies) in Slovakia. Recently, we linked the disease in this population to the GLC3A locus on 2p21. At this locus, mutations in the cytochrome P4501B1 (CYP1B1) gene have been identified as a molecular basis for this condition. Here, we report the results of CYP1B1 mutation screening of 43 PCG patients from 26 Slovak Rom families. A homozygous G→A transition at nucleotide 1505 in the highly conserved region of exon 3 was detected in all families. This mutation results in the E387K substitution, which affects the conserved K helix region of the cytochrome P450 molecule. Determination of the CYP1B1 polymorphic background showed a common DNA haplotype in all patients, thus indicating that the E387K mutation in Roms has originated from a single ancestral mutational event. The Slovak Roms represent the first population in which PCG is found to result from a single mutation in the CYP1B1 gene, so that a founder effect is the most plausible explanation of its increased incidence. An ARMS-PCR assay has been developed for fast detection of this mutation, thus allowing direct DNA based prenatal diagnosis as well as gene carrier detection in this particular population. Screening of 158 healthy Roms identified 17 (10.8%) mutation carriers, indicating that the frequency of PCG in this population may be even higher than originally estimated.


Keywords: primary congenital glaucoma (PCG); cytochrome P4501B1; Roms (Gypsies); founder effect PMID:10227395

  18. Biallelic mutations in huntington disease: A new case with just one affected parent, review of the literature and terminology.

    PubMed

    Uhlmann, Wendy R; Peñaherrera, Maria S; Robinson, Wendy P; Milunsky, Jeff M; Nicholson, Jane M; Albin, Roger L

    2015-05-01

    Patients with biallelic mutations for Huntington disease (HD) are rare. We present a 46-year-old female with two expanded Huntingtin (HTT) alleles with just one known affected parent. This is the first reported patient with molecular studies performed to exclude HTT uniparental disomy (UPD). The proband had biparental inheritance of HTT alleles (42/44 CAG repeats). Given the negative UPD results, the proband's unaffected mother either had a reduced penetrance allele that expanded into the full mutation range during transmission to our patient or an unknown full HTT mutation and died before symptom onset, unlikely given no family history of HD and asymptomatic at age 59. We made the novel observation in our literature review that most patients with biallelic HD did not have two full HTT mutations. Most had one HTT allele that was in the intermediate or reduced penetrance ranges or 40 CAG repeats, the lowest limit of the full mutation range. Although the number of patients is small, when an allele in these size ranges was present, generally the age of HD onset was in the 50s. If the second HTT allele had >45 repeats, then onset was typically 20s-30s. While similar ages of onset have been reported for patients with one or two HTT mutations, patients with biallelic mutations may have later onset if an expanded HTT allele has ≤40 CAG repeats. Finally, we propose that "biallelic mutations" or "compound heterozygosity" are more accurate descriptive terms than "homozygosity" when there are two non-identical expanded HTT alleles.

  19. Isolation of Mutations Affecting Neural Circuitry Required for Grooming Behavior in Drosophila Melanogaster

    PubMed Central

    Phillis, R. W.; Bramlage, A. T.; Wotus, C.; Whittaker, A.; Gramates, L. S.; Seppala, D.; Farahanchi, F.; Caruccio, P.; Murphey, R. K.

    1993-01-01

    We have developed a screen for the isolation of mutations that produce neural defects in adult Drosophila melanogaster. In this screen, we identify mutants as flies unable to remove a light coating of applied dust in a 2-hr period. We have recovered and characterized six mutations and have found that they produce coordination defects and some have reduced levels of reflex responsiveness to the stimulation of single tactile sensory bristles. The grooming defects produced by all six of the mutations are recessive, and each of the mutations has been genetically mapped. We have also used our assay to test the grooming ability of stocks containing mutations that produce known neural defects. PMID:8454205

  20. Identification of a novel mutation in the PAX9 gene in a family affected by oligodontia and other dental anomalies.

    PubMed

    Tallón-Walton, Victòria; Manzanares-Céspedes, Maria Cristina; Arte, Sirpa; Carvalho-Lobato, Patricia; Valdivia-Gandur, Ivan; Garcia-Susperregui, Antonio; Ventura, Francesc; Nieminen, Pekka

    2007-12-01

    The objective of the present work was to study the phenotype and the genotype of three generations of a family affected by oligodontia and other dental anomalies. These family members also presented systemic conditions such as hypercholesterolemia, hypothyroidism, diabetes mellitus, scoliosis, and congenital cardiovascular anomalies. Clinical evaluation, panoramic radiographs, and anamnestic data were used for dental analysis. DNA extraction was carried out from gum samples or buccal swabs. A mutation was identified in six subjects across three generations affected by oligodontia, as well as different phenotypical manifestations, both systemic and oral. The previously undescribed PAX9 mutation was observed in the paired box (exon 2); this was a heterozygote transition of C175 to T, implying the change of arginine 59 for a termination codon. These results strongly suggested that the identified mutation was the etiological cause of the oligodontia. However, in two family members affected by both hypodontia and peg-shaped upper lateral incisors, no mutations in the PAX9 and MSX1 genes were identified. This fact underscores the importance that other presently unknown genes and developmental factors have in tooth development and in the etiology of dental anomalies.

  1. Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

    PubMed

    Mistri, Mehul; Tamhankar, Parag M; Sheth, Frenny; Sanghavi, Daksha; Kondurkar, Pratima; Patil, Swapnil; Idicula-Thomas, Susan; Gupta, Sarita; Sheth, Jayesh

    2012-01-01

    Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

  2. Recurrent somatic mutations affecting B-cell receptor signaling pathway genes in follicular lymphoma

    PubMed Central

    Matlock, Matthew; Trani, Lee; Fronick, Catrina C.; Fulton, Robert S.; Kreisel, Friederike; Cashen, Amanda F.; Carson, Kenneth R.; Bartlett, Nancy L.

    2017-01-01

    Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma, yet it remains only partially characterized at the genomic level. To improve our understanding of the genetic underpinnings of this incurable and clinically heterogeneous disease, whole-exome sequencing was performed on tumor/normal pairs from a discovery cohort of 24 patients with FL. Using these data and mutations identified in other B-cell malignancies, 1716 genes were sequenced in 113 FL tumor samples from 105 primarily treatment-naive individuals. We identified 39 genes that were mutated significantly above background mutation rates. CREBBP mutations were associated with inferior PFS. In contrast, mutations in previously unreported HVCN1, a voltage-gated proton channel-encoding gene and B-cell receptor signaling modulator, were associated with improved PFS. In total, 47 (44.8%) patients harbor mutations in the interconnected B-cell receptor (BCR) and CXCR4 signaling pathways. Histone gene mutations were more frequent than previously reported (identified in 43.8% of patients) and often co-occurred (17.1% of patients). A novel, recurrent hotspot was identified at a posttranslationally modified residue in the histone H2B family. This study expands the number of mutated genes described in several known signaling pathways and complexes involved in lymphoma pathogenesis (BCR, Notch, SWitch/sucrose nonfermentable (SWI/SNF), vacuolar ATPases) and identified novel recurrent mutations (EGR1/2, POU2AF1, BTK, ZNF608, HVCN1) that require further investigation in the context of FL biology, prognosis, and treatment. PMID:28064239

  3. Metabolic symbiosis at the origin of eukaryotes.

    PubMed

    López-Garćia, P; Moreira, D

    1999-03-01

    Thirty years after Margulis revived the endosymbiosis theory for the origin of mitochondria and chloroplasts, two novel symbiosis hypotheses for the origin of eukaryotes have been put forward. Both propose that eukaryotes arose through metabolic symbiosis (syntrophy) between eubacteria and methanogenic Archaea. They also propose that this was mediated by interspecies hydrogen transfer and that, initially, mitochondria were anaerobic. These hypotheses explain the mosaic character of eukaryotes (i.e. an archaeal-like genetic machinery and a eubacterial-like metabolism), as well as distinct eukaryotic characteristics (which are proposed to be products of symbiosis). Combined data from comparative genomics, microbial ecology and the fossil record should help to test their validity.

  4. The Rhizobium-plant symbiosis.

    PubMed Central

    van Rhijn, P; Vanderleyden, J

    1995-01-01

    Rhizobium, Bradyrhizobium, and Azorhizobium species are able to elicit the formation of unique structures, called nodules, on the roots or stems of the leguminous host. In these nodules, the rhizobia convert atmospheric N2 into ammonia for the plant. To establish this symbiosis, signals are produced early in the interaction between plant and rhizobia and they elicit discrete responses by the two symbiotic partners. First, transcription of the bacterial nodulation (nod) genes is under control of the NodD regulatory protein, which is activated by specific plant signals, flavonoids, present in the root exudates. In return, the nod-encoded enzymes are involved in the synthesis and excretion of specific lipooligosaccharides, which are able to trigger on the host plant the organogenic program leading to the formation of nodules. An overview of the organization, regulation, and function of the nod genes and their participation in the determination of the host specificity is presented. PMID:7708010

  5. Identification of polymerase gene mutations that affect viral replication in H5N1 influenza viruses isolated from pigeons.

    PubMed

    Elgendy, Emad Mohamed; Arai, Yasuha; Kawashita, Norihito; Daidoji, Tomo; Takagi, Tatsuya; Ibrahim, Madiha Salah; Nakaya, Takaaki; Watanabe, Yohei

    2017-01-01

    Highly pathogenic avian influenza virus H5N1 infects a wide range of host species, with a few cases of sporadic pigeon infections reported in the Middle East and Asia. However, the role of pigeons in the ecology and evolution of H5N1 viruses remains unclear. We previously reported two H5N1 virus strains, isolated from naturally infected pigeons in Egypt, that have several unique mutations in their viral polymerase genes. Here, we investigated the effect of these mutations on H5N1 polymerase activity and viral growth and identified three mutations that affected viral polymerase activity. The results showed that the PB1-V3D mutation significantly decreased polymerase activity and viral growth in both mammalian and avian cells. In contrast, the PB2-K627E and PA-K158R mutations had moderate effects: PB2-K627E decreased and PA-K158R increased polymerase activity. Structural homology modelling indicated that the PB1-V3D residue was located in the PB1 core region that interacts with PA, predicting that the PB1 mutation would produce a stronger interaction between PB1 and PA that results in decreased replication of pigeon-derived H5N1 viruses. Our results identified several unique mutations responsible for changes in polymerase activity in H5N1 virus strains isolated from infected pigeons, emphasizing the importance of avian influenza surveillance in pigeons and in studying the possible role of pigeon-derived H5N1 viruses in avian influenza virus evolution.

  6. Identification of a novel synonymous mutation in the human β -Ureidopropionase Gene UPB1 affecting pre-mRNA splicing.

    PubMed

    Meijer, J; Nakajima, Y; Zhang, C; Meinsma, R; Ito, T; Van Kuilenburg, A B P

    2013-01-01

    β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyzes the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, and ammonia and CO2. To date, only 16 genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report the clinical, biochemical, and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Mutation analysis of the UPB1 gene showed that the patient was compound heterozygous for a novel synonymous mutation c.93C >T (p.Gly31Gly) in exon 1 and a previously described missense mutation c.977G >A (p.Arg326Gln) in exon 9. The in silico predicted effect of the synonymous mutation p.Gly31Gly on pre-mRNA splicing was investigated using a minigene approach. Wild-type and the mutated minigene constructs, containing the entire exon 1, intron 1, and exon 2 of UPB1, yielded different splicing products after expression in HEK293 cells. The c.93C >T (p.Gly31Gly) mutation resulted in altered pre-mRNA splicing of the UPB1 minigene construct and a deletion of the last 13 nucleotides of exon 1. This deletion (r.92_104delGCAAGGAACTCAG) results in a frame shift and the generation of a premature stop codon (p.Lys32SerfsX31). Using a minigene approach, we have thus identified the first synonymous mutation in the UPB1 gene, creating a cryptic splice-donor site affecting pre-mRNA splicing.

  7. Evolution of symbiosis with resource allocation from fecundity to survival.

    PubMed

    Fukui, Shin

    2014-05-01

    Symbiosis is one of the most fundamental relationships between or among organisms and includes parasitism (which has negative effects on the fitness of the interacting partner), commensalism (no effect), and mutualism (positive effects). The effects of these interactions are usually assumed to influence a single component of a species' fitness, either survival or fecundity, even though in reality the interaction can simultaneously affect both of these components. I used a dual lattice model to investigate the process of evolution of mutualistic symbiosis in the presence of interactive effects on both survival and fecundity. I demonstrate that a positive effect on survival and a negative effect on fecundity are key to the establishment of mutualism. Furthermore, both the parasitic and the mutualistic behaviour must carry large costs for mutualism to evolve. This helps develop a new understanding of symbiosis as a function of resource allocation, in which resources are shifted from fecundity to survival. The simultaneous establishment of mutualism from parasitism never occurs in two species, but can do so in one of the species as long as the partner still behaves parasitically. This suggests that one of the altruistic behaviours in a mutualistic unit consisting of two species must originate as a parasitic behaviour.

  8. Shoot- and root-borne cytokinin influences arbuscular mycorrhizal symbiosis.

    PubMed

    Cosme, Marco; Ramireddy, Eswarayya; Franken, Philipp; Schmülling, Thomas; Wurst, Susanne

    2016-10-01

    The arbuscular mycorrhizal (AM) symbiosis is functionally important for the nutrition and growth of most terrestrial plants. Nearly all phytohormones are employed by plants to regulate the symbiosis with AM fungi, but the regulatory role of cytokinin (CK) is not well understood. Here, we used transgenic tobacco (Nicotiana tabacum) with a root-specific or constitutive expression of CK-degrading CKX genes and the corresponding wild-type to investigate whether a lowered content of CK in roots or in both roots and shoots influences the interaction with the AM fungus Rhizophagus irregularis. Our data indicates that shoot CK has a positive impact on AM fungal development in roots and on the root transcript level of an AM-responsive phosphate transporter gene (NtPT4). A reduced CK content in roots caused shoot and root growth depression following AM colonization, while neither the uptake of phosphorus or nitrogen nor the root transcript levels of NtPT4 were significantly affected. This suggests that root CK may restrict the C availability from the roots to the fungus thus averting parasitism by AM fungi. Taken together, our study indicates that shoot- and root-borne CK have distinct roles in AM symbiosis. We propose a model illustrating how plants may employ CK to regulate nutrient exchange with the ubiquitous AM fungi.

  9. Evolution of symbiosis with resource allocation from fecundity to survival

    NASA Astrophysics Data System (ADS)

    Fukui, Shin

    2014-05-01

    Symbiosis is one of the most fundamental relationships between or among organisms and includes parasitism (which has negative effects on the fitness of the interacting partner), commensalism (no effect), and mutualism (positive effects). The effects of these interactions are usually assumed to influence a single component of a species' fitness, either survival or fecundity, even though in reality the interaction can simultaneously affect both of these components. I used a dual lattice model to investigate the process of evolution of mutualistic symbiosis in the presence of interactive effects on both survival and fecundity. I demonstrate that a positive effect on survival and a negative effect on fecundity are key to the establishment of mutualism. Furthermore, both the parasitic and the mutualistic behaviour must carry large costs for mutualism to evolve. This helps develop a new understanding of symbiosis as a function of resource allocation, in which resources are shifted from fecundity to survival. The simultaneous establishment of mutualism from parasitism never occurs in two species, but can do so in one of the species as long as the partner still behaves parasitically. This suggests that one of the altruistic behaviours in a mutualistic unit consisting of two species must originate as a parasitic behaviour.

  10. Search for mutations affecting protein structure in children of atomic bomb survivors: preliminary report

    SciTech Connect

    Neel, J.V.; Satoh, C.; Hamilton, H.B.; Otake, M.; Goriki, K.; Kageoka, T.; Fujita, M.; Neriishi, S.; Asakawa J.

    1980-07-01

    A total of 289,868 locus tests, based on 28 different protein phenotypes and using one-dimensional electrophoresis to detect variant proteins, has yielded one probable mutation in the offspring of proximally exposed parents, who received an estimated average gonadal exposure of 31 to 39 rem in the atomic bombings of Hiroshima and Nagasaki. There were no mutations in 208,196 locus tests involving children of distally exposed parents, who had essentially no radiation exposure.

  11. Search for mutations affecting protein structure in children of atomic bomb survivors: preliminary report.

    PubMed

    Neel, J V; Satoh, C; Hamilton, H B; Otake, M; Goriki, K; Kageoka, T; Fujita, M; Neriishi, S; Asakawa, J

    1980-07-01

    A total of 289,868 locus tests, based on 28 different protein phenotypes and using one-dimensional electrophoresis to detect variant proteins, has yielded one probable mutation in the offspring of "proximally exposed" parents, who received an estimated average gonadal exposure of 31 to 39 rem in the atomic bombings of Hiroshima and Nagasaki. There were no mutations in 208,196 locus tests involving children of "distally exposed" parents, who had essentially no radiation exposure.

  12. Mutations in MCT8 in patients with Allan-Herndon-Dudley-syndrome affecting its cellular distribution.

    PubMed

    Kersseboom, Simone; Kremers, Gert-Jan; Friesema, Edith C H; Visser, W Edward; Klootwijk, Wim; Peeters, Robin P; Visser, Theo J

    2013-05-01

    Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellular distribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins.

  13. Molecular modeling studies demonstrate key mutations that could affect the ligand recognition by influenza AH1N1 neuraminidase.

    PubMed

    Ramírez-Salinas, Gema L; García-Machorro, J; Quiliano, Miguel; Zimic, Mirko; Briz, Verónica; Rojas-Hernández, Saul; Correa-Basurto, J

    2015-11-01

    The goal of this study was to identify neuraminidase (NA) residue mutants from human influenza AH1N1 using sequences from 1918 to 2012. Multiple alignment studies of complete NA sequences (5732) were performed. Subsequently, the crystallographic structure of the 1918 influenza (PDB ID: 3BEQ-A) was used as a wild-type structure and three-dimensional (3-D) template for homology modeling of the mutated selected NA sequences. The 3-D mutated NAs were refined using molecular dynamics (MD) simulations (50 ns). The refined 3-D models were used to perform docking studies using oseltamivir. Multiple sequence alignment studies showed seven representative mutations (A232V, K262R, V263I, T264V, S367L, S369N, and S369K). MD simulations applied to 3-D NAs showed that each NA had different active-site shapes according to structural surface visualization and docking results. Moreover, Cartesian principal component analyses (cPCA) show structural differences among these NA structures caused by mutations. These theoretical results suggest that the selected mutations that are located outside of the active site of NA could affect oseltamivir recognition and could be associated with resistance to oseltamivir.

  14. A critical functional missense mutation (H173R) in the bovine PROP1 gene significantly affects growth traits in cattle.

    PubMed

    Pan, Chuanying; Wu, Chongyang; Jia, Wenchao; Xu, Yao; Lei, Chuzhao; Hu, Shenrong; Lan, Xianyong; Chen, Hong

    2013-12-01

    The PROP1 protein, encoded by the prophet of Pit-1 (PROP1) gene, exhibits both DNA-binding and transcriptional activation abilities. Its expression leads to the ontogenesis of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), and pituitary hormone. The missense mutation H173R in PROP1 may result in deficiencies of GH, PRL, TSH, and Pit-1, thereby affecting growth traits. The objective of this study was to characterize the H173R mutation within the PROP1 gene and examine its associations with growth traits in cattle. Accordingly, the H173R mutation was genotyped in 1207 cows belonging to five Chinese native breeds. Three genotypes were identified among the specimens, with genotype AA being the major one. Consequently, the "G" allele was the minor allele. Association testing revealed that the H173R mutation was significantly associated with body weight, average daily weight gain and physical parameters in the analyzed breeds. Interestingly, the cows with genotype AG and/or AA had superior growth traits compared with those expressing the GG genotype, in all tested breeds. These findings revealed that the "A" allele had positive effects on growth traits, which was consistent with the increasing binding ability and enhanced activation capacity associated with the bovine isoform PROP1-173H, representing the "A" allele. Therefore, the H173R mutation can be considered as a DNA marker for selecting individuals with superior growth traits, thereby contributing to research on breeding and genetics in the beef industry.

  15. A Novel Inducer of Roseobacter Motility Is Also a Disruptor of Algal Symbiosis

    PubMed Central

    Sule, Preeti

    2013-01-01

    Silicibacter sp. strain TM1040, a member of the Roseobacter clade, forms a symbiosis with unicellular phytoplankton, which is inextricably linked to the biphasic “swim or stick” lifestyle of the bacteria. Mutations in flaC bias the population toward the motile phase. Renewed examination of the FlaC− strain (HG1016) uncovered that it is composed of two different cells: a pigmented type, PS01, and a nonpigmented cell, PS02, each of which has an identical mutation in flaC. While monocultures of PS01 and PS02 had few motile cells (0.6 and 6%, respectively), coculturing the two strains resulted in a 10-fold increase in the number of motile cells. Cell-free supernatants from coculture or wild-type cells were fully capable of restoring motility to PS01 and PS02, which was due to increased fliC3 (flagellin) transcription, FliC3 protein levels per cell, and flagella synthesis. The motility-inducing compound has an estimated mass of 226 Da, as determined by mass spectrometry, and is referred to as Roseobacter Motility Inducer (RMI). Mutations affecting genes involved in phenyl acetic acid synthesis significantly reduced RMI, while defects in tropodithietic acid (TDA) synthesis had marginal or no effect on RMI. RMI biosynthesis is induced by p-coumaric acid, a product of algal lignin degradation. When added to algal cultures, RMI caused loss of motility, cell enlargement, and vacuolization in the algal cells. RMI is a new member of the roseobacticide family of troponoid compounds whose activities affect roseobacters, by shifting their population toward motility, as well as their phytoplankton hosts, through an algicidal effect. PMID:23161030

  16. HERC 1 Ubiquitin Ligase Mutation Affects Neocortical, CA3 Hippocampal and Spinal Cord Projection Neurons: An Ultrastructural Study

    PubMed Central

    Ruiz, Rocío; Pérez-Villegas, Eva María; Bachiller, Sara; Rosa, José Luis; Armengol, José Angel

    2016-01-01

    The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tambaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic loss of cerebellar Purkinje cells. Interestingly, signs of autophagy are absent in both interneurons and neuroglia cells. Affected neurons have in common that they are projection neurons which receive strong and varied synaptic inputs, and possess the highest degree of neuronal activity. Therefore, because the integrity of the ubiquitin-proteasome system is essential for protein degradation and hence, for normal protein turnover, it could be hypothesized that the deleterious effects of the misrouting of these pathways would depend directly on the neuronal activity. PMID:27147983

  17. Novel missense mutation in the GALNS gene in an affected patient with severe form of mucopolysaccharidosis type IVA.

    PubMed

    Seyedhassani, Seyed Mohammad; Hashemi-Gorji, Feyzollah; Yavari, Mahdieh; Mirfakhraie, Reza

    2015-10-23

    Mucopolysaccharidosis type IVA (MPS IVA), also known as Morquio A, is an autosomal recessive disorder characterized by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes major skeletal and connective tissue abnormalities and affects multiple organ systems. In this study, one MPS IVA patient with a severe form from consanguine large Iranian family has been investigated. To find a mutation, all of the 14 exons and intron-exon junctions of GALNS gene were sequenced. Sequencing results were analyzed using bioinformatic analysis in order to predict probable pathogenic effect of the variant. One novel homozygous missense mutation in exon 5, c.542A>G (p.Y181C), was found in the proband. That was predicted as being probably pathogenic by bioinformatics analysis. Segregation and familial study confirmed this pathogenic mutation. In conclusion, we have identified the novel mutation responsible for MPS IVA in an Iranian patient to assist in the diagnosis, genetic counseling and prenatal diagnosis of the affected families.

  18. HERC 1 Ubiquitin Ligase Mutation Affects Neocortical, CA3 Hippocampal and Spinal Cord Projection Neurons: An Ultrastructural Study.

    PubMed

    Ruiz, Rocío; Pérez-Villegas, Eva María; Bachiller, Sara; Rosa, José Luis; Armengol, José Angel

    2016-01-01

    The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tambaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic loss of cerebellar Purkinje cells. Interestingly, signs of autophagy are absent in both interneurons and neuroglia cells. Affected neurons have in common that they are projection neurons which receive strong and varied synaptic inputs, and possess the highest degree of neuronal activity. Therefore, because the integrity of the ubiquitin-proteasome system is essential for protein degradation and hence, for normal protein turnover, it could be hypothesized that the deleterious effects of the misrouting of these pathways would depend directly on the neuronal activity.

  19. C-Nap1 mutation affects centriole cohesion and is associated with a Seckel-like syndrome in cattle

    PubMed Central

    Floriot, Sandrine; Vesque, Christine; Rodriguez, Sabrina; Bourgain-Guglielmetti, Florence; Karaiskou, Anthi; Gautier, Mathieu; Duchesne, Amandine; Barbey, Sarah; Fritz, Sébastien; Vasilescu, Alexandre; Bertaud, Maud; Moudjou, Mohammed; Halliez, Sophie; Cormier-Daire, Valérie; E.L. Hokayem, Joyce; Nigg, Erich A.; Manciaux, Luc; Guatteo, Raphaël; Cesbron, Nora; Toutirais, Geraldine; Eggen, André; Schneider-Maunoury, Sylvie; Boichard, Didier; Sobczak-Thépot, Joelle; Schibler, Laurent

    2015-01-01

    Caprine-like Generalized Hypoplasia Syndrome (SHGC) is an autosomal-recessive disorder in Montbéliarde cattle. Affected animals present a wide range of clinical features that include the following: delayed development with low birth weight, hind limb muscular hypoplasia, caprine-like thin head and partial coat depigmentation. Here we show that SHGC is caused by a truncating mutation in the CEP250 gene that encodes the centrosomal protein C-Nap1. This mutation results in centrosome splitting, which neither affects centriole ultrastructure and duplication in dividing cells nor centriole function in cilium assembly and mitotic spindle organization. Loss of C-Nap1-mediated centriole cohesion leads to an altered cell migration phenotype. This discovery extends the range of loci that constitute the spectrum of autosomal primary recessive microcephaly (MCPH) and Seckel-like syndromes. PMID:25902731

  20. Mutations in the passenger polypeptide can affect its partitioning between mitochondria and cytoplasm: mutations can impair the mitochondrial import of DsRed.

    PubMed

    Pastukh, Viktoriya; Shokolenko, Inna N; Wilson, Glenn L; Alexeyev, Mikhail F

    2008-06-01

    In this study, we report that the partitioning between mitochondria and cytoplasm of two variants, mCherry and DsRed Express (DRE), of the red fluorescent protein, DsRed, fused to one of the six matrix targeting sequences (MTSs) can be affected by both MTS and amino acid substitutions in DsRed. Of the six MTSs tested, MTSs from superoxide dismutase and DNA polymerase gamma failed to direct mCherry, but not DRE to mitochondria. By evaluating a series of chimeras between mCherry and DRE fused to the MTS of superoxide dismutase, we attribute the differences in the mitochondrial partitioning to differences in the primary amino acid sequence of the passenger polypeptide. The impairment of mitochondrial partitioning closely parallels the number of mCherry-specific mutations, and is not specific to mutations located in any particular region of the polypeptide. These observations suggest that both MTS and the passenger polypeptide affect the efficiency of mitochondrial import and provide a rationale for the observed diversity in the primary amino acid sequences of natural MTSs.

  1. The shiverer mutation affects the persistence of Theiler's virus in the central nervous system.

    PubMed Central

    Bihl, F; Pena-Rossi, C; Guénet, J L; Brahic, M; Bureau, J F

    1997-01-01

    Theiler's virus persists in the white matter of the spinal cord of genetically susceptible mice and causes primary demyelination. The virus persists in macrophages/microglial cells, but also in oligodendrocytes, the myelin-forming cells. Susceptibility/resistance to this chronic infection has been mapped to several loci including one tentatively located in the telomeric region of chromosome 18, close to the myelin basic protein locus (Mbp locus). To determine if the MBP gene influences viral persistence, we inoculated C3H mice bearing the shiverer mutation, a 20-kb deletion in the gene. Whereas control C3H mice were of intermediate susceptibility, C3H mice heterozygous for the mutation were very susceptible, and those homozygous for the mutation were completely resistant. This resistance was not immune mediated. Furthermore, C3H/101H mice homozygous for a point mutation in the gene coding for the proteolipid protein of myelin, the rumpshaker mutation, were resistant. These results strongly support the view that oligodendrocytes are a necessary viral target for the establishment of a persistent infection by Theiler's virus. PMID:9188567

  2. Heritable symbiosis: The advantages and perils of an evolutionary rabbit hole.

    PubMed

    Bennett, Gordon M; Moran, Nancy A

    2015-08-18

    Many eukaryotes have obligate associations with microorganisms that are transmitted directly between generations. A model for heritable symbiosis is the association of aphids, a clade of sap-feeding insects, and Buchnera aphidicola, a gammaproteobacterium that colonized an aphid ancestor 150 million years ago and persists in almost all 5,000 aphid species. Symbiont acquisition enables evolutionary and ecological expansion; aphids are one of many insect groups that would not exist without heritable symbiosis. Receiving less attention are potential negative ramifications of symbiotic alliances. In the short run, symbionts impose metabolic costs. Over evolutionary time, hosts evolve dependence beyond the original benefits of the symbiosis. Symbiotic partners enter into an evolutionary spiral that leads to irreversible codependence and associated risks. Host adaptations to symbiosis (e.g., immune-system modification) may impose vulnerabilities. Symbiont genomes also continuously accumulate deleterious mutations, limiting their beneficial contributions and environmental tolerance. Finally, the fitness interests of obligate heritable symbionts are distinct from those of their hosts, leading to selfish tendencies. Thus, genes underlying the host-symbiont interface are predicted to follow a coevolutionary arms race, as observed for genes governing host-pathogen interactions. On the macroevolutionary scale, the rapid evolution of interacting symbiont and host genes is predicted to accelerate host speciation rates by generating genetic incompatibilities. However, degeneration of symbiont genomes may ultimately limit the ecological range of host species, potentially increasing extinction risk. Recent results for the aphid-Buchnera symbiosis and related systems illustrate that, whereas heritable symbiosis can expand ecological range and spur diversification, it also presents potential perils.

  3. Heritable symbiosis: The advantages and perils of an evolutionary rabbit hole

    PubMed Central

    Bennett, Gordon M.; Moran, Nancy A.

    2015-01-01

    Many eukaryotes have obligate associations with microorganisms that are transmitted directly between generations. A model for heritable symbiosis is the association of aphids, a clade of sap-feeding insects, and Buchnera aphidicola, a gammaproteobacterium that colonized an aphid ancestor 150 million years ago and persists in almost all 5,000 aphid species. Symbiont acquisition enables evolutionary and ecological expansion; aphids are one of many insect groups that would not exist without heritable symbiosis. Receiving less attention are potential negative ramifications of symbiotic alliances. In the short run, symbionts impose metabolic costs. Over evolutionary time, hosts evolve dependence beyond the original benefits of the symbiosis. Symbiotic partners enter into an evolutionary spiral that leads to irreversible codependence and associated risks. Host adaptations to symbiosis (e.g., immune-system modification) may impose vulnerabilities. Symbiont genomes also continuously accumulate deleterious mutations, limiting their beneficial contributions and environmental tolerance. Finally, the fitness interests of obligate heritable symbionts are distinct from those of their hosts, leading to selfish tendencies. Thus, genes underlying the host–symbiont interface are predicted to follow a coevolutionary arms race, as observed for genes governing host–pathogen interactions. On the macroevolutionary scale, the rapid evolution of interacting symbiont and host genes is predicted to accelerate host speciation rates by generating genetic incompatibilities. However, degeneration of symbiont genomes may ultimately limit the ecological range of host species, potentially increasing extinction risk. Recent results for the aphid–Buchnera symbiosis and related systems illustrate that, whereas heritable symbiosis can expand ecological range and spur diversification, it also presents potential perils. PMID:25713367

  4. Oncogenic Mutations Differentially Affect Bax Monomer, Dimer, and Oligomeric Pore Formation in the Membrane

    NASA Astrophysics Data System (ADS)

    Zhang, Mingzhen; Zheng, Jie; Nussinov, Ruth; Ma, Buyong

    2016-09-01

    Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.

  5. Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses, the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example, the Bmr6 gene in sorghum (Sorghum bicolor) has b...

  6. The glabra1 mutation affects cuticle formation and plant responses to microbes.

    PubMed

    Xia, Ye; Yu, Keshun; Navarre, Duroy; Seebold, Kenneth; Kachroo, Aardra; Kachroo, Pradeep

    2010-10-01

    Systemic acquired resistance (SAR) is a form of defense that provides resistance against a broad spectrum of pathogens in plants. Previous work indicates a role for plastidial glycerolipid biosynthesis in SAR. Specifically, mutations in FATTY ACID DESATURASE7 (FAD7), which lead to reduced trienoic fatty acid levels and compromised plastidial lipid biosynthesis, have been associated with defective SAR. We show that the defective SAR in Arabidopsis (Arabidopsis thaliana) fad7-1 plants is not associated with a mutation in FAD7 but rather with a second-site mutation in GLABRA1 (GL1), a gene well known for its role in trichome formation. The compromised SAR in gl1 plants is associated with impairment in their cuticles. Furthermore, mutations in two other components of trichome development, GL3 and TRANSPARENT TESTA GLABRA1, also impaired cuticle development and SAR. This suggests an overlap in the biochemical pathways leading to cuticle and trichome development. Interestingly, exogenous application of gibberellic acid (GA) not only enhanced SAR in wild-type plants but also restored SAR in gl1 plants. In contrast to GA, the defense phytohoromes salicylic acid and jasmonic acid were unable to restore SAR in gl1 plants. GA application increased levels of cuticular components but not trichome formation on gl1 plants, thus implicating cuticle, but not trichomes, as an important component of SAR. Our findings question the prudence of using mutant backgrounds for genetic screens and underscore a need to reevaluate phenotypes previously studied in the gl1 background.

  7. Oncogenic Mutations Differentially Affect Bax Monomer, Dimer, and Oligomeric Pore Formation in the Membrane

    PubMed Central

    Zhang, Mingzhen; Zheng, Jie; Nussinov, Ruth; Ma, Buyong

    2016-01-01

    Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail. PMID:27630059

  8. Structural analysis of tissues affected by cytochrome C oxidase deficiency due to mutations in the SCO2 gene.

    PubMed

    Vesela, Katerina; Hulkova, Helena; Hansikova, Hana; Zeman, Jiri; Elleder, Milan

    2008-01-01

    Structural and histochemical studies carried out in a series of seven cases (from five families) with isolated cytochrome c oxidase (COX) deficiency caused by mutations in the SCO2 gene (1, 2) disclosed changes concentrated in the nervous system, skeletal muscle and myocardium. In five patients homozygous for the E140K mutation, the phenotype was predominantly neuromuscular and the average life span ranged between 9 and 15 months. In two cases, the course was more rapid (death at 7 and 11 weeks of life) and featured marked cardiac hypertrophy (3- and 4-fold increase in heart weight). This predominantly cardiomyopathic phenotype was associated with compound heterozygosity (E140K with another nonsense mutation) in the SCO2 gene. Polioencephalopathy with neurodegeneration and neuronal drop out was present in all cases with evidence that retinal neurons might be seriously affected too. Involvement of spinal motoneurons together with cytochrome c oxidase deficiency in muscle represents a "double hit" for the skeletal muscle. The mitochondrial population was not found to be significantly increased or structurally altered, with the exception of two compound heterozygotes in which the cardiac mitochondria were increased in number and size. Our report extends knowledge of the pathology of COX deficiency caused by mutations in the SCO2 gene.

  9. Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport.

    PubMed Central

    Tripodi, G; Valtorta, F; Torielli, L; Chieregatti, E; Salardi, S; Trusolino, L; Menegon, A; Ferrari, P; Marchisio, P C; Bianchi, G

    1996-01-01

    The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage. PMID:8675693

  10. Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins.

    PubMed

    De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2012-12-01

    The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

  11. Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus–Mesorhizobium loti symbiosis

    PubMed Central

    Fukudome, Mitsutaka; Calvo-Begueria, Laura; Kado, Tomohiro; Osuki, Ken-ichi; Rubio, Maria Carmen; Murakami, Ei-ichi; Nagata, Maki; Kucho, Ken-ichi; Sandal, Niels; Stougaard, Jens; Becana, Manuel; Uchiumi, Toshiki

    2016-01-01

    Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia–legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5′-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo. The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels. PMID:27443280

  12. Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis.

    PubMed

    Fukudome, Mitsutaka; Calvo-Begueria, Laura; Kado, Tomohiro; Osuki, Ken-Ichi; Rubio, Maria Carmen; Murakami, Ei-Ichi; Nagata, Maki; Kucho, Ken-Ichi; Sandal, Niels; Stougaard, Jens; Becana, Manuel; Uchiumi, Toshiki

    2016-09-01

    Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels.

  13. Mutations in Nonconserved Domains of Ty3 Integrase Affect Multiple Stages of the Ty3 Life Cycle

    PubMed Central

    Nymark-McMahon, M. Henrietta; Sandmeyer, Suzanne B.

    1999-01-01

    Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3′ ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3′ ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle. PMID:9847351

  14. Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells.

    PubMed

    Kim, Euysoo; Hyrc, Krzysztof L; Speck, Judith; Salles, Felipe T; Lundberg, Yunxia W; Goldberg, Mark P; Kachar, Bechara; Warchol, Mark E; Ornitz, David M

    2011-03-01

    Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.

  15. Paxillin mutations affect focal adhesions and lead to altered mitochondrial dynamics

    PubMed Central

    Kawada, Ichiro; Hasina, Rifat; Lennon, Frances E; Bindokas, Vytautas P; Usatyuk, Peter; Tan, Yi-Hung C; Krishnaswamy, Soundararajan; Arif, Qudsia; Carey, George; Hseu, Robyn D; Robinson, Matthew; Tretiakova, Maria; Brand, Toni M; Iida, Mari; Ferguson, Mark K; Wheeler, Deric L; Husain, Aliya N; Natarajan, Viswanathan; Vokes, Everett E; Singleton, Patrick A; Salgia, Ravi

    2013-01-01

    Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets. PMID:23792636

  16. Rhizobium-legume symbiosis shares an exocytotic pathway required for arbuscule formation.

    PubMed

    Ivanov, Sergey; Fedorova, Elena E; Limpens, Erik; De Mita, Stephane; Genre, Andrea; Bonfante, Paola; Bisseling, Ton

    2012-05-22

    Endosymbiotic interactions are characterized by the formation of specialized membrane compartments, by the host in which the microbes are hosted, in an intracellular manner. Two well-studied examples, which are of major agricultural and ecological importance, are the widespread arbuscular mycorrhizal symbiosis and the Rhizobium-legume symbiosis. In both symbioses, the specialized host membrane that surrounds the microbes forms a symbiotic interface, which facilitates the exchange of, for example, nutrients in a controlled manner and, therefore, forms the heart of endosymbiosis. Despite their key importance, the molecular and cellular mechanisms underlying the formation of these membrane interfaces are largely unknown. Recent studies strongly suggest that the Rhizobium-legume symbiosis coopted a signaling pathway, including receptor, from the more ancient arbuscular mycorrhizal symbiosis to form a symbiotic interface. Here, we show that two highly homologous exocytotic vesicle-associated membrane proteins (VAMPs) are required for formation of the symbiotic membrane interface in both interactions. Silencing of these Medicago VAMP72 genes has a minor effect on nonsymbiotic plant development and nodule formation. However, it blocks symbiosome as well as arbuscule formation, whereas root colonization by the microbes is not affected. Identification of these VAMP72s as common symbiotic regulators in exocytotic vesicle trafficking suggests that the ancient exocytotic pathway forming the periarbuscular membrane compartment has also been coopted in the Rhizobium-legume symbiosis.

  17. A new Gsdma3 mutation affecting anagen phase of first hair cycle

    SciTech Connect

    Tanaka, Shigekazu; Tamura, Masaru; Aoki, Aya; Fujii, Tomoaki; Komiyama, Hiromitsu; Sagai, Tomoko; Shiroishi, Toshihiko . E-mail: tshirois@lab.nig.ac.jp

    2007-08-10

    Recombination-induced mutation 3 (Rim3) is a spontaneous mouse mutation that exhibits dominant phenotype of hyperkeratosis and hair loss. Fine linkage analysis of Rim3 and sequencing revealed a novel single point mutation, G1124A leading to Ala348Thr, in Gsdma3 in chromosome 11. Transgenesis with BAC DNA harboring the Rim3-type Gsdma3 recaptured the Rim3 phenotype, providing direct evidence that Gsdma3 is the causative gene of Rim3. We examined the spatial expression of Gsdma3 and characterized the Rim3 phenotype in detail. Gsdma3 is expressed in differentiated epidermal cells in the skin, but not in the proliferating epidermal cells. Histological analysis of Rim3 mutant showed hyperplasia of the epidermal cells in the upper hair follicles and abnormal anagen phase at the first hair cycle. Furthermore, immunohistochemical analysis revealed hyperproliferation and misdifferentiation of the upper follicular epidermis in Rim3 mutant. These results suggest that Gsdma3 is involved in the proliferation and differentiation of epidermal stem cells.

  18. Mutations in Durum Wheat SBEII Genes affect Grain Yield Components, Quality, and Fermentation Responses in Rats.

    PubMed

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Hamilton, M Kristina; Rust, Bret; Raybould, Helen E; Newman, John W; Martin, Roy; Dubcovsky, Jorge

    2015-01-01

    Increased amylose in wheat (Triticum ssp.) starch is associated with increased resistant starch, a fermentable dietary fiber. Fermentation of resistant starch in the large intestine produces short-chain fatty acids that are associated with human health benefits. Since wheat foods are an important component of the human diet, increases in amylose and resistant starch in wheat grains have the potential to deliver health benefits to a large number of people. In three replicated field trials we found that mutations in starch branching enzyme II genes (SBEIIa and SBEIIb) in both A and B genomes (SBEIIa/b-AB) of durum wheat [T. turgidum L. subsp. durum (Desf.) Husn.] resulted in large increases of amylose and resistant starch content. The presence of these four mutations was also associated with an average 5% reduction in kernel weight (P = 0.0007) and 15% reduction in grain yield (P = 0.06) compared to the wild type. Complete milling and pasta quality analysis showed that the mutant lines have an acceptable quality with positive effects on pasta firmness and negative effects on semolina extraction and pasta color. Positive fermentation responses were detected in rats (Rattus spp.) fed with diets incorporating mutant wheat flour. This study quantifies benefits and limitations associated with the deployment of the SBEIIa/b-AB mutations in durum wheat and provides the information required to develop realistic strategies to deploy durum wheat varieties with increased levels of amylose and resistant starch.

  19. Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

    PubMed

    Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

    2014-01-01

    Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

  20. Mutations in Durum Wheat SBEII Genes affect Grain Yield Components, Quality, and Fermentation Responses in Rats

    PubMed Central

    Hazard, Brittany; Zhang, Xiaoqin; Naemeh, Mahmoudreza; Hamilton, M. Kristina; Rust, Bret; Raybould, Helen E.; Newman, John W.; Martin, Roy; Dubcovsky, Jorge

    2016-01-01

    Increased amylose in wheat (Triticum ssp.) starch is associated with increased resistant starch, a fermentable dietary fiber. Fermentation of resistant starch in the large intestine produces short-chain fatty acids that are associated with human health benefits. Since wheat foods are an important component of the human diet, increases in amylose and resistant starch in wheat grains have the potential to deliver health benefits to a large number of people. In three replicated field trials we found that mutations in starch branching enzyme II genes (SBEIIa and SBEIIb) in both A and B genomes (SBEIIa/b-AB) of durum wheat [T. turgidum L. subsp. durum (Desf.) Husn.] resulted in large increases of amylose and resistant starch content. The presence of these four mutations was also associated with an average 5% reduction in kernel weight (P = 0.0007) and 15% reduction in grain yield (P = 0.06) compared to the wild type. Complete milling and pasta quality analysis showed that the mutant lines have an acceptable quality with positive effects on pasta firmness and negative effects on semolina extraction and pasta color. Positive fermentation responses were detected in rats (Rattus spp.) fed with diets incorporating mutant wheat flour. This study quantifies benefits and limitations associated with the deployment of the SBEIIa/b-AB mutations in durum wheat and provides the information required to develop realistic strategies to deploy durum wheat varieties with increased levels of amylose and resistant starch. PMID:27134286

  1. Mutations in two regions upstream of the A gamma globin gene canonical promoter affect gene expression.

    PubMed Central

    Lloyd, J A; Lee, R F; Lingrel, J B

    1989-01-01

    Two regions upstream of the human fetal (A gamma) globin gene, which interact with protein factors from K562 and HeLa nuclear extracts, have functional significance in gene expression. One binding site (site I) is at a position -290 to -267 bp upstream of the transcription initiation site, the other (site II) is at -182 to -168 bp. Site II includes the octamer sequence (ATGCAAAT) found in an immunoglobulin enhancer and the histone H2b gene promoter. A point mutation (T----C) at -175, within the octamer sequence, is characteristic of a naturally occurring HPFH (hereditary persistence of fetal hemoglobin), and decreases factor binding to an oligonucleotide containing the octamer motif. Expression assays using a A gamma globin promoter-CAT (chloramphenicol acetyl transferase) fusion gene show that the point mutation at -175 increases expression in erythroid, but not non-erythroid cells when compared to a wild-type construct. This correlates with the actual effect of the HPFH mutation in humans. This higher expression may result from a mechanism more complex than reduced binding of a negative regulator. A site I clustered-base substitution gives gamma-CAT activity well below wild-type, suggesting that this factor is a positive regulator. Images PMID:2472607

  2. Colon cancer metastasis in mouse liver is not affected by hypercoagulability due to Factor V Leiden mutation

    PubMed Central

    Klerk, CPW; Smorenburg, SM; Spek, CA; Van Noorden, CJF

    2007-01-01

    Abstract Clinical trials have shown life-prolonging effects of antithrombotics in cancer patients, but the molecular mechanisms remain unknown due to the multitude of their effects. We investigated in a mouse model whether one of the targets of antithrombotic therapy, fibrin deposition, stimulates tumour development. Fibrin may provide either protection of cancer cells in the circulation against mechanical stress and the immune system, or form a matrix for tumours and/or angiogenesis in tumours to develop. Mice homozygous for Factor V Leiden (FVL), a mutation in one of the coagulation factors that facilitates fibrin formation, were used to investigate whether hypercoagulability affects tumour development in an experimental metastasis model. Liver metastases of colon cancer were induced in mice with the FVL mutation and wild-type littermates. At day 21, number and size of tumours at the liver surface, fibrin/fibrinogen distribution, vessel density and the presence of newly formed vessels in tumours were analysed. Number and size of tumours did not differ between mice with and without the FVL mutation. Fibrin/fibrinogen was found in the cytoplasm of hepatocytes and cancer cells, in blood vessels in liver and tumour tissue and diffusely distributed outside vessels in tumours, indicating leaky vessels. Vessel density and angiogenesis varied widely between tumours, but a pre-dominance for vessel-rich or vessel-poor tumours or vessel formation could not be found in either genotype. In conclusion, the FVL mutation has no effect on the development of secondary tumours of colon cancer in livers of mice. Fibrin deposition and thus inhibition of fibrin formation by anticoagulants do not seem to affect tumour development in this model. PMID:17635646

  3. Transient congenital hypothyroidism caused by compound heterozygous mutations affecting the NADPH-oxidase domain of DUOX2.

    PubMed

    Yoshizawa-Ogasawara, Atsuko; Abe, Kiyomi; Ogikubo, Sayaka; Narumi, Satoshi; Hasegawa, Tomonobu; Satoh, Mari

    2016-03-01

    Here, we describe three cases of loss-of-function mutations in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase (NOX) domain of dual oxidase 2 (DUOX2) occurring along with concurrent missense mutations in thyroid peroxidase (TPO), leading to transient congenital hypothyroidism (CH). Three Japanese boys with nonconsanguineous parents were diagnosed with CH during their neonatal screenings. All patients presented with moderate-to-severe neonatal hypothyroidism and were diagnosed with transient CH after re-evaluation of thyroid function. Two siblings were compound heterozygous for p.[R1110Q]+[Y1180X] in DUOX2; one of them was also heterozygous for p.[R361L] in TPO. The third patient was compound heterozygous for p.[L1160del]+[R1334W] in DUOX2 and heterozygous for p.[P883S] in TPO. This is the first report of a de novo L1160del mutation affecting the DUOX2 gene and of the novel mutations Y1180X in DUOX2 and R361L in TPO. R1110Q and L1160del were found to reduce H2O2 production (5%-9%, p<0.01), while Y1180X, which introduces a premature stop codon, did not confer detectable H2O2 production (-0.7%±0.6%, p<0.01). Moreover, R1334W, a missense mutation possibly affecting electron transfer, led to reduced H2O2 production (24%±0.9%, p<0.01) in vitro, and R1110Q and R1334W resulted in reduced protein expression. Y1180X was detected in a 120 kDa truncated form, whereas L1160del expression was maintained. Further, R361L, a novel missense mutation in TPO, caused partial reduction in peroxidase activity (20.6%±0.8%, p=0.01), whereas P883S, a missense variant, increased it (133.7%±2.8%, p=0.02). The protein expression levels in the case of R361L and P883S were maintained. In conclusion, we provide clinical and in vitro demonstrations of different functional defects and phenotypic heterogeneity in the same thyroid hormonogenesis pathway.

  4. Ocean acidification alters fish-jellyfish symbiosis.

    PubMed

    Nagelkerken, Ivan; Pitt, Kylie A; Rutte, Melchior D; Geertsma, Robbert C

    2016-06-29

    Symbiotic relationships are common in nature, and are important for individual fitness and sustaining species populations. Global change is rapidly altering environmental conditions, but, with the exception of coral-microalgae interactions, we know little of how this will affect symbiotic relationships. We here test how the effects of ocean acidification, from rising anthropogenic CO2 emissions, may alter symbiotic interactions between juvenile fish and their jellyfish hosts. Fishes treated with elevated seawater CO2 concentrations, as forecast for the end of the century on a business-as-usual greenhouse gas emission scenario, were negatively affected in their behaviour. The total time that fish (yellowtail scad) spent close to their jellyfish host in a choice arena where they could see and smell their host was approximately three times shorter under future compared with ambient CO2 conditions. Likewise, the mean number of attempts to associate with jellyfish was almost three times lower in CO2-treated compared with control fish, while only 63% (high CO2) versus 86% (control) of all individuals tested initiated an association at all. By contrast, none of three fish species tested were attracted solely to jellyfish olfactory cues under present-day CO2 conditions, suggesting that the altered fish-jellyfish association is not driven by negative effects of ocean acidification on olfaction. Because shelter is not widely available in the open water column and larvae of many (and often commercially important) pelagic species associate with jellyfish for protection against predators, modification of the fish-jellyfish symbiosis might lead to higher mortality and alter species population dynamics, and potentially have flow-on effects for their fisheries.

  5. Effects of nano-ZnO on the agronomically relevant Rhizobium-legume symbiosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The impact of nano-ZnO (nZnO) on Rhizobium-legume symbiosis was studied with garden pea and its compatible bacterial partner Rhizobium leguminosarum bv. viciae 3841. Exposure of peas to nZnO had no impact on germination, but significantly affected root length. Chronic exposure of plant to nZnO impac...

  6. Effects of nano-TiO2 on the agronomically-relevant Rhizobium-legume symbiosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The impact of nano-TiO2 on Rhizobium-legume symbiosis was studied using garden peas and the compatible bacterial partner Rhizobium leguminosarum bv. viciae 3841. Exposure to nano-TiO2 did not affect the germination of peas grown aseptically, nor did it impact the gross root structure. However, nano-...

  7. A novel COL11A1 mutation affecting splicing in a patient with Stickler syndrome

    PubMed Central

    Kohmoto, Tomohiro; Naruto, Takuya; Kobayashi, Haruka; Watanabe, Miki; Okamoto, Nana; Masuda, Kiyoshi; Imoto, Issei; Okamoto, Nobuhiko

    2015-01-01

    Stickler syndrome is a clinically and genetically heterogeneous collagenopathy characterized by ocular, auditory, skeletal and orofacial abnormalities, commonly occurring as an autosomal dominant trait. We conducted target resequencing to analyze candidate genes associated with known clinical phenotypes from a 4-year-old girl with Stickler syndrome. We detected a novel heterozygous intronic mutation (NM_001854.3:c.3168+5G>A) in COL11A1 that may impair splicing, which was suggested by in silico prediction and a minigene assay. PMID:27081549

  8. A novel COL11A1 mutation affecting splicing in a patient with Stickler syndrome.

    PubMed

    Kohmoto, Tomohiro; Naruto, Takuya; Kobayashi, Haruka; Watanabe, Miki; Okamoto, Nana; Masuda, Kiyoshi; Imoto, Issei; Okamoto, Nobuhiko

    2015-01-01

    Stickler syndrome is a clinically and genetically heterogeneous collagenopathy characterized by ocular, auditory, skeletal and orofacial abnormalities, commonly occurring as an autosomal dominant trait. We conducted target resequencing to analyze candidate genes associated with known clinical phenotypes from a 4-year-old girl with Stickler syndrome. We detected a novel heterozygous intronic mutation (NM_001854.3:c.3168+5G>A) in COL11A1 that may impair splicing, which was suggested by in silico prediction and a minigene assay.

  9. Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

    SciTech Connect

    Argaw, Takele; Wilson, Carolyn A.

    2015-01-15

    Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.

  10. Skeletal muscle sodium channel is affected by an epileptogenic beta1 subunit mutation.

    PubMed

    Moran, O; Conti, F

    2001-03-23

    The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology.

  11. pigk Mutation underlies macho behavior and affects Rohon-Beard cell excitability

    PubMed Central

    Carmean, V.; Yonkers, M. A.; Tellez, M. B.; Willer, J. R.; Willer, G. B.; Gregg, R. G.; Geisler, R.; Neuhauss, S. C.

    2015-01-01

    The study of touch-evoked behavior allows investigation of both the cells and circuits that generate a response to tactile stimulation. We investigate a touch-insensitive zebrafish mutant, macho (maco), previously shown to have reduced sodium current amplitude and lack of action potential firing in sensory neurons. In the genomes of mutant but not wild-type embryos, we identify a mutation in the pigk gene. The encoded protein, PigK, functions in attachment of glycophosphatidylinositol anchors to precursor proteins. In wild-type embryos, pigk mRNA is present at times when mutant embryos display behavioral phenotypes. Consistent with the predicted loss of function induced by the mutation, knock-down of PigK phenocopies maco touch insensitivity and leads to reduced sodium current (INa) amplitudes in sensory neurons. We further test whether the genetic defect in pigk underlies the maco phenotype by overexpressing wild-type pigk in mutant embryos. We find that ubiquitous expression of wild-type pigk rescues the touch response in maco mutants. In addition, for maco mutants, expression of wild-type pigk restricted to sensory neurons rescues sodium current amplitudes and action potential firing in sensory neurons. However, expression of wild-type pigk limited to sensory cells of mutant embryos does not allow rescue of the behavioral touch response. Our results demonstrate an essential role for pigk in generation of the touch response beyond that required for maintenance of proper INa density and action potential firing in sensory neurons. PMID:26133798

  12. pigk Mutation underlies macho behavior and affects Rohon-Beard cell excitability.

    PubMed

    Carmean, V; Yonkers, M A; Tellez, M B; Willer, J R; Willer, G B; Gregg, R G; Geisler, R; Neuhauss, S C; Ribera, A B

    2015-08-01

    The study of touch-evoked behavior allows investigation of both the cells and circuits that generate a response to tactile stimulation. We investigate a touch-insensitive zebrafish mutant, macho (maco), previously shown to have reduced sodium current amplitude and lack of action potential firing in sensory neurons. In the genomes of mutant but not wild-type embryos, we identify a mutation in the pigk gene. The encoded protein, PigK, functions in attachment of glycophosphatidylinositol anchors to precursor proteins. In wild-type embryos, pigk mRNA is present at times when mutant embryos display behavioral phenotypes. Consistent with the predicted loss of function induced by the mutation, knock-down of PigK phenocopies maco touch insensitivity and leads to reduced sodium current (INa) amplitudes in sensory neurons. We further test whether the genetic defect in pigk underlies the maco phenotype by overexpressing wild-type pigk in mutant embryos. We find that ubiquitous expression of wild-type pigk rescues the touch response in maco mutants. In addition, for maco mutants, expression of wild-type pigk restricted to sensory neurons rescues sodium current amplitudes and action potential firing in sensory neurons. However, expression of wild-type pigk limited to sensory cells of mutant embryos does not allow rescue of the behavioral touch response. Our results demonstrate an essential role for pigk in generation of the touch response beyond that required for maintenance of proper INa density and action potential firing in sensory neurons.

  13. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  14. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

    PubMed Central

    Garbers, C; DeLong, A; Deruére, J; Bernasconi, P; Söll, D

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis. Images PMID:8641277

  15. Novel Familial Dilated Cardiomyopathy Mutation in MYL2 Affects the Structure and Function of Myosin Regulatory Light Chain

    PubMed Central

    Huang, Wenrui; Liang, Jingsheng; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Zhou, Zhiqun; Morales, Ana; McBride, Kim L.; Fitzgerald-Butt, Sara M.; Hershberger, Ray E.; Szczesna-Cordary, Danuta

    2015-01-01

    Dilated Cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. In this report we describe a novel DCM mutation identified for the first time in the myosin regulatory light chain (RLC), replacing Aspartic Acid at position 94 with Alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To gain insight into the functional significance of this pathogenic MYL2 variant, we have cloned and purified the human ventricular RLC wild-type (WT) and D94A-mutant proteins and performed in vitro experiments using RLC-exchanged porcine cardiac preparations. The mutation was observed to induce a reduction in the α-helical content of the RLC and imposed intra-molecular rearrangements. The Ca2+-calmodulin-activated myosin light chain kinase phosphorylation of RLC was not affected by D94A. The mutation was seen to impair the binding of RLC to the MHC (myosin heavy chain), and its incorporation into the RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared to ATPase of WT. No changes in myofibrillar ATPase-pCa relationship were observed in WT- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle with no change in calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with WT. Our data indicate that subtle structural rearrangements in the RLC molecule followed by its impaired interaction with the MHC may trigger functional abnormalities contributing to the DCM phenotype. PMID:25825243

  16. Two novel pathogenic mitochondrial DNA mutations affecting organelle number and protein synthesis. Is the tRNA(Leu(UUR)) gene an etiologic hot spot?

    PubMed Central

    Moraes, C T; Ciacci, F; Bonilla, E; Jansen, C; Hirano, M; Rao, N; Lovelace, R E; Rowland, L P; Schon, E A; DiMauro, S

    1993-01-01

    We identified two patients with pathogenic single nucleotide changes in two different mitochondrial tRNA genes: the first mutation in the tRNA(Asn) gene, and the ninth known mutation in the tRNA(Leu(UUR)) gene. The mutation in tRNA(Asn) was associated with isolated ophthalmoplegia, whereas the mutation in tRNA(Leu(UUR)) caused a neurological syndrome resembling MERRF (myoclonus epilepsy and ragged-red fibers) plus optic neuropathy, retinopathy, and diabetes. Both mutations were heteroplasmic, with higher percentages of mutant mtDNA in affected tissues, and undetectable levels in maternal relatives. Analysis of single muscle fibers indicated that morphological and biochemical alterations appeared only when the proportions of mutant mtDNA exceeded 90% of the total cellular mtDNA pool. The high incidence of mutations in the tRNA(Leu(UUR)) gene suggests that this region is an "etiologic hot spot" in mitochondrial disease. Images PMID:8254046

  17. Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

    PubMed Central

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng-Hua

    2016-01-01

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls. PMID:26745802

  18. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    DOE PAGES

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; ...

    2016-01-08

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reductionmore » in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.« less

  19. Experimental and molecular dynamics studies showed that CBP KIX mutation affects the stability of CBP:c-Myb complex.

    PubMed

    Odoux, Anne; Jindal, Darren; Tamas, Tamara C; Lim, Benjamin W H; Pollard, Drake; Xu, Wu

    2016-06-01

    The coactivators CBP (CREBBP) and its paralog p300 (EP300), two conserved multi-domain proteins in eukaryotic organisms, regulate gene expression in part by binding DNA-binding transcription factors. It was previously reported that the CBP/p300 KIX domain mutant (Y650A, A654Q, and Y658A) altered both c-Myb-dependent gene activation and repression, and that mice with these three point mutations had reduced numbers of platelets, B cells, T cells, and red blood cells. Here, our transient transfection assays demonstrated that mouse embryonic fibroblast cells containing the same mutations in the KIX domain and without a wild-type allele of either CBP or p300, showed decreased c-Myb-mediated transcription. Dr. Wright's group solved a 3-D structure of the mouse CBP:c-Myb complex using NMR. To take advantage of the experimental structure and function data and improved theoretical calculation methods, we performed MD simulations of CBP KIX, CBP KIX with the mutations, and c-Myb, as well as binding energy analysis for both the wild-type and mutant complexes. The binding between CBP and c-Myb is mainly mediated by a shallow hydrophobic groove in the center where the side-chain of Leu302 of c-Myb plays an essential role and two salt bridges at the two ends. We found that the KIX mutations slightly decreased stability of the CBP:c-Myb complex as demonstrated by higher binding energy calculated using either MM/PBSA or MM/GBSA methods. More specifically, the KIX mutations affected the two salt bridges between CBP and c-Myb (CBP-R646 and c-Myb-E306; CBP-E665 and c-Myb-R294). Our studies also revealed differing dynamics of the hydrogen bonds between CBP-R646 and c-Myb-E306 and between CBP-E665 and c-Myb-R294 caused by the CBP KIX mutations. In the wild-type CBP:c-Myb complex, both of the hydrogen bonds stayed relatively stable. In contrast, in the mutant CBP:c-Myb complex, hydrogen bonds between R646 and E306 showed an increasing trend followed by a decreasing trend, and hydrogen

  20. Unethical and Deadly Symbiosis in Higher Education

    ERIC Educational Resources Information Center

    Crumbley, D. Larry; Flinn, Ronald; Reichelt, Kenneth J.

    2012-01-01

    As administrators are pressured to increase retention rates in accounting departments, and higher education in general, a deadly symbiosis is occurring. Most students and parents only wish for high grades, so year after year many educators engage in unethical grade inflation and course work deflation. Since administrators use the students to audit…

  1. Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore.

    PubMed

    Zhang, Zhifen; Park, Minyoung; Tam, John; Auger, Anick; Beilhartz, Greg L; Lacy, D Borden; Melnyk, Roman A

    2014-03-11

    Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues--clustered between amino acids 1,035 and 1,107--that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming "hotspots" of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.

  2. Mutations Affecting Donor Preference during Mating Type Interconversion in Saccharomyces Cerevisiae

    PubMed Central

    Weiler, K. S.; Szeto, L.; Broach, J. R.

    1995-01-01

    Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to α or α to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MATα cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MATα background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference. PMID:7789755

  3. Characterization and genetic mapping of a mutation affecting apurinic endonuclease activity in Staphylococcus aureus.

    PubMed Central

    Tam, J E; Pattee, P A

    1986-01-01

    Protoplast fusion between the Rec- mutant RN981 (L. Wyman, R. V. Goering, and R. P. Novick, Genetics 76:681-702, 1974) of Staphylococcus aureus NCTC 8325 and a Rec+ NCTC 8325 derivative yielded Rec+ recombinants that exhibited the increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine characteristic of RN981. Transformation analyses identified a specific mutation, designated ngr-374, that was responsible not only for N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, but also sensitivity to methyl methanesulfonate, ethyl methanesulfonate, nitrous acid, and UV irradiation. However, ngr-374-carrying recombinants showed no significant increase in their sensitivity to mitomycin C or 4-nitroquinoline 1-oxide and were unaffected in recombination proficiency. In vitro assays showed that ngr-374-carrying strains had lower apurinic/apyrimidinic endonuclease activities than the wild type. The chromosomal locus occupied by ngr-374 was shown to exist in the gene order omega(Chr::Tn551)40-ngr-374-thrB106. PMID:2430940

  4. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  5. Point mutation of H3/H4 histones affects acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Liu, Xiangyong; Zhang, Xiaohua; Zhang, Zhaojie

    2014-10-10

    The molecular mechanism of acetic acid tolerance in yeast remains unclear despite of its importance for efficient cellulosic ethanol production. In this study, we examined the effects of histone H3/H4 point mutations on yeast acetic acid tolerance by comprehensively screening a histone H3/H4 mutant library. A total of 24 histone H3/H4 mutants (six acetic acid resistant and 18 sensitive) were identified. Compared to the wild-type strain, the histone acetic acid-resistant mutants exhibited improved ethanol fermentation performance under acetic acid stress. Genome-wide transcriptome analysis revealed that changes in the gene expression in the acetic acid-resistant mutants H3 K37A and H4 K16Q were mainly related to energy production, antioxidative stress. Our results provide novel insights into yeast acetic acid tolerance on the basis of histone, and suggest a novel approach to improve ethanol production by altering the histone H3/H4 sequences.

  6. Arbuscular mycorrhizal symbiosis induces strigolactone biosynthesis under drought and improves drought tolerance in lettuce and tomato.

    PubMed

    Ruiz-Lozano, Juan Manuel; Aroca, Ricardo; Zamarreño, Ángel María; Molina, Sonia; Andreo-Jiménez, Beatriz; Porcel, Rosa; García-Mina, José María; Ruyter-Spira, Carolien; López-Ráez, Juan Antonio

    2016-02-01

    Arbuscular mycorrhizal (AM) symbiosis alleviates drought stress in plants. However, the intimate mechanisms involved, as well as its effect on the production of signalling molecules associated with the host plant-AM fungus interaction remains largely unknown. In the present work, the effects of drought on lettuce and tomato plant performance and hormone levels were investigated in non-AM and AM plants. Three different water regimes were applied, and their effects were analysed over time. AM plants showed an improved growth rate and efficiency of photosystem II than non-AM plants under drought from very early stages of plant colonization. The levels of the phytohormone abscisic acid, as well as the expression of the corresponding marker genes, were influenced by drought stress in non-AM and AM plants. The levels of strigolactones and the expression of corresponding marker genes were affected by both AM symbiosis and drought. The results suggest that AM symbiosis alleviates drought stress by altering the hormonal profiles and affecting plant physiology in the host plant. In addition, a correlation between AM root colonization, strigolactone levels and drought severity is shown, suggesting that under these unfavourable conditions, plants might increase strigolactone production in order to promote symbiosis establishment to cope with the stress.

  7. Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

    PubMed Central

    Morin, N; Delsert, C; Klessig, D F

    1989-01-01

    The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed. Images PMID:2585602

  8. Mutation increasing β-carotene concentrations does not adversely affect concentrations of essential mineral elements in pepper fruit

    PubMed Central

    Thompson, Jacqueline A.; Penchev, Emil A.; Nielen, Stephan

    2017-01-01

    Vitamin and mineral deficiencies are prevalent in human populations throughout the world. Vitamin A deficiency affects hundreds of millions of pre-school age children in low income countries. Fruits of pepper (Capsicum annuum L.) can be a major dietary source of precursors to Vitamin A biosynthesis, such as β-carotene. Recently, pepper breeding programs have introduced the orange-fruited (of) trait of the mutant variety Oranzheva kapiya, which is associated with high fruit β-carotene concentrations, to the mutant variety Albena. In this manuscript, concentrations of β-carotene and mineral elements (magnesium, phosphorus, sulphur, potassium, zinc, calcium, manganese, iron and copper) were compared in fruit from P31, a red-fruited genotype derived from the variety Albena, and M38, a genotype developed by transferring the orange-fruited mutation (of) into Albena. It was observed that fruit from M38 plants had greater β-carotene concentration at both commercial and botanical maturity (4.9 and 52.7 mg / kg fresh weight, respectively) than fruit from P31 plants (2.3 and 30.1 mg / kg fresh weight, respectively). The mutation producing high β-carotene concentrations in pepper fruits had no detrimental effect on the concentrations of mineral elements required for human nutrition. PMID:28207797

  9. Mutation increasing β-carotene concentrations does not adversely affect concentrations of essential mineral elements in pepper fruit.

    PubMed

    Tomlekova, Nasya B; White, Philip J; Thompson, Jacqueline A; Penchev, Emil A; Nielen, Stephan

    2017-01-01

    Vitamin and mineral deficiencies are prevalent in human populations throughout the world. Vitamin A deficiency affects hundreds of millions of pre-school age children in low income countries. Fruits of pepper (Capsicum annuum L.) can be a major dietary source of precursors to Vitamin A biosynthesis, such as β-carotene. Recently, pepper breeding programs have introduced the orange-fruited (of) trait of the mutant variety Oranzheva kapiya, which is associated with high fruit β-carotene concentrations, to the mutant variety Albena. In this manuscript, concentrations of β-carotene and mineral elements (magnesium, phosphorus, sulphur, potassium, zinc, calcium, manganese, iron and copper) were compared in fruit from P31, a red-fruited genotype derived from the variety Albena, and M38, a genotype developed by transferring the orange-fruited mutation (of) into Albena. It was observed that fruit from M38 plants had greater β-carotene concentration at both commercial and botanical maturity (4.9 and 52.7 mg / kg fresh weight, respectively) than fruit from P31 plants (2.3 and 30.1 mg / kg fresh weight, respectively). The mutation producing high β-carotene concentrations in pepper fruits had no detrimental effect on the concentrations of mineral elements required for human nutrition.

  10. Interleukin-6 Deficiency Does Not Affect Motor Neuron Disease Caused by Superoxide Dismutase 1 Mutation

    PubMed Central

    Han, Yongmei; Ripley, Barry; Serada, Satoshi; Naka, Tetsuji; Fujimoto, Minoru

    2016-01-01

    Background & Aim Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive, motor neuron degenerative disease. Recent evidence indicates that inflammation is associated with many neurodegenerative diseases including ALS. Previously, abnormal levels of inflammatory cytokines including IL-1β, IL-6 and TNF-α were described in ALS patients and/or in mouse ALS models. In addition, one study showed that blocking IL-1β could slow down progression of ALS-like symptoms in mice. In this study, we examined a role for IL-6 in ALS, using an animal model for familial ALS. Methods Mice with mutant SOD1 (G93A) transgene, a model for familial ALS, were used in this study. The expression of the major inflammatory cytokines, IL-6, IL-1β and TNF-α, in spinal cords of these SOD1 transgenic (TG) mice were assessed by real time PCR. Mice were then crossed with IL-6(-/-) mice to generate SOD1TG/IL-6(-/-) mice. SOD1 TG/IL-6(-/-) mice (n = 17) were compared with SOD1 TG/IL-6(+/-) mice (n = 18), SOD1 TG/IL-6(+/+) mice (n = 11), WT mice (n = 15), IL-6(+/-) mice (n = 5) and IL-6(-/-) mice (n = 8), with respect to neurological disease severity score, body weight and the survival. We also histologically compared the motor neuron loss in lumber spinal cords and the atrophy of hamstring muscles between these mouse groups. Results Levels of IL-6, IL-1β and TNF-α in spinal cords of SOD1 TG mice was increased compared to WT mice. However, SOD1 TG/IL-6(-/-) mice exhibited weight loss, deterioration in motor function and shortened lifespan (167.55 ± 11.52 days), similarly to SOD1 TG /IL-6(+/+) mice (164.31±12.16 days). Motor neuron numbers and IL-1β and TNF-α levels in spinal cords were not significantly different in SOD1 TG /IL-6(-/-) mice and SOD1 TG /IL-6 (+/+) mice. Conclusion These results provide compelling preclinical evidence indicating that IL-6 does not directly contribute to motor neuron disease caused by SOD1 mutations. PMID:27070121

  11. Mutations that affect structure and assembly of light-harvesting proteins in the cyanobacterium Synechocystis sp. strain 6701

    SciTech Connect

    Anderson, L.K.; Rayner, M.C.; Eiserling, F.A.

    1987-01-01

    The unicellular cyanobacterium Synechocystis sp. strain 6701 was mutagenized with UV irradiation and screened for pigment changes that indicated genetic lesions involving the light-harvesting proteins of the phycobilisome. A previous examination of the pigment mutant UV16 showed an assembly defect in the phycocyanin component of the phycobilisome. Mutagenesis of UV16 produced an additional double mutant, UV16-40, with decreased phycoerythrin content. Phycocyanin and phycoerythrin were isolated from UV16-40 and compared with normal biliproteins. The results suggested that the UV16 mutation affected the alpha subunit of phycocyanin, while the phycoerythrin beta subunit from UV16-40 had lost one of its three chromophores. Characterization of the unassembled phycobilisome components in these mutants suggests that these strains will be useful for probing in vivo the regulated expression and assembly of phycobilisomes.

  12. Systematic screening for mutations in the human serotonin 1F receptor gene in patients with bipolar affective disorder and schizophrenia

    SciTech Connect

    Shimron-Abarbanell, D.; Harms, H.; Erdmann, J.; Propping, P.; Noethen, M.M.

    1996-04-09

    Using single strand conformational analysis we screened the complete coding sequence of the serotonin 1F (5-HT{sub 1F}) receptor gene for the presence of DNA sequence variation in a sample of 137 unrelated individuals including 45 schizophrenic patients, 46 bipolar patients, as well as 46 healthy controls. We detected only three rare sequence variants which are characterized by single base pair substitutions, namely a silent T{r_arrow}A transversion in the third position of codon 261 (encoding isoleucine), a silent C{r_arrow}T transition in the third position of codon 176 (encoding histidine), and a C{r_arrow}T transition in position -78 upstream from the start codon. The lack of significant mutations in patients suffering from schizophrenia and bipolar affective disorder indicates that the 5-HT{sub 1F} receptor is not commonly involved in the etiology of these diseases. 12 refs., 1 fig., 2 tabs.

  13. BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age

    PubMed Central

    Bergthorsson, J; Ejlertsen, B; Olsen, J; Borg, A; Nielsen, K; Barkardottir, R; Klausen, S; Mouridsen, H; Winther, K; Fenger, K; Niebuhr, A; Harboe, T; Niebuhr, E

    2001-01-01

    INTRODUCTION—A small fraction of breast cancer is the result of germline mutations in the BRCA1 and BRCA2 cancer susceptibility genes. Mutation carriers frequently have a positive family history of breast and ovarian cancer, are often diagnosed at a young age, and may have a higher incidence of double or multiple primary breast tumours than breast cancer patients in general.
OBJECTIVES—To estimate the prevalence and spectrum of BRCA1 and BRCA2 mutations in young Danish patients affected with bilateral or multifocal breast cancer and to determine the relationship of mutation status to family history of cancer.
SUBJECTS—From the files of the Danish Breast Cancer Cooperative Group (DBCG), we selected 119 breast cancer patients diagnosed before the age of 46 years with either bilateral (n=59) or multifocal (n=61) disease.
METHODS—DNA from the subjects was screened for BRCA1 and BRCA2 mutations using single strand conformation analysis (SSCA) and the protein truncation test (PTT). Observed and expected cancer incidence in first degree relatives of the patients was estimated using data from the Danish Cancer Registry.
RESULTS—Twenty four mutation carriers were identified (20%), of whom 13 had a BRCA1 mutation and 11 carried a BRCA2 mutation. Two mutations in BRCA1 were found repeatedly in the material and accounted for seven of the 24 (29%) mutation carriers. The mutation frequency was about equal in patients with bilateral (22%) and multifocal breast cancer (18%). The incidence of breast and ovarian cancer was greatly increased in first degree relatives of BRCA1 and BRCA2 mutation carriers, but to a much lesser degree in relatives of non-carriers. An increased risk of cancer was also noted in brothers of non-carriers.
CONCLUSIONS—A relatively broad spectrum of germline mutations was observed in BRCA1 and BRCA2 and most of the mutations are present in other populations. Our results indicate that a diagnosis of bilateral and multifocal breast

  14. The Impact of Nitrogen Limitation and Mycorrhizal Symbiosis on Aspen Tree Growth and Development

    SciTech Connect

    Tran, Bich Thi Ngoc

    2014-08-18

    Nitrogen deficiency is the most common and widespread nutritional deficiency affecting plants worldwide. Ectromycorrhizal symbiosis involves the beneficial interaction of plants with soil fungi and plays a critical role in nutrient cycling, including the uptake of nitrogen from the environment. The main goal of this study is to understand how limiting nitrogen in the presence or absence of an ectomycorrhizal fungi, Laccaria bicolor, affects the health of aspen trees, Populus temuloides.

  15. An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

    PubMed

    Ziv, L; Muto, A; Schoonheim, P J; Meijsing, S H; Strasser, D; Ingraham, H A; Schaaf, M J M; Yamamoto, K R; Baier, H

    2013-06-01

    Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

  16. Interacting genes that affect microtubule function in Drosophila melanogaster: Two classes of mutation revert the failure to complement between hay sup nc2 and mutations in tubulin genes

    SciTech Connect

    Regan, C.L.; Fuller, M.T. )

    1990-05-01

    The recessive male sterile mutation hay{sup nc2} of Drosophila melanogaster fails to complement certain {beta}{sub 2}-tubulin and {alpha}-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by hay{sup nc2}, which may act as a structural poison. Based on this observation, the authors have isolated ten new mutations with EMS that revert the failure to complement between hay{sup nc2} and B2t{sup n}. The revertants tested behaved as intragenic mutations of hay in recombination tests, and feel into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than hay{sup nc2} in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the hay{sup nc2} allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywire{sup nc2} product to interact structurally with microtubules. Flies heterozygous for the original hay{sup nc2} allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle.

  17. Interacting Genes That Affect Microtubule Function in Drosophila Melanogaster: Two Classes of Mutation Revert the Failure to Complement between Hay(nc2) and Mutations in Tubulin Genes

    PubMed Central

    Regan, C. L.; Fuller, M. T.

    1990-01-01

    The recessive male sterile mutation hay(nc2) of Drosophila melanogaster fails to complement certain β(2)-tubulin and α-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by hay(nc2), which may act as a structural poison. Based on this observation, we have isolated ten new mutations that revert the failure to complement between hay(nc2) and B2t(n). The revertants tested behaved as intragenic mutations of hay in recombination tests, and fell into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than hay(nc2) in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the hay(nc2) allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywire(nc2) product to interact structurally with microtubules. Flies heterozygous for the original hay(nc2) allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle. PMID:2111265

  18. Molecular analysis of HEXA gene in Argentinean patients affected with Tay-Sachs disease: possible common origin of the prevalent c.459+5A>G mutation.

    PubMed

    Zampieri, Stefania; Montalvo, Annalisa; Blanco, Mariana; Zanin, Irene; Amartino, Hernan; Vlahovicek, Kristian; Szlago, Marina; Schenone, Andrea; Pittis, Gabriela; Bembi, Bruno; Dardis, Andrea

    2012-05-15

    Tay-Sachs disease (TSD) is a recessively inherited disorder caused by the deficient activity of hexosaminidase A due to mutations in the HEXA gene. Up to date there is no information regarding the molecular genetics of TSD in Argentinean patients. In the present study we have studied 17 Argentinean families affected by TSD, including 20 patients with the acute infantile form and 3 with the sub-acute form. Overall, we identified 14 different mutations accounting for 100% of the studied alleles. Eight mutations were novel: 5 were single base changes leading to drastic residue changes or truncated proteins, 2 were small deletions and one was an intronic mutation that may cause a splicing defect. Although the spectrum of mutations was highly heterogeneous, a high frequency of the c.459+5G>A mutation, previously described in different populations was found among the studied cohort. Haplotype analysis suggested that in these families the c.459+5G>A mutation might have arisen by a single mutational event.

  19. Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

    PubMed

    Faà, Valeria; Incani, Federica; Meloni, Alessandra; Corda, Denise; Masala, Maddalena; Baffico, A Maria; Seia, Manuela; Cao, Antonio; Rosatelli, M Cristina

    2009-10-30

    Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.

  20. NF1 mutation rather than individual genetic variability is the main determinant of the NF1-transcriptional profile of mutations affecting splicing.

    PubMed

    Pros, Eva; Larriba, Sara; López, Eva; Ravella, Anna; Gili, M Lluïsa; Kruyer, Helena; Valls, Joan; Serra, Eduard; Lázaro, Conxi

    2006-11-01

    A significant number of neurofibromatosis type 1 (NF1) mutations result in exon skipping. The majority of these mutations do not occur in the canonical splice sites and can produce different aberrant transcripts whose proportions have not been well studied. It has been hypothesized that differences in the mutation-determined NF1-transcriptional profile could partially explain disease variability among patients bearing the same NF1 splice defect. In order to gain insight into these aspects, we analyzed the proportion of the different transcripts generated by nine NF1-splicing mutations in 30 patients. We assessed the influence of the mutation in the NF1-related transcriptional profiles and investigated the existence of individual differences in a global manner. We analyzed potential differences in tissue-specific transcriptional profiles and evaluated the influence of sample processing and mRNA nonsense-mediated decay (NMD). Small transcriptional differences were found in neurofibromas and neurofibroma-derived Schwann cells (SC) compared to blood. We also detected a higher cell culture-dependent NMD. We observed that mutation per se explains 93.5% of the profile variability among mutations studied. However, despite the importance of mutation in determining the proportion of NF1 transcripts generated, we found certain variability among patients with the same mutation. From our results, it seems that genetic factors influencing RNA processing play a minor role in determining the NF1-transcriptional profile. Nevertheless neurofibromin studies would clarify whether these small differences translate into significant functional changes that could explain the great clinical expressivity observed in the disease or any of the disease-related traits.

  1. X chromosome exome sequencing reveals a novel ALG13 mutation in a nonsyndromic intellectual disability family with multiple affected male siblings.

    PubMed

    Bissar-Tadmouri, Nesrine; Donahue, Whithey L; Al-Gazali, Lihadh; Nelson, Stanley F; Bayrak-Toydemir, Pinar; Kantarci, Sibel

    2014-01-01

    X-linked intellectual disability (XLID) is a heterogeneous condition associated with mutations in >100 genes, accounting for over 10% of all cases of intellectual impairment. The majority of XLID cases show nonsyndromic forms (NSXLID), in which intellectual disability is the sole clinically consistent manifestation. Here we performed X chromosome exome (X-exome) sequencing to identify the causative mutation in an NSXLID family with four affected male siblings and five unaffected female siblings. The X-exome sequencing at 88× coverage in one affected male sibling revealed a novel missense mutation (p.Tyr1074Cys) in the asparagine-linked glycosylation 13 homolog (ALG13) gene. Segregation analysis by Sanger sequencing showed that the all affected siblings were hemizygous and the mother was heterozygous for the mutation. Recently, a de novo missense mutation in ALG13 has been reported in a patient with X-linked congenital disorders of glycosylation type I. Our study reports the first case of NSXLID caused by a mutation in ALG13 involved in protein N-glycosylation.

  2. Widespread fitness alignment in the legume-rhizobium symbiosis.

    PubMed

    Friesen, Maren L

    2012-06-01

    Although 'cheaters' potentially destabilize the legume-rhizobium mutualism, we lack a comprehensive review of host-symbiont fitness correlations. Studies measuring rhizobium relative or absolute fitness and host benefit are surveyed. Mutant studies are tallied for evidence of pleiotropy; studies of natural strains are analyzed with meta-analysis. Of 80 rhizobium mutations, 19 decrease both partners' fitness, four increase both, two increase host fitness but decrease symbiont fitness and none increase symbiont fitness at the host's expense. The pooled correlation between rhizobium nodulation competitiveness and plant aboveground biomass is 0.65 across five experiments that compete natural strains against a reference, whereas, across 14 experiments that compete rhizobia against soil populations or each other, the pooled correlation is 0.24. Pooled correlations between aboveground biomass and nodule number and nodule biomass are 0.76 and 0.83. Positive correlations between legume and rhizobium fitness imply that most ineffective rhizobia are 'defective' rather than 'defectors'; this extends to natural variants, with only one significant fitness conflict. Most studies involve non-coevolved associations, indicating that fitness alignment is the default state. Rhizobium mutations that increase both host and symbiont fitness suggest that some plants maladaptively restrict symbiosis with novel strains.

  3. [Effect of five fungicides on growth of Glycyrrhiza uralensis and efficiency of mycorrhizal symbiosis].

    PubMed

    Li, Peng-ying; Yang, Guang; Zhou, Xiu-teng; Zhou, Liane-yun; Shao, Ai-juan; Chen, Mei-lan

    2015-12-01

    In order to obtain the fungicides with minimal impact on efficiency of mycorrhizal symbiosis, the effect of five fungicides including polyoxins, jinggangmycins, thiophanate methylate, chlorothalonil and carbendazim on the growth of medicinal plant and efficiency of mycorrhizal symbiosis were studied. Pot cultured Glycyrrhiza uralensis was treated with different fungicides with the concentration that commonly used in the field. 60 d after treated with fungicides, infection rate, infection density, biomass indexes, photosyn- thetic index and the content of active component were measured. Experimental results showed that carbendazim had the strongest inhibition on mycorrhizal symbiosis effect. Carbendazim significantly inhibited the mycorrhizal infection rate, significantly suppressed the actual photosynthetic efficiency of G. uralensis and the most indicators of biomass. Polyoxins showed the lowest inhibiting affection. Polyoxins had no significant effect on mycorrhizal infection rate, the actual photosynthetic efficiency of G. uralensis and the most indicators of biomass. The other three fungicides also had an inhibitory effect on efficiency of mycorrhizal symbiosis, and the inhibition degrees were all between polyoxins's and carbendazim's. The author considered that fungicide's inhibition degree on mycorrhizal effect might be related with the species of fungicides, so the author suggested that the farmer should try to choose bio-fungicides like polyoxins.

  4. The effect of pseudo-microgravity on the symbiosis of plants and microorganisms

    NASA Astrophysics Data System (ADS)

    Tomita-Yokotani, Kaori; Maki, Asano; Aoki, Toshio; Tamura, Kenji; Wada, Hidenori; Hashimoto, Hirofumi; Yamashita, Masamichi

    The symbiosis of plants and microorganisms is important to conduct agriculture under space environment. However, we have less knowledge on whether this kind of symbiosis can be established under space condition. We examined the functional compounds responsible to symbiosis between rhizobiaum and Lotus japonicus as a model of symbiotic combination. The existence of the substances for their symbiosis, some flavonoids, have already been known from the study of gene expression, but the detail structures have not yet been elucidated. Pseudomicrogravity was generated by the 3D-clinorotation. Twenty flavonoids were found in the extracts of 16 days plants of Lotus japonicus grown under the normal gravity by HPLC. Content of two flavonoids among them was affected by the infection of Mesorhizobium loti to them. It has a possibility that the two flavonoids were key substances for their combination process. The productions of those flavonoids were confirmed also under the pseudo-microgravity. The amount of one flavonoid was increased by both infection of rhizobium and exposure to the normal and pseudo-micro gravity. Chemical species of these flavonoids were identified by LC- ESI/MS and spectroscopic analysis. To show the effects of pseudo-microgravity on the gene expression, enzymic activities related to the functional compounds are evaluated after the rhizobial infection.

  5. Insights on the Impact of Arbuscular Mycorrhizal Symbiosis on Tomato Tolerance to Water Stress1[OPEN

    PubMed Central

    Siciliano, Ilenia

    2016-01-01

    Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers, whose exploitation could represent a promising avenue for the development in the future of a more sustainable next-generation agriculture. The best understood function in symbiosis is an improvement in plant mineral nutrient acquisition, as exchange for carbon compounds derived from the photosynthetic process: this can enhance host growth and tolerance to environmental stresses, such as water stress (WS). However, physiological and molecular mechanisms occurring in arbuscular mycorrhiza-colonized plants and directly involved in the mitigation of WS effects need to be further investigated. The main goal of this work is to verify the potential impact of AM symbiosis on the plant response to WS. To this aim, the effect of two AM fungi (Funneliformis mosseae and Rhizophagus intraradices) on tomato (Solanum lycopersicum) under the WS condition was studied. A combined approach, involving ecophysiological, morphometric, biochemical, and molecular analyses, has been used to highlight the mechanisms involved in plant response to WS during AM symbiosis. Gene expression analyses focused on a set of target genes putatively involved in the plant response to drought, and in parallel, we considered the expression changes induced by the imposed stress on a group of fungal genes playing a key role in the water-transport process. Taken together, the results show that AM symbiosis positively affects the tolerance to WS in tomato, with a different plant response depending on the AM fungi species involved. PMID:27208301

  6. Mutation of AREA affects growth, sporulation, nitrogen regulation, and pathogenicity in Colletotrichum gloeosporioides.

    PubMed

    Bi, Fangcheng; Ment, Dana; Luria, Neta; Meng, Xiangchun; Prusky, Dov

    2017-02-01

    The GATA transcription factor AreA is a global nitrogen regulator that restricts the utilization of complex and poor nitrogen sources in the presence of good nitrogen sources in microorganisms. In this study, we report the biological function of an AreA homolog (the CgareA gene) in the fruit postharvest pathogen Colletotrichum gloeosporioides. Targeted gene deletion mutants of areA exhibited significant reductions in vegetative growth, increases in conidia production, and slight decreases in conidial germination rates. Quantitative RT-PCR (qRT-PCR) analysis revealed that the expression of AreA was highly induced under nitrogen-limiting conditions. Moreover, compared to wild-type and complemented strains, nitrogen metabolism-related genes were misregulated in ΔareA mutant strains. Pathogenicity assays indicated that the virulence of ΔareA mutant strains were affected by the nitrogen content, but not the carbon content, of fruit hosts. Taken together, our results indicate that CgareA plays a critical role in fungal development, conidia production, regulation of nitrogen metabolism and virulence in Colletotrichum gloeosporioides.

  7. High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype–Phenotype Correlation

    PubMed Central

    Rojnueangnit, Kitiwan; Xie, Jing; Gomes, Alicia; Sharp, Angela; Callens, Tom; Chen, Yunjia; Liu, Ying; Cochran, Meagan; Abbott, Mary‐Alice; Atkin, Joan; Babovic‐Vuksanovic, Dusica; Barnett, Christopher P.; Crenshaw, Melissa; Bartholomew, Dennis W.; Basel, Lina; Bellus, Gary; Ben‐Shachar, Shay; Bialer, Martin G.; Bick, David; Blumberg, Bruce; Cortes, Fanny; David, Karen L.; Destree, Anne; Duat‐Rodriguez, Anna; Earl, Dawn; Escobar, Luis; Eswara, Marthanda; Ezquieta, Begona; Frayling, Ian M.; Frydman, Moshe; Gardner, Kathy; Gripp, Karen W.; Hernández‐Chico, Concepcion; Heyrman, Kurt; Ibrahim, Jennifer; Janssens, Sandra; Keena, Beth A; Llano‐Rivas, Isabel; Leppig, Kathy; McDonald, Marie; Misra, Vinod K.; Mulbury, Jennifer; Narayanan, Vinodh; Orenstein, Naama; Galvin‐Parton, Patricia; Pedro, Helio; Pivnick, Eniko K.; Powell, Cynthia M.; Randolph, Linda; Raskin, Salmo; Rosell, Jordi; Rubin, Karol; Seashore, Margretta; Schaaf, Christian P.; Scheuerle, Angela; Schultz, Meredith; Schorry, Elizabeth; Schnur, Rhonda; Siqveland, Elizabeth; Tkachuk, Amanda; Tonsgard, James; Upadhyaya, Meena; Verma, Ishwar C.; Wallace, Stephanie; Williams, Charles; Zackai, Elaine; Zonana, Jonathan; Lazaro, Conxi; Claes, Kathleen; Korf, Bruce; Martin, Yolanda; Legius, Eric

    2015-01-01

    ABSTRACT Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype–phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café‐au‐lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan‐like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1‐patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi‐exon deletion, providing genetic evidence that p.Arg1809Cys is a loss‐of‐function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype–phenotype correlation will affect counseling and management of a significant number of patients. PMID:26178382

  8. High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

    PubMed

    Rojnueangnit, Kitiwan; Xie, Jing; Gomes, Alicia; Sharp, Angela; Callens, Tom; Chen, Yunjia; Liu, Ying; Cochran, Meagan; Abbott, Mary-Alice; Atkin, Joan; Babovic-Vuksanovic, Dusica; Barnett, Christopher P; Crenshaw, Melissa; Bartholomew, Dennis W; Basel, Lina; Bellus, Gary; Ben-Shachar, Shay; Bialer, Martin G; Bick, David; Blumberg, Bruce; Cortes, Fanny; David, Karen L; Destree, Anne; Duat-Rodriguez, Anna; Earl, Dawn; Escobar, Luis; Eswara, Marthanda; Ezquieta, Begona; Frayling, Ian M; Frydman, Moshe; Gardner, Kathy; Gripp, Karen W; Hernández-Chico, Concepcion; Heyrman, Kurt; Ibrahim, Jennifer; Janssens, Sandra; Keena, Beth A; Llano-Rivas, Isabel; Leppig, Kathy; McDonald, Marie; Misra, Vinod K; Mulbury, Jennifer; Narayanan, Vinodh; Orenstein, Naama; Galvin-Parton, Patricia; Pedro, Helio; Pivnick, Eniko K; Powell, Cynthia M; Randolph, Linda; Raskin, Salmo; Rosell, Jordi; Rubin, Karol; Seashore, Margretta; Schaaf, Christian P; Scheuerle, Angela; Schultz, Meredith; Schorry, Elizabeth; Schnur, Rhonda; Siqveland, Elizabeth; Tkachuk, Amanda; Tonsgard, James; Upadhyaya, Meena; Verma, Ishwar C; Wallace, Stephanie; Williams, Charles; Zackai, Elaine; Zonana, Jonathan; Lazaro, Conxi; Claes, Kathleen; Korf, Bruce; Martin, Yolanda; Legius, Eric; Messiaen, Ludwine

    2015-11-01

    Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.

  9. Speciation by Symbiosis: the Microbiome and Behavior

    PubMed Central

    Shropshire, J. Dylan

    2016-01-01

    ABSTRACT Species are fundamental units of comparison in biology. The newly discovered importance and ubiquity of host-associated microorganisms are now stimulating work on the roles that microbes can play in animal speciation. We previously synthesized the literature and advanced concepts of speciation by symbiosis with notable attention to hybrid sterility and lethality. Here, we review recent studies and relevant data on microbes as players in host behavior and behavioral isolation, emphasizing the patterns seen in these analyses and highlighting areas worthy of additional exploration. We conclude that the role of microbial symbionts in behavior and speciation is gaining exciting traction and that the holobiont and hologenome concepts afford an evolving intellectual framework to promote research and intellectual exchange between disciplines such as behavior, microbiology, genetics, symbiosis, and speciation. Given the increasing centrality of microbiology in macroscopic life, microbial symbiosis is arguably the most neglected aspect of animal and plant speciation, and studying it should yield a better understanding of the origin of species. PMID:27034284

  10. Speciation by Symbiosis: the Microbiome and Behavior.

    PubMed

    Shropshire, J Dylan; Bordenstein, Seth R

    2016-03-31

    Species are fundamental units of comparison in biology. The newly discovered importance and ubiquity of host-associated microorganisms are now stimulating work on the roles that microbes can play in animal speciation. We previously synthesized the literature and advanced concepts of speciation by symbiosis with notable attention to hybrid sterility and lethality. Here, we review recent studies and relevant data on microbes as players in host behavior and behavioral isolation, emphasizing the patterns seen in these analyses and highlighting areas worthy of additional exploration. We conclude that the role of microbial symbionts in behavior and speciation is gaining exciting traction and that the holobiont and hologenome concepts afford an evolving intellectual framework to promote research and intellectual exchange between disciplines such as behavior, microbiology, genetics, symbiosis, and speciation. Given the increasing centrality of microbiology in macroscopic life, microbial symbiosis is arguably the most neglected aspect of animal and plant speciation, and studying it should yield a better understanding of the origin of species.

  11. Culture Volume and Vessel Affect Long-Term Survival, Mutation Frequency, and Oxidative Stress of Escherichia coli

    PubMed Central

    Kram, Karin E.

    2014-01-01

    Bacteria such as Escherichia coli are frequently studied during exponential- and stationary-phase growth. However, many strains can survive in long-term stationary phase (LTSP), without the addition of nutrients, from days to several years. During LTSP, cells experience a variety of stressors, including reactive oxidative species, nutrient depletion, and metabolic toxin buildup, that lead to physiological responses and changes in genetic stability. In this study, we monitored survival during LTSP, as well as reporters of genetic and physiological change, to determine how the physical environment affects E. coli during long-term batch culture. We demonstrate differences in yield during LTSP in cells incubated in LB medium in test tubes versus Erlenmeyer flasks, as well as growth in different volumes of medium. We determined that these differences are only partially due to differences in oxygen levels by incubating the cells in different volumes of media under anaerobic conditions. Since we hypothesized that differences in long-term survival are the result of changes in physiological outputs during the late log and early stationary phases, we monitored alkalization, mutation frequency, oxidative stress response, and glycation. Although initial cell yields are essentially equivalent under each condition tested, physiological responses vary greatly in response to culture environment. Incubation in lower-volume cultures leads to higher oxyR expression but lower mutation frequency and glycation levels, whereas incubation in high-volume cultures has the opposite effect. We show here that even under commonly used experimental conditions that are frequently treated as equivalent, the stresses experienced by cells can differ greatly, suggesting that culture vessel and incubation conditions should be carefully considered in the planning or analysis of experiments. PMID:24375138

  12. DHPLC Screening of ATM Gene in Italian Patients Affected by Ataxia-Telangiectasia: Fourteen Novel ATM Mutations

    PubMed Central

    Magliozzi, Monia; Piane, Maria; Torrente, Isabella; Sinibaldi, Lorenzo; Rizzo, Giovanni; Savio, Camilla; Lulli, Patrizia; De Luca, Alessandro; Dallapiccola, Bruno; Chessa, Luciana

    2006-01-01

    The gene for ataxia-telangiectasia (A-T:MIM:#208900), ATM, spans about 150~kb of genomic DNA and is composed of 62 coding exons. ATM mutations are found along the entire coding sequence of the gene, without evidence of mutational hot spots. Using DNA as the starting material, we used denaturing high performance liquid chromatography (DHPLC) technique to search for ATM gene mutations. Initially, DHPLC was validated in a retrospective study of 16 positive control samples that included 19 known mutations; 100% of mutations were detected. Subsequently, DHPLC was used to screen for mutations a cohort of 22 patients with the classical form of A-T. A total of 27 different mutations were identified on 38 of the 44 alleles, corresponding to a 86% detection rate. Fourteen of the mutations were novel. In addition, 15 different variants and polymorphisms of unknown functional significance were found. The high incidence of new and individual A-T mutations in our cohort of patients demonstrates marked mutational heterogeneity of A-T in Italy and corroborate the efficiency of DHPLC as a method for the mutation screening of A-T patients. PMID:17124347

  13. Helical mutations in type I collagen that affect the processing of the amino-propeptide result in an Osteogenesis Imperfecta/Ehlers-Danlos Syndrome overlap syndrome

    PubMed Central

    2013-01-01

    Background Whereas mutations affecting the helical domain of type I procollagen classically cause Osteogenesis Imperfecta (OI), helical mutations near the amino (N)-proteinase cleavage site have been suggested to result in a mixed OI/Ehlers-Danlos syndrome (EDS)-phenotype. Methods We performed biochemical and molecular analysis of type I (pro-) collagen in a cohort of seven patients referred with a clinical diagnosis of EDS and showing only subtle signs of OI. Transmission electron microscopy of the dermis was available for one patient. Results All of these patients harboured a COL1A1 / COL1A2 mutation residing within the most N-terminal part of the type I collagen helix. These mutations affect the rate of type I collagen N-propeptide cleavage and disturb normal collagen fibrillogenesis. Importantly, patients with this type of mutation do not show a typical OI phenotype but mainly present as EDS patients displaying severe joint hyperlaxity, soft and hyperextensible skin, abnormal wound healing, easy bruising, and sometimes signs of arterial fragility. In addition, they show subtle signs of OI including blue sclerae, relatively short stature and osteopenia or fractures. Conclusion Recognition of this distinct phenotype is important for accurate genetic counselling, clinical management and surveillance, particularly in relation to the potential risk for vascular rupture associated with these mutations. Because these patients present clinical overlap with other EDS subtypes, biochemical collagen analysis is necessary to establish the correct diagnosis. PMID:23692737

  14. Whole-Genome Sequencing and iPLEX MassARRAY Genotyping Map an EMS-Induced Mutation Affecting Cell Competition in Drosophila melanogaster

    PubMed Central

    Lee, Chang-Hyun; Rimesso, Gerard; Reynolds, David M.; Cai, Jinlu; Baker, Nicholas E.

    2016-01-01

    Cell competition, the conditional loss of viable genotypes only when surrounded by other cells, is a phenomenon observed in certain genetic mosaic conditions. We conducted a chemical mutagenesis and screen to recover new mutations that affect cell competition between wild-type and RpS3 heterozygous cells. Mutations were identified by whole-genome sequencing, making use of software tools that greatly facilitate the distinction between newly induced mutations and other sources of apparent sequence polymorphism, thereby reducing false-positive and false-negative identification rates. In addition, we utilized iPLEX MassARRAY for genotyping recombinant chromosomes. These approaches permitted the mapping of a new mutation affecting cell competition when only a single allele existed, with a phenotype assessed only in genetic mosaics, without the benefit of complementation with existing mutations, deletions, or duplications. These techniques expand the utility of chemical mutagenesis and whole-genome sequencing for mutant identification. We discuss mutations in the Atm and Xrp1 genes identified in this screen. PMID:27574103

  15. A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor*

    PubMed Central

    Zhu, Li; Jang, Geeng-Fu; Jastrzebska, Beata; Filipek, Sławomir; Pearce-Kelling, Susan E.; Aguirre, Gustavo D.; Stenkamp, Ronald E.; Acland, Gregory M.; Palczewski, Krzysztof

    2005-01-01

    Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHOT4R/T4R dog retina, we found that the mutation abolished glycosylation at Asn2, whereas glycosylation at Asn15 was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho* lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (Gt). Structurally, the mutation affected mainly the “plug” at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity. PMID:15459196

  16. A naturally occurring mutation of the opsin gene (T4R) in dogs affects glycosylation and stability of the G protein-coupled receptor.

    PubMed

    Zhu, Li; Jang, Geeng-Fu; Jastrzebska, Beata; Filipek, Slawomir; Pearce-Kelling, Susan E; Aguirre, Gustavo D; Stenkamp, Ronald E; Acland, Gregory M; Palczewski, Krzysztof

    2004-12-17

    Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

  17. G364R mutation of MCM4 detected in human skin cancer cells affects DNA helicase activity of MCM4/6/7 complex.

    PubMed

    Ishimi, Yukio; Irie, Daiki

    2015-06-01

    A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30-50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.

  18. Exclusion of PAX9 and MSX1 mutation in six families affected by tooth agenesis. A genetic study and literature review

    PubMed Central

    Manzanares-Céspedes, Maria C.; Carvalho-Lobato, Patricia; Valdivia-Gandur, Ivan; Arte, Sirpa; Nieminen, Pekka

    2014-01-01

    Objectives: In the present study, it is described the phenotypical analysis and the mutational screening, for genes PAX9 and MSX1, of six families affected by severe forms of tooth agenesis associated with other dental anomalies and systemic entities. Study Design: Six families affected by severe tooth agenesis associated with other dental anomalies and systemic entities were included. Oral exploration, radiological examination, medical antecedents consideration and mutational screening for PAX9 and MSX1 were carried out. Results: No mutations were discovered despite the fact that numerous teeth were missing. An important phenotypical variability was observed within the probands, not being possible to establish a parallelism with the patterns associated to previously described PAX9 and MSX1 mutations. Conclusions: These results bring us to conclude that probably other genes can determine phenotypical patterns of dental agenesis in the families studied, different than the ones described in the mutations of PAX9 and MSX1. Moreover, epigenetic factors can be involved, as those that can reduce gene dosage and other post-transcriptional modulation agents, causing dental agenesis associated or not with systemic anomalies. Key words:Maxillofacial development, tooth agenesis, PAX9 gene, MSX1 gene, gene mutation. PMID:24316698

  19. Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley☆

    PubMed Central

    Howard, Thomas P.; Fahy, Brendan; Leigh, Fiona; Howell, Phil; Powell, Wayne; Greenland, Andy; Trafford, Kay; Smith, Alison M.

    2014-01-01

    The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent. PMID:24748716

  20. Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley.

    PubMed

    Howard, Thomas P; Fahy, Brendan; Leigh, Fiona; Howell, Phil; Powell, Wayne; Greenland, Andy; Trafford, Kay; Smith, Alison M

    2014-03-01

    The effects on barley starch and grain properties of four starch synthesis mutations were studied during the introgression of the mutations from diverse backgrounds into an elite variety. The lys5f (ADPglucose transporter), wax (granule-bound starch synthase), isa1 (debranching enzyme isoamylase 1) and sex6 (starch synthase IIa) mutations were introgressed into NFC Tipple to give mutant and wild-type BC2F4 families with different genomic contributions of the donor parent. Comparison of starch and grain properties between the donor parents, the BC2F4 families and NFC Tipple allowed the effects of the mutations to be distinguished from genetic background effects. The wax and sex6 mutations had marked effects on starch properties regardless of genetic background. The sex6 mutation conditioned low grain weight and starch content, but the wax mutation did not. The lys5 mutation conditioned low grain weight and starch content, but exceptionally high β-glucan contents. The isa1 mutation promotes synthesis of soluble α-glucan (phytoglycogen). Its introgression into NFC Tipple increased grain weight and total α-glucan content relative to the donor parent, but reduced the ratio of phytoglycogen to starch. This study shows that introgression of mutations into a common, commercial background provides new insights that could not be gained from the donor parent.

  1. Regulation of lux Genes in Vibrio fischeri: Control of Symbiosis-Related Gene Expression System in a Marine Bacterium

    DTIC Science & Technology

    1989-11-04

    The pool of mutagenized plasmids was used to transform E . coli cells containing pHIK555 a plasmid compatible with pHK724 which possesses functional...inclusion bodies form upon overexpression of a foreign protein in E . coli . WORK PLAN (Year 2): The mutations described define two regions in the terminal...RR04106 411d019 11 TITLE (Include Security Classification) U. Regulation of lux Genes in Vibrio fischeri : Control of a Symbiosis-Related Gene Expression

  2. Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat.

    PubMed

    Kaur, Jagdeep; Fellers, John; Adholeya, Alok; Velivelli, Siva L S; El-Mounadi, Kaoutar; Nersesian, Natalya; Clemente, Thomas; Shah, Dilip

    2017-02-01

    Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.

  3. Molecular characterization of a mutation affecting abscisic acid biosynthesis and consequently stomatal responses to humidity in an agriculturally important species

    PubMed Central

    McAdam, Scott A. M.; Sussmilch, Frances C.; Brodribb, Timothy J.; Ross, John J.

    2015-01-01

    Mutants deficient in the phytohormone abscisic acid (ABA) have been instrumental in determining not only the biosynthetic pathway for this hormone, but also its physiological role in land plants. The wilty mutant of Pisum sativum is one of the classical, well-studied ABA-deficient mutants; however, this mutant remains uncharacterized at a molecular level. Using a candidate gene approach, we show that the wilty mutation affects the xanthoxin dehydrogenase step in ABA biosynthesis. To date, this step has only been represented by mutants in the ABA2 gene of Arabidopsis thaliana. Functional ABA biosynthesis appears to be critical for normal stomatal responses to changes in humidity in angiosperms, with wilty mutant plants having no increase in foliar ABA levels in response to a doubling in vapour pressure deficit, and no closure of stomata. Phylogenetic analysis of the ABA2 gene family from diverse land plants indicates that an ABA-biosynthesis-specific short-chain dehydrogenase (ABA2) evolved in the earliest angiosperms. The relatively recent origin of specificity in this step has important implications for both the evolution of ABA biosynthesis and action in land plants. PMID:26216469

  4. The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific.

    PubMed

    Raule, Nicola; Sevini, Federica; Li, Shengting; Barbieri, Annalaura; Tallaro, Federica; Lomartire, Laura; Vianello, Dario; Montesanto, Alberto; Moilanen, Jukka S; Bezrukov, Vladyslav; Blanché, Hélène; Hervonen, Antti; Christensen, Kaare; Deiana, Luca; Gonos, Efstathios S; Kirkwood, Tom B L; Kristensen, Peter; Leon, Alberta; Pelicci, Pier Giuseppe; Poulain, Michel; Rea, Irene M; Remacle, Josè; Robine, Jean Marie; Schreiber, Stefan; Sikora, Ewa; Eline Slagboom, Peternella; Spazzafumo, Liana; Antonietta Stazi, Maria; Toussaint, Olivier; Vaupel, James W; Rose, Giuseppina; Majamaa, Kari; Perola, Markus; Johnson, Thomas E; Bolund, Lars; Yang, Huanming; Passarino, Giuseppe; Franceschi, Claudio

    2014-06-01

    To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se have a low, or very low, impact. In particular, the analysis of the mutations occurring in different OXPHOS complex showed a complex scenario with a different mutation burden in 90+ subjects with respect to controls. These findings suggested that mutations in subunits of the OXPHOS complex I had a beneficial effect on longevity, while the simultaneous presence of mutations in complex I and III (which also occurs in J subhaplogroups involved in LHON) and in complex I and V seemed to be detrimental, likely explaining previous contradictory results. On the whole, our study, which goes beyond haplogroup analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes.

  5. Molecular characterization of 7 patients affected by dys- or hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation.

    PubMed

    Asselta, Rosanna; Robusto, Michela; Platé, Manuela; Santoro, Cristina; Peyvandi, Flora; Duga, Stefano

    2015-07-01

    Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes. In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene.

  6. Factors that affect the molecular nature of germ-line mutations recovered in the mouse specific-locus test

    SciTech Connect

    Russell, L.B. )

    1991-01-01

    The morphological specific locus test (SLT), which allows the scoring of 2,000 loci/hr/person, has been in use for four decades for measuring mammalian germ-line mutation rates under various conditions of exposure. More recently, the SLT's capabilities for the qualitative characterization of mutations have been exploited. The large sets of mutations centered on specific loci that have been accumulated over the years, including sets of nested deletions, have provided prime material for fine-structure genetic analyses. Subsequent molecular entry to these regions has led to intensive physical/functional mapping of megabase segments of the genome. In turn, these investigations have generated genetic and molecular tools for analyzing individual mutations as to extent and nature of the genomic lesion. These and related quantitative findings now make it possible to optimize conditions for the use of mutagens in providing desired types of mutations as tools.

  7. Variable Autosomal and X Divergence Near and Far from Genes Affects Estimates of Male Mutation Bias in Great Apes.

    PubMed

    Narang, Pooja; Wilson Sayres, Melissa A

    2016-12-31

    Male mutation bias, when more mutations are passed on via the male germline than via the female germline, is observed across mammals. One common way to infer the magnitude of male mutation bias, α, is to compare levels of neutral sequence divergence between genomic regions that spend different amounts of time in the male and female germline. For great apes, including human, we show that estimates of divergence are reduced in putatively unconstrained regions near genes relative to unconstrained regions far from genes. Divergence increases with increasing distance from genes on both the X chromosome and autosomes, but increases faster on the X chromosome than autosomes. As a result, ratios of X/A divergence increase with increasing distance from genes and corresponding estimates of male mutation bias are significantly higher in intergenic regions near genes versus far from genes. Future studies in other species will need to carefully consider the effect that genomic location will have on estimates of male mutation bias.

  8. The E89K Mutation in the Matrix Protein of the Measles Virus Affects In Vitro Cell Death and Virus Replication Efficiency in Human PBMC

    PubMed Central

    Dong, Jianbao; Zhu, Wei; Saito, Akatsuki; Goto, Yoshitaka; Iwata, Hiroyuki; Haga, Takeshi

    2012-01-01

    Matrix protein is known to have an important role in the process of virus assembly and virion release during measles virus replication. In the present in vitro study, a single mutation of E89K in the matrix protein was shown to affect cell death and virus replication efficiency in human PBMC. One strain with this mutation caused less cell death than the parental virus, and possessed high virus replication efficiency. Moreover, by Annexin V-FITC staining, polycaspase FLICA staining, and double labeling with poly-caspase FLICA and the Hoechst stain, the cell death seen was shown to be apoptosis. PMID:22715352

  9. Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation.

    PubMed

    Cheng, Haiming; Rashid, Shayan; Yu, Zhuoxin; Yoshizumi, Ayumi; Hwang, Eileen; Brodsky, Barbara

    2011-01-21

    The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

  10. Mutations in epidermis-specific HD-ZIP IV genes affect floral organ identity in Arabidopsis thaliana.

    PubMed

    Kamata, Naoko; Okada, Hitomi; Komeda, Yoshibumi; Takahashi, Taku

    2013-08-01

    Development of the epidermis involves members of the class-IV homeodomain-leucine zipper (HD-ZIP IV) transcription factors. The Arabidopsis HD-ZIP IV family consists of 16 members, among which PROTODERMAL FACTOR 2 (PDF2) and ARABIDOPSIS THALIANA MERISTEM LAYER 1 (ATML1) play an indispensable role in the differentiation of shoot epidermal cells; however, the functions of other HD-ZIP IV genes that are also expressed specifically in the shoot epidermis remain to be fully elucidated. We constructed double mutant combinations of these HD-ZIP IV mutant alleles and found that the double mutants of pdf2-1 with homeodomain glabrous1-1 (hdg1-1), hdg2-3, hdg5-1 and hdg12-2 produced abnormal flowers with sepaloid petals and carpelloid stamens in association with the reduced expression of the petal and stamen identity gene APETALA 3 (AP3). Expression of another petal and stamen identity gene PISTILATA (PI) was less affected in these mutants. We confirmed that AP3 expression in pdf2-1 hdg2-3 was normally induced at the initial stages of flower development, but was attenuated both in the epidermis and internal cell layers of developing flowers. As the expression of PDF2 and these HD-ZIP IV genes during floral organ formation is exclusively limited to the epidermal cell layer, these double mutations may have non-cell-autonomous effects on AP3 expression in the internal cell layers. Our results suggest that cooperative functions of PDF2 and other members of the HD-ZIP IV family in the epidermis are crucial for normal development of floral organs in Arabidopsis.

  11. Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder ( Paralichthys olivaceus)

    NASA Astrophysics Data System (ADS)

    Ma, Ruiqin; He, Feng; Wen, Haishen; Li, Jifang; Shi, Bao; Shi, Dan; Liu, Miao; Mu, Weijie; Zhang, Yuanqing; Hu, Jian; Han, Weiguo; Zhang, Jianan; Wang, Qingqing; Yuan, Yuren; Liu, Qun

    2012-03-01

    As a specific gene of fish, cytochrome P450c17-II ( CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder ( Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI ( P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

  12. Network analysis of eight industrial symbiosis systems

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Zheng, Hongmei; Shi, Han; Yu, Xiangyi; Liu, Gengyuan; Su, Meirong; Li, Yating; Chai, Yingying

    2016-06-01

    Industrial symbiosis is the quintessential characteristic of an eco-industrial park. To divide parks into different types, previous studies mostly focused on qualitative judgments, and failed to use metrics to conduct quantitative research on the internal structural or functional characteristics of a park. To analyze a park's structural attributes, a range of metrics from network analysis have been applied, but few researchers have compared two or more symbioses using multiple metrics. In this study, we used two metrics (density and network degree centralization) to compare the degrees of completeness and dependence of eight diverse but representative industrial symbiosis networks. Through the combination of the two metrics, we divided the networks into three types: weak completeness, and two forms of strong completeness, namely "anchor tenant" mutualism and "equality-oriented" mutualism. The results showed that the networks with a weak degree of completeness were sparse and had few connections among nodes; for "anchor tenant" mutualism, the degree of completeness was relatively high, but the affiliated members were too dependent on core members; and the members in "equality-oriented" mutualism had equal roles, with diverse and flexible symbiotic paths. These results revealed some of the systems' internal structure and how different structures influenced the exchanges of materials, energy, and knowledge among members of a system, thereby providing insights into threats that may destabilize the network. Based on this analysis, we provide examples of the advantages and effectiveness of recent improvement projects in a typical Chinese eco-industrial park (Shandong Lubei).

  13. Mastering ectomycorrhizal symbiosis: the impact of carbohydrates.

    PubMed

    Nehls, Uwe

    2008-01-01

    Mycorrhiza formation is the consequence of a mutualistic interaction between certain soil fungi and plant roots that helps to overcome nutritional limitations faced by the respective partners. In symbiosis, fungi contribute to tree nutrition by means of mineral weathering and mobilization of nutrients from organic matter, and obtain plant-derived carbohydrates as a response. Support with easily degradable carbohydrates seems to be the driving force for fungi to undergo this type of interaction. As a consequence, the fungal hexose uptake capacity is strongly increased in Hartig net hyphae of the model fungi Amanita muscaria and Laccaria bicolor. Next to fast carbohydrate uptake and metabolism, storage carbohydrates are of special interest. In functional A. muscaria ectomycorrhizas, expression and activity of proteins involved in trehalose biosynthesis is mainly localized in hyphae of the Hartig net, indicating an important function of trehalose in generation of a strong carbon sink by fungal hyphae. In symbiosis, fungal partners receive up to approximately 19 times more carbohydrates from their hosts than normal leakage of the root system would cause, resulting in a strong carbohydrate demand of infected roots and, as a consequence, a more efficient plant photosynthesis. To avoid fungal parasitism, the plant seems to have developed mechanisms to control carbohydrate drain towards the fungal partner and link it to the fungus-derived mineral nutrition. In this contribution, current knowledge on fungal strategies to obtain carbohydrates from its host and plant strategies to enable, but also to control and restrict (under certain conditions), carbon transfer are summarized.

  14. Microfungal "weeds" in the leafcutter ant symbiosis.

    PubMed

    Rodrigues, A; Bacci, M; Mueller, U G; Ortiz, A; Pagnocca, F C

    2008-11-01

    Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as "weeds" in the ant-fungus symbiosis.

  15. Nitrate regulates rhizobial and mycorrhizal symbiosis in common bean (Phaseolus vulgaris L.).

    PubMed

    Nanjareddy, Kalpana; Blanco, Lourdes; Arthikala, Manoj-Kumar; Affantrange, Xochitl Alvarado; Sánchez, Federico; Lara, Miguel

    2014-03-01

    Nitrogen-limited conditions are considered to be a prerequisite for legume-rhizobial symbiosis, but the effects of nitrate-rich conditions on symbiotic status remain poorly understood. We addressed this issue by examining rhizobial (Rhizobim tropici) and arbusclar mycorrhizal (Glomus intraradices) symbiosis in Phaseolus vulgaris L. cv. Negro Jamapa under nitrate pre-incubation and continuous nitrate conditions. Our results indicate that nitrate pre-incubation, independent of the concentration, did not affect nodule development. However, the continuous supply of nitrate at high concentrations impaired nodule maturation and nodule numbers. Low nitrate conditions, in addition to positively regulating nodule number, biomass, and nitrogenase activity, also extended the span of nitrogen-fixing activity. By contrast, for arbuscular mycorrhizae, continuous 10 and 50 mmol/L nitrate increased the percent root length colonization, concomitantly reduced arbuscule size, and enhanced ammonia transport without affecting phosphate transport. Therefore, in this manuscript, we have proposed the importance of nitrate as a positive regulator in promoting both rhizobial and mycorrhizal symbiosis in the common bean.

  16. Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) rapidly identified a critical missense mutation (P236T) of bovine ACADVL gene affecting growth traits.

    PubMed

    Zhang, Sihuan; Dang, Yonglong; Zhang, Qingfeng; Qin, Qiaomei; Lei, Chuzhao; Chen, Hong; Lan, Xianyong

    2015-04-01

    Acyl-CoA dehydrogenase, very long chain (ACADVL), encoding ACADVL protein, targets the inner mitochondrial membrane where it catalyzes the first step of the mitochondrial fatty acid beta-oxidation pathway and plays an important role in body metabolism and oxidation of long chain fatty acid releasing energy. Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is an easy-to-operate, rapid, inexpensive, and exact method for SNP genotyping. Herein, T-ARMS-PCR was carried out to detect a critical missense mutation (AC_000176:g.2885C>A; Pro236Thr) within the ACADVL gene in 644 individuals from two cattle breeds. In order to evaluate the accuracy of the T-ARMS-PCR at this locus, the genotype of the sampled individuals was also identified by PCR-RFLP. The concordance between these two methods was 98.76%. Statistical analysis showed that the bovine ACADVL gene had a significant effect on chest width (P<0.05), chest depth (P<0.05), and hip width (P<0.05) in the Qinchuan breed. The cattle with AA genotype had superior growth traits compared to cattle with AC and/or CC genotypes. The "A" allele had positive effects on growth traits. Therefore, T-ARMS-PCR can replace PCR-RFLP for rapid genotyping of this mutation, which could be used as a DNA marker for selecting individuals with superior growth traits in the Qinchuan breed. These findings contribute to breeding and genetics in beef cattle industry.

  17. Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing.

    PubMed

    van Kuilenburg, André B P; Meijer, Judith; Maurer, Dirk; Dobritzsch, Doreen; Meinsma, Rutger; Los, Maartje; Knegt, Lia C; Zoetekouw, Lida; Jansen, Rob L H; Dezentjé, Vincent; van Huis-Tanja, Lieke H; van Kampen, Roel J W; Hertz, Jens Michael; Hennekam, Raoul C M

    2017-03-01

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

  18. Pseudohypoaldosteronism type 1 and Liddle’s syndrome mutations that affect the single-channel properties of the epithelial Na+ channel

    PubMed Central

    Boiko, Nina; Kucher, Volodymyr; Stockand, James D

    2015-01-01

    These studies test whether three disease-causing mutations in genes (SCNN1A and SCNN1G) encoding subunits of the epithelial Na+ channel, ENaC, affect the biophysical and gating properties of this important renal ion channel. The S562P missense mutation in αENaC and the K106_S108delinsN mutation in γENaC are associated with pseudohypoaldosteronism type 1 (PHA1). The N530S missense mutation in γENaC causes Liddle’s syndrome. Incorporation of S562P into αENaC and K106_S108N into γENaC resulted in significant decreases in macroscopic ENaC currents. Conversely, incorporation of N530S into γENaC increased macroscopic ENaC current. The S562P substitution resulted in a nonfunctional channel. The K106_S108N mutation produced a functional channel having a normal macroscopic current–voltage relation, there was a slight but significant decrease in unitary conductance and a marked decrease in single-channel open probability. The N530S substitution increased single-channel open probability having no effect on the macroscopic current–voltage relation or unitary conductance of the channel. These findings are consistent with mutation of residues at 562 in αENaC and 530 in γENaC, and a 3′ splice site in SCNN1G (318-1 G→A; K106_108SdelinsN) resulting in aberrant ENaC activity due to changes in the biophysical and gating properties of the channel. Such changes likely contribute to the cellular mechanism underpinning the PHA1 and Liddle’s syndrome caused by these mutations in ENaC subunits. PMID:26537344

  19. Publisher's note. Identification of a novel synonymous mutation in the human β-ureidopropionase gene UPB1 affecting pre-mRNA splicing.

    PubMed

    Meijer, J; Nakajima, Y; Zhang, C; Meinsma, R; Ito, T; Van Kuilenburg, A B P

    2014-01-01

    β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyzes the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, and ammonia and CO2. To date, only 16 genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report the clinical, biochemical, and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Mutation analysis of the UPB1 gene showed that the patient was compound heterozygous for a novel synonymous mutation c.93C>T (p.Gly31Gly) in exon 1 and a previously described missense mutation c.977G>A (p.Arg326Gln) in exon 9. The in silico predicted effect of the synonymous mutation p.Gly31Gly on pre-mRNA splicing was investigated using a minigene approach. Wild-type and the mutated minigene constructs, containing the entire exon 1, intron 1, and exon 2 of UPB1, yielded different splicing products after expression in HEK293 cells. The c.93C>T (p.Gly31Gly) mutation resulted in altered pre-mRNA splicing of the UPB1 minigene construct and a deletion of the last 13 nucleotides of exon 1. This deletion (r.92 104delGCAAGGAACTCAG) results in a frame shift and the generation of a premature stop codon (p.Lys32SerfsX31). Using a minigene approach, we have thus identified the first synonymous mutation in the UPB1 gene, creating a cryptic splice-donor site affecting pre-mRNA splicing.

  20. Correspondence regarding Ballana et al., "Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment".

    PubMed

    Abreu-Silva, R S; Batissoco, A C; Lezirovitz, K; Romanos, J; Rincon, D; Auricchio, M T B M; Otto, P A; Mingroni-Netto, R C

    2006-05-12

    Ballana et al. [E. Ballana, E. Morales, R. Rabionet, B. Montserrat, M. Ventayol, O. Bravo, P. Gasparini, X. Estivill, Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment, Biochem. Biophys. Res. Commun. 341 (2006) 950-957] detected a T1291C mutation segregating in a Cuban pedigree with hearing impairment. They interpreted it as probably pathogenic, based on family history, RNA conformation prediction and its absence in a control group of 95 Spanish subjects. We screened a sample of 203 deaf subjects and 300 hearing controls (110 "European-Brazilians" and 190 "African-Brazilians") for the mitochondrial mutations A1555G and T1291C. Five deaf subjects had the T1291C substitution, three isolated cases and two familial cases. In the latter, deafness was paternally inherited or segregated with the A1555G mutation. This doesn't support the hypothesis of T1291C mutation being pathogenic. Two "African-Brazilian" controls also had the T1291C substitution. Six of the seven T1291C-carriers (five deaf and two controls) had mitochondrial DNA of African origin, belonging to macrohaplogroup L1/L2. Therefore, these data point to T1291C substitution as most probably an African non-pathogenic polymorphism.

  1. Relationship between a point mutation S97C in CK1δ protein and its affect on ATP-binding affinity.

    PubMed

    Kumar, Ambuj; Rajendran, Vidya; Sethumadhavan, Rao; Purohit, Rituraj

    2014-01-01

    CK1δ (Casein kinase I isoform delta) is a member of CK1 kinase family protein that mediates neurite outgrowth and the function as brain-specific microtubule-associated protein. ATP binding kinase domain of CK1δ is essential for regulating several key cell cycle signal transduction pathways. Mutation in CK1δ protein is reported to cause cancers and affects normal brain development. S97C mutation in kinase domain of CK1δ protein has been involved to induce breast cancer and ductal carcinoma. We performed molecular docking studies to examine the effect of this mutation on its ATP-binding affinity. Further, we conducted molecular dynamics simulations to understand the structural consequences of S97C mutation over the kinase domain of CK1δ protein. Docking results indicated the loss of ATP-binding affinity of mutant structure, which were further rationalized by molecular dynamics simulations, where a notable loss in 3-D conformation of CK1δ kinase domain was observed in mutant as compared to native. Our results explained the underlying molecular mechanism behind the observed cancer associated phenotype caused by S97C mutation in CK1δ protein.

  2. Patient affected by neurofibromatosis type 1 and thyroid C-cell hyperplasia harboring pathogenic germ-line mutations in both NF1 and RET genes.

    PubMed

    Ercolino, Tonino; Lai, Roberta; Giachè, Valentino; Melchionda, Salvatore; Carella, Massimo; Delitala, Alessandro; Mannelli, Massimo; Fanciulli, Giuseppe

    2014-02-25

    Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease with an estimated incidence of 1 in 3000/3500 live births. NF1 is caused by a mutation in a gene which encodes a protein known as neurofibromin. In up to 5% of cases, NF1 is associated with pheochromocytomas. RET proto-oncogene encodes a member of the receptor tyrosine kinase family involved in the normal development or the neoplastic growth of neural crest cell lineages. Germ-line RET mutations account for cases of Multiple Endocrine Neoplasia type 2 (MEN2), an autosomal dominant genetic syndrome where medullary thyroid carcinoma (MTC) is the major and more clinically severe feature, with nearly complete penetrance. C-cell hyperplasia (CCH) is described in MEN2 patients, and it has been implicated as the precursor of in situ MTC. Patients with RET mutations develop pheochromocytomas in 50% of cases. Rarely, patients with NF1 have been found to present, in addition to the NF1 clinical picture, other lesions, such as parathyroid hyperplasia/adenoma and/or medullary thyroid carcinoma. In spite of the presence of these MEN2 lesions, in none of these patients mutations of gene RET have been found so far. In this report, we describe the first case of a patient affected by a germ-line mutation in both NF1 and RET genes.

  3. Characterization of an acromesomelic dysplasia, Grebe type case: novel mutation affecting the recognition motif at the processing site of GDF5.

    PubMed

    Martinez-Garcia, Monica; Garcia-Canto, Eva; Fenollar-Cortes, Maria; Aytes, Antonio Perez; Trujillo-Tiebas, María José

    2016-09-01

    Acromesomelic dysplasia, Grebe type is a very rare skeletal dysplasia characterized by severe dwarfism with marked micromelia and deformation of the upper and lower limbs, with a proximodistal gradient of severity. CDMP1 gene mutations have been associated with Grebe syndrome, Hunter-Thompson syndrome, Du Pan syndrome and brachydactyly type C. The proband is a 4-year-old boy, born of consanguineous Pakistani parents. Radiographic imaging revealed features typical of Grebe syndrome: severe shortening of the forearms with an acromesomelic pattern following a proximodistal gradient, with distal parts more severely affected than medial parts; hypoplastic hands, with the phalangeal zone more affected than the metacarpal zone; and severe hypoplastic tibial/femoral zones in both limbs. After molecular analyses, the p.Arg377Trp variant in a homozygous pattern was identified in the CDMP1 gene in the affected child. In silico and structural analyses predicted the p.Arg377Trp amino acid change to be pathogenic. Of the 34 mutations described in the CDMP1 gene, four different missense mutations have been associated with Grebe syndrome. The CDMP1 gene encodes growth differentiation factor 5 (GDF5), which plays a role in regulation of limb patterning, joint formation and distal bone growth. Homozygous mutations in the mature domain of GDF5 result in severe limb malformations such as the Grebe type or the Hunter-Thompson type of acromesomelic chondrodysplasia. The p.Arg377Trp mutation is located within the recognition motif at the processing site of GDF5 where the sequence RRKRR changes to WRKRR. The genotype-phenotype correlation allowed not only confirmation of the clinical diagnosis but also appropriate genetic counselling to be offered to this family.

  4. Dominant-negative mutation in the beta2 and beta6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage.

    PubMed

    Schweisguth, F

    1999-09-28

    In Drosophila, dominant-negative mutations in the beta2 and beta6 proteasome catalytic subunit genes have been identified as dominant temperature-sensitive (DTS) mutations. At restrictive temperature, beta2 and beta6 DTS mutations confer lethality at the pupal stage. I investigate here the role of proteasome activity in regulating cell fate decisions in the sense organ lineage at the early pupal stage. Temperature-shift experiments in beta2 and beta6 DTS mutant pupae occasionally resulted in external sense organs with two sockets and no shaft. This double-socket phenotype was strongly enhanced in conditions in which Notch signaling was up-regulated. Furthermore, conditional overexpression of the beta6 dominant-negative mutant subunit led to shaft-to-socket and to neuron-to-sheath cell fate transformations, which are both usually associated with increased Notch signaling activity. Finally, expression of the beta6 dominant-negative mutant subunit led to the stabilization of an ectopically expressed nuclear form of Notch in imaginal wing discs. This study demonstrates that mutations affecting two distinct proteasome catalytic subunits affect two alternative cell fate decisions and enhance Notch signaling activity in the sense organ lineage. These findings raise the possibility that the proteasome targets an active form of the Notch receptor for degradation in Drosophila.

  5. The LPS O-Antigen in Photosynthetic Bradyrhizobium Strains Is Dispensable for the Establishment of a Successful Symbiosis with Aeschynomene Legumes

    PubMed Central

    Busset, Nicolas; De Felice, Antonia; Chaintreuil, Clémence; Gully, Djamel; Fardoux, Joël; Romdhane, Sana; Molinaro, Antonio; Silipo, Alba; Giraud, Eric

    2016-01-01

    The photosynthetic bradyrhizobia are able to use a Nod-factor independent process to induce nitrogen-fixing nodules on some semi-aquatic Aeschynomene species. These bacteria display a unique LPS O-antigen composed of a new sugar, the bradyrhizose that is regarded as a key symbiotic factor due to its non-immunogenic character. In this study, to check this hypothesis, we isolated mutants affected in the O-antigen synthesis by screening a transposon mutant library of the ORS285 strain for clones altered in colony morphology. Over the 10,000 mutants screened, five were selected and found to be mutated in two genes, rfaL, encoding for a putative O-antigen ligase and gdh encoding for a putative dTDP-glucose 4,6-dehydratase. Biochemical analysis confirmed that the LPS of these mutants completely lack the O-antigen region. However, no effect of the mutations could be detected on the symbiotic properties of the mutants indicating that the O-antigen region of photosynthetic Bradyrhizobium strains is not required for the establishment of symbiosis with Aeschynomene. PMID:26849805

  6. Nitrogen metabolism in Sinorhizobium meliloti-alfalfa symbiosis: dissecting the role of GlnD and PII proteins.

    PubMed

    Yurgel, Svetlana N; Rice, Jennifer; Kahn, Michael L

    2012-03-01

    To contribute nitrogen for plant growth and establish an effective symbiosis with alfalfa, Sinorhizobium meliloti Rm1021 needs normal operation of the GlnD protein, a bifunctional uridylyltransferase/uridylyl-cleavage enzyme that measures cellular nitrogen status and initiates a nitrogen stress response (NSR). However, the only two known targets of GlnD modification in Rm1021, the PII proteins GlnB and GlnK, are not necessary for effectiveness. We introduced a Tyr→Phe variant of GlnB, which cannot be uridylylated, into a glnBglnK background to approximate the expected state in a glnD-sm2 mutant, and this strain was effective. These results suggested that unmodified PII does not inhibit effectiveness. We also generated a glnBglnK-glnD triple mutant and used this and other mutants to dissect the role of these proteins in regulating the free-living NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation was dominant to the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes. The data show that the GlnD protein has a role in free-living growth and in symbiotic nitrogen exchange that does not depend on the PII proteins, suggesting that S. meliloti GlnD can communicate with the cell by alternate mechanisms.

  7. Mutations in the helper component protease gene of zucchini yellow mosaic virus affect its ability to mediate aphid transmissibility.

    PubMed

    Huet, H; Gal-On, A; Meir, E; Lecoq, H; Raccah, B

    1994-06-01

    The nucleotide sequence of the helper component protease (HC-Pro) genes of three zucchini yellow mosaic virus (ZYMV) strains has been compared with that of a helper-deficient strain of ZYMV-HC. The comparisons revealed three unique deduced amino acid differences. Two of these mutations were located in regions which are conserved in other potyviruses. The role of these mutations in aphid transmissibility was examined by exchanging DNA fragments of part of the deficient HC-Pro gene with the respective section within the gene of the infectious full-length clone of the aphid-transmissible ZYMV. The first exchange included two of the three mutations, the first coding for a change from Asp to Gly (in a non-conserved region) and the second coding for a change from Arg to Ile [within the Phe-Arg-Asp-Lys (FRNK) conserved box]. This exchange resulted in a reduced transmission (20.6% for the mutated virus compared with 57.4% in the normal ZYMV when acquired from plants and 37.2% compared with 83.1%, respectively, when acquired from membranes). The second exchange incorporated a single mutation [conferring a change from Thr to Ala within the Pro-Thr-Lys (PTK) conserved box]. This single mutation resulted in almost total loss of HC activity in aphid transmission both from plants and from membranes. The Lys residue in the conserved Lys-Ile-Thr-Cys (KITC) box, which is related to loss of HC activity in potato virus Y, tobacco vein mottling virus and in the Michigan strain of ZYMV, is unchanged in the helper-deficient ZYMV. It is therefore proposed that more than one site in HC-Pro may be functionally related to aphid transmissibility. The possible reasons for the role of these mutations in helper activity in aphid transmission of ZYMV are discussed.

  8. NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

    PubMed Central

    DeDiego, Marta L.; Nogales, Aitor; Lambert-Emo, Kris; Martinez-Sobrido, Luis

    2016-01-01

    ABSTRACT Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence

  9. Variable Autosomal and X Divergence Near and Far from Genes Affects Estimates of Male Mutation Bias in Great Apes

    PubMed Central

    Narang, Pooja; Wilson Sayres, Melissa A.

    2016-01-01

    Male mutation bias, when more mutations are passed on via the male germline than via the female germline, is observed across mammals. One common way to infer the magnitude of male mutation bias, α, is to compare levels of neutral sequence divergence between genomic regions that spend different amounts of time in the male and female germline. For great apes, including human, we show that estimates of divergence are reduced in putatively unconstrained regions near genes relative to unconstrained regions far from genes. Divergence increases with increasing distance from genes on both the X chromosome and autosomes, but increases faster on the X chromosome than autosomes. As a result, ratios of X/A divergence increase with increasing distance from genes and corresponding estimates of male mutation bias are significantly higher in intergenic regions near genes versus far from genes. Future studies in other species will need to carefully consider the effect that genomic location will have on estimates of male mutation bias. PMID:27702816

  10. An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster.

    PubMed Central

    Luschnig, Stefan; Moussian, Bernard; Krauss, Jana; Desjeux, Isabelle; Perkovic, Josip; Nüsslein-Volhard, Christiane

    2004-01-01

    Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning. PMID:15166158

  11. SUMF1 mutations affecting stability and activity of formylglycine generating enzyme predict clinical outcome in multiple sulfatase deficiency.

    PubMed

    Schlotawa, Lars; Ennemann, Eva Charlotte; Radhakrishnan, Karthikeyan; Schmidt, Bernhard; Chakrapani, Anupam; Christen, Hans-Jürgen; Moser, Hugo; Steinmann, Beat; Dierks, Thomas; Gärtner, Jutta

    2011-03-01

    Multiple Sulfatase Deficiency (MSD) is caused by mutations in the sulfatase-modifying factor 1 gene encoding the formylglycine-generating enzyme (FGE). FGE post translationally activates all newly synthesized sulfatases by generating the catalytic residue formylglycine. Impaired FGE function leads to reduced sulfatase activities. Patients display combined clinical symptoms of single sulfatase deficiencies. For ten MSD patients, we determined the clinical phenotype, FGE expression, localization and stability, as well as residual FGE and sulfatase activities. A neonatal, very severe clinical phenotype resulted from a combination of two nonsense mutations leading to almost fully abrogated FGE activity, highly unstable FGE protein and nearly undetectable sulfatase activities. A late infantile mild phenotype resulted from FGE G263V leading to unstable protein but high residual FGE activity. Other missense mutations resulted in a late infantile severe phenotype because of unstable protein with low residual FGE activity. Patients with identical mutations displayed comparable clinical phenotypes. These data confirm the hypothesis that the phenotypic outcome in MSD depends on both residual FGE activity as well as protein stability. Predicting the clinical course in case of molecularly characterized mutations seems feasible, which will be helpful for genetic counseling and developing therapeutic strategies aiming at enhancement of FGE.

  12. Brassinosteroids Regulate Root Growth, Development, and Symbiosis.

    PubMed

    Wei, Zhuoyun; Li, Jia

    2016-01-04

    Brassinosteroids (BRs) are natural plant hormones critical for growth and development. BR deficient or signaling mutants show significantly shortened root phenotypes. However, for a long time, it was thought that these phenotypes were solely caused by reduced cell elongation in the mutant roots. Functions of BRs in regulating root development have been largely neglected. Nonetheless, recent detailed analyses, revealed that BRs are not only involved in root cell elongation but are also involved in many aspects of root development, such as maintenance of meristem size, root hair formation, lateral root initiation, gravitropic response, mycorrhiza formation, and nodulation in legume species. In this review, current findings on the functions of BRs in mediating root growth, development, and symbiosis are discussed.

  13. Symbiosis and the origin of eukaryotic motility

    NASA Technical Reports Server (NTRS)

    Margulis, L.; Hinkle, G.

    1991-01-01

    Ongoing work to test the hypothesis of the origin of eukaryotic cell organelles by microbial symbioses is discussed. Because of the widespread acceptance of the serial endosymbiotic theory (SET) of the origin of plastids and mitochondria, the idea of the symbiotic origin of the centrioles and axonemes for spirochete bacteria motility symbiosis was tested. Intracellular microtubular systems are purported to derive from symbiotic associations between ancestral eukaryotic cells and motile bacteria. Four lines of approach to this problem are being pursued: (1) cloning the gene of a tubulin-like protein discovered in Spirocheata bajacaliforniesis; (2) seeking axoneme proteins in spirochets by antibody cross-reaction; (3) attempting to cultivate larger, free-living spirochetes; and (4) studying in detail spirochetes (e.g., Cristispira) symbiotic with marine animals. Other aspects of the investigation are presented.

  14. Robust homology-directed repair within mouse mammary tissue is not specifically affected by Brca2 mutation

    PubMed Central

    Kass, Elizabeth M.; Lim, Pei Xin; Helgadottir, Hildur R.; Moynahan, Mary Ellen; Jasin, Maria

    2016-01-01

    The mammary gland undergoes significant proliferative stages after birth, but little is known about how the developmental changes impact DNA double-strand break (DSB) repair. Mutations in multiple genes involved in homology-directed repair (HDR), considered a particularly accurate pathway for repairing DSBs, are linked to breast cancer susceptibility, including BRCA2. Using reporter mice that express an inducible endonuclease, we find that HDR is particularly robust in mammary tissue during puberty and pregnancy, accounting for 34–40% of detected repair events, more than in other tissues examined. Brca2 hypomorphic mutation leads to HDR defects in mammary epithelium during puberty and pregnancy, including in different epithelial lineages. Notably, a similar dependence on Brca2 is observed in other proliferative tissues, including small intestine epithelium. Our results suggest that the greater reliance on HDR in the proliferating mammary gland, rather than a specific dependence on BRCA2, may increase its susceptibility to tumorigenesis incurred by BRCA2 mutation. PMID:27779185

  15. Do the costs and benefits of fungal endophyte symbiosis vary with light availability?

    PubMed

    Davitt, Andrew J; Stansberry, Marcus; Rudgers, Jennifer A

    2010-11-01

    • Here, we examined whether fungal endophytes modulated host plant responses to light availability. First, we conducted a literature review to evaluate whether natural frequencies of endophyte symbiosis in grasses from shaded habitats were higher than frequencies in grasses occupying more diverse light environments. Then, in a glasshouse experiment, we assessed how four levels of light and the presence of endophyte symbioses affected the growth of six grass species. • In our literature survey, endophytes were more commonly present in grasses restricted to shaded habitats than in grasses from diverse light environments. • In the glasshouse, endophyte symbioses did not mediate plant growth in response to light availability. However, in the host grass, Agrostis perennans, symbiotic plants produced 53% more inflorescences than nonsymbiotic plants at the highest level of shade. In addition, under high shade, symbiotic Poa autumnalis invested more in specific leaf area than symbiont-free plants. Finally, shade increased the density of the endophyte in leaf tissues across all six grass species. • Our results highlight the potential for symbiosis to alter the plasticity of host physiological traits, demonstrate a novel benefit of endophyte symbiosis under shade stress for one host species, and show a positive association between shade-restricted grass species and fungal endophytes.

  16. Role of arbuscular mycorrhizal symbiosis in root mineral uptake under CaCO3 stress.

    PubMed

    Labidi, Sonia; Ben Jeddi, Fayçal; Tisserant, Benoit; Debiane, Djouher; Rezgui, Salah; Grandmougin-Ferjani, Anne; Lounès-Hadj Sahraoui, Anissa

    2012-07-01

    This study investigated the effects of increasing CaCO(3) concentrations (0, 5, 10, 20 mM) on arbuscular mycorrhizal (AM) symbiosis establishment as well as on chicory root growth and mineral nutrient uptake in a monoxenic system. Although CaCO(3) treatments significantly decreased root growth and altered the symbiosis-related development steps of the AM fungus Rhizophagus irregularis (germination, germination hypha elongation, root colonization rate, extraradical hyphal development, sporulation), the fungus was able to completely fulfill its life cycle. Even when root growth decreased more drastically in mycorrhizal roots than in non-mycorrhizal ones in the presence of high CaCO(3) levels, the AM symbiosis was found to be beneficial for root mineral uptake. Significant increases in P, N, Fe, Zn and Cu concentrations were recorded in the mycorrhizal roots. Whereas acid and alkaline phosphatase enzymatic activities remained constant in mycorrhizal roots, they were affected in non-mycorrhizal roots grown in the presence of CaCO(3) when compared with the control.

  17. The site-directed mutation I(L177)H in Rhodobacter sphaeroides reaction center affects coordination of P(A) and B(B) bacteriochlorophylls.

    PubMed

    Vasilieva, L G; Fufina, T Y; Gabdulkhakov, A G; Leonova, M M; Khatypov, R A; Shuvalov, V A

    2012-08-01

    To explore the influence of the I(L177)H single mutation on the properties of the nearest bacteriochlorophylls (BChls), three reaction centers (RCs) bearing double mutations were constructed in the photosynthetic purple bacterium Rhodobacter sphaeroides, and their properties and pigment content were compared with those of the correspondent single mutant RCs. Each pair of the mutations comprised the amino acid substitution I(L177)H and another mutation altering histidine ligand of BChl P(A) or BChl B(B). Contrary to expectations, the double mutation I(L177)H+H(L173)L does not bring about a heterodimer RC but causes a 46nm blue shift of the long-wavelength P absorbance band. The histidine L177 or a water molecule were suggested as putative ligands for P(A) in the RC I(L177)H+H(L173)L although this would imply a reorientation of the His backbone and additional rearrangements in the primary donor environment or even a repositioning of the BChl dimer. The crystal structure of the mutant I(L177)H reaction center determined to a resolution of 2.9Å shows changes at the interface region between the BChl P(A) and the monomeric BChl B(B). Spectral and pigment analysis provided evidence for β-coordination of the BChl B(B) in the double mutant RC I(L177)H+H(M182)L and for its hexacoordination in the mutant reaction center I(L177)H. Computer modeling suggests involvement of two water molecules in the β-coordination of the BChl B(B). Possible structural consequences of the L177 mutation affecting the coordination of the two BChls P(A) and B(B) are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

  18. TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia

    PubMed Central

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R.; Kantarjian, Hagop M.; Liang, Shoudan; Estecio, Marcos R.; Godley, Lucy A.; Issa, Jean-Pierre J.

    2015-01-01

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently-modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here we report on DNA methylomes in TET2 wild type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites (28,114 sites in CpG islands (CGIs) and 57,020 in non-CpG islands (NCGIs)). TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. By contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and non-promoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1, and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor binding sites. PMID:25972343

  19. Segregation of the fragile-X mutation from an affected male: Evidence of unusual somatic instability in the FMR-1 locus

    SciTech Connect

    Kambouris, M.; Bluhm, D.; Feldman, G.L.

    1994-09-01

    Fragile X syndrome is associated with an unstable CGG-repeat in the FMR-1 gene. There are few reports of affected males transmitting the FMR-1 gene to offspring. We report a family in which the paternal grandfather has an unusual FMR-1 pattern, with allele sizes ranging from premutation to full mutation. The family was initially ascertained because of a diagnosis of fragile X syndrome in this individual`s grandson. For Southern blot analyses, the samples were digested with Pst 1 and hybridized to the pE5.1 probe or digested with HindIII and hybridized to the StB12.3 probe. The proband had a high molecular weight allele, indicating significant amplifications, and an abnormal methylation pattern, consistent with a full mutation. His twin sister, who also had features of fragile X syndrome, had a similar pattern in addition to her normal allele (30 repeats). Their mother had one normal allele (33 repeats) and a premutation allele (>130 CGG repeats), with a normal methylation pattern. The maternal grandmother had alleles of 32 and 33 CGG repeats. These findings support the hypothesis that transmission of a full fragile-X mutation does not occur through a male, even if that male has clincial and molecular evidence of a full mutation. Gonadal mosaicism is an alternative explanation. Thus, an affected male with extensive FMR-1 somatic mosaicism transmitted a large premutation to his daughter, who in turn transmitted a full mutation to both of her offspring. FMR-1 protein studies on this individual, which are in progress, should help to determine the correlation, if any, of the molecular findings with the phenotypic effects.

  20. Arbuscular mycorrhizal symbiosis influences strigolactone production under salinity and alleviates salt stress in lettuce plants.

    PubMed

    Aroca, Ricardo; Ruiz-Lozano, Juan Manuel; Zamarreño, Angel María; Paz, José Antonio; García-Mina, José María; Pozo, María José; López-Ráez, Juan Antonio

    2013-01-01

    Arbuscular mycorrhizal (AM) symbiosis can alleviate salt stress in plants. However the intimate mechanisms involved, as well as the effect of salinity on the production of signalling molecules associated to the host plant-AM fungus interaction remains largely unknown. In the present work, we have investigated the effects of salinity on lettuce plant performance and production of strigolactones, and assessed its influence on mycorrhizal root colonization. Three different salt concentrations were applied to mycorrhizal and non-mycorrhizal plants, and their effects, over time, analyzed. Plant biomass, stomatal conductance, efficiency of photosystem II, as well as ABA content and strigolactone production were assessed. The expression of ABA biosynthesis genes was also analyzed. AM plants showed improved growth rates and a better performance of physiological parameters such as stomatal conductance and efficiency of photosystem II than non-mycorrhizal plants under salt stress since very early stages - 3 weeks - of plant colonization. Moreover, ABA levels were lower in those plants, suggesting that they were less stressed than non-colonized plants. On the other hand, we show that both AM symbiosis and salinity influence strigolactone production, although in a different way in AM and non-AM plants. The results suggest that AM symbiosis alleviates salt stress by altering the hormonal profiles and affecting plant physiology in the host plant. Moreover, a correlation between strigolactone production, ABA content, AM root colonization and salinity level is shown. We propose here that under these unfavourable conditions, plants increase strigolactone production in order to promote symbiosis establishment to cope with salt stress.

  1. Identification and Characterization of a Mutation Affecting the Division Arrest Signaling of the Pheromone Response Pathway in Saccharomyces Cerevisiae

    PubMed Central

    Fujimura, H. A.

    1990-01-01

    Mating pheromones, a- and α-factors, arrest the division of cells of opposite mating types, α and a cells, respectively. I have isolated a sterile mutant of Saccharomyces cerevisiae that is defective in division arrest in response to α-factor but not defective in morphological changes and agglutinin induction. The mutation was designated dac2 for division arrest control by mating pheromones. The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18 and dac1). Although dac2 cells had no phenotype in the absence of pheromones, they showed morphological alterations and divided continuously in the presence of pheromones. As a result, dac2 cells had a mating defect. The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene (previously shown to encode a protein with similarity to the α subunit of mammalian G proteins). In addition, dac2 cells formed prezygotes with wild-type cells of opposite mating types, although they could not undergo cell fusion. These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation. PMID:2407613

  2. Differential secretion of the mutated protein is a major component affecting phenotypic severity in CRLF1-associated disorders

    PubMed Central

    Herholz, Jana; Meloni, Alessandra; Marongiu, Mara; Chiappe, Francesca; Deiana, Manila; Herrero, Carmen Roche; Zampino, Giuseppe; Hamamy, Hanan; Zalloum, Yusra; Waaler, Per Erik; Crisponi, Giangiorgio; Crisponi, Laura; Rutsch, Frank

    2011-01-01

    Crisponi syndrome (CS) and cold-induced sweating syndrome type 1 (CISS1) are disorders caused by mutations in CRLF1. The two syndromes share clinical characteristics, such as dysmorphic features, muscle contractions, scoliosis and cold-induced sweating, with CS patients showing a severe clinical course in infancy involving hyperthermia, associated with death in most cases in the first years of life. To evaluate a potential genotype/phenotype correlation and whether CS and CISS1 represent two allelic diseases or manifestations at different ages of the same disorder, we carried out a detailed clinical analysis of 19 patients carrying mutations in CRLF1. We studied the functional significance of the mutations found in CRLF1, providing evidence that phenotypic severity of the two disorders mainly depends on altered kinetics of secretion of the mutated CRLF1 protein. On the basis of these findings, we believe that the two syndromes, CS and CISS1, represent manifestations of the same disorder, with different degrees of severity. We suggest renaming the two genetic entities CS and CISS1 with the broader term of Sohar–Crisponi syndrome. PMID:21326283

  3. The hunt for a functional mutation affecting conformation and calving traits on chromosome 18 in Holstein cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequence data from 11 US Holstein bulls were analyzed to identify putative causal mutations associated with calving and conformation traits. The SNP ARS-BFGL-NGS-109285 at 57,589,121 bp (UMD 3.1 assembly) on BTA18 has large effects on 4 measures of body shape and size, 2 measures of dystocia, longev...

  4. Mutations Affecting Starch Synthase III in Arabidopsis Alter Leaf Starch Structure and Increase the Rate of Starch Synthesis1

    PubMed Central

    Zhang, Xiaoli; Myers, Alan M.; James, Martha G.

    2005-01-01

    The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis. PMID:15908598

  5. Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1.

    PubMed Central

    Clark, R M; Marker, P C; Roessler, E; Dutra, A; Schimenti, J C; Muenke, M; Kingsley, D M

    2001-01-01

    The major locus for dominant preaxial polydactyly in humans has been mapped to 7q36. In mice the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) mutations map to a homologous chromosomal region and cause similar limb defects. The Lmbr1 gene is entirely within the small critical intervals recently defined for both the mouse and human mutations and is misexpressed at the exact time that the mouse Hx phenotype becomes apparent during limb development. This result suggests that Lmbr1 may underlie preaxial polydactyly in both mice and humans. We have used deletion chromosomes to demonstrate that the dominant mouse and human limb defects arise from gain-of-function mutations and not from haploinsufficiency. Furthermore, we created a loss-of-function mutation in the mouse Lmbr1 gene that causes digit number reduction (oligodactyly) on its own and in trans to a deletion chromosome. The loss of digits that we observed in mice with reduced Lmbr1 activity is in contrast to the gain of digits observed in Hx mice and human polydactyly patients. Our results suggest that the Lmbr1 gene is required for limb formation and that reciprocal changes in levels of Lmbr1 activity can lead to either increases or decreases in the number of digits in the vertebrate limb. PMID:11606546

  6. The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger.

    PubMed Central

    Kudla, B; Caddick, M X; Langdon, T; Martinez-Rossi, N M; Bennett, C F; Sibley, S; Davies, R W; Arst, H N

    1990-01-01

    The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans has been sequenced and its transcript mapped and orientated. A single ORF can encode a protein of 719 amino acids. A 52 amino acid region including a putative 'zinc finger' strongly resembles putative DNA binding regions of the major regulatory protein of erythroid cells. The derived protein sequence also contains a highly acidic region possibly involved in gene activation and 22 copies of the motif S(T)PXX, abundant in DNA binding proteins. Analysis of chromosomal rearrangements and transformation with deletion clones identified 342 N-terminal and 124 C-terminal residues as inessential and localized a C-terminal region required for nitrogen metabolite repressibility. A -1 frameshift eliminating the inessential 122 C-terminal amino acids is a surprising loss-of-function mutation. Extraordinary basicity of the replacement C terminus might explain its phenotype. Mutant sequencing also identified a polypeptide chain termination and several missense mutations, but most interesting are sequence changes associated with specificity mutations. A mutation elevating expression of some structural genes under areA control whilst reducing or not affecting expression of others is a leucine to valine change in the zinc finger loop. It reverts to a partly reciprocal phenotype by replacing the mutant valine by methionine. Images Fig.2 Fig.4 Fig.5 Fig. 8. Fig. 9. PMID:1970293

  7. Exome and deep sequencing of clinically aggressive neuroblastoma reveal somatic mutations that affect key pathways involved in cancer progression

    PubMed Central

    Lasorsa, Vito Alessandro; Formicola, Daniela; Pignataro, Piero; Cimmino, Flora; Calabrese, Francesco Maria; Mora, Jaume; Esposito, Maria Rosaria; Pantile, Marcella; Zanon, Carlo; De Mariano, Marilena; Longo, Luca; Hogarty, Michael D.; de Torres, Carmen; Tonini, Gian Paolo; Iolascon, Achille; Capasso, Mario

    2016-01-01

    The spectrum of somatic mutation of the most aggressive forms of neuroblastoma is not completely determined. We sought to identify potential cancer drivers in clinically aggressive neuroblastoma. Whole exome sequencing was conducted on 17 germline and tumor DNA samples from high-risk patients with adverse events within 36 months from diagnosis (HR-Event3) to identify somatic mutations and deep targeted sequencing of 134 genes selected from the initial screening in additional 48 germline and tumor pairs (62.5% HR-Event3 and high-risk patients), 17 HR-Event3 tumors and 17 human-derived neuroblastoma cell lines. We revealed 22 significantly mutated genes, many of which implicated in cancer progression. Fifteen genes (68.2%) were highly expressed in neuroblastoma supporting their involvement in the disease. CHD9, a cancer driver gene, was the most significantly altered (4.0% of cases) after ALK. Other genes (PTK2, NAV3, NAV1, FZD1 and ATRX), expressed in neuroblastoma and involved in cell invasion and migration were mutated at frequency ranged from 4% to 2%. Focal adhesion and regulation of actin cytoskeleton pathways, were frequently disrupted (14.1% of cases) thus suggesting potential novel therapeutic strategies to prevent disease progression. Notably BARD1, CHEK2 and AXIN2 were enriched in rare, potentially pathogenic, germline variants. In summary, whole exome and deep targeted sequencing identified novel cancer genes of clinically aggressive neuroblastoma. Our analyses show pathway-level implications of infrequently mutated genes in leading neuroblastoma progression. PMID:27009842

  8. Study of cnidarian-algal symbiosis in the "omics" age.

    PubMed

    Meyer, Eli; Weis, Virginia M

    2012-08-01

    The symbiotic associations between cnidarians and dinoflagellate algae (Symbiodinium) support productive and diverse ecosystems in coral reefs. Many aspects of this association, including the mechanistic basis of host-symbiont recognition and metabolic interaction, remain poorly understood. The first completed genome sequence for a symbiotic anthozoan is now available (the coral Acropora digitifera), and extensive expressed sequence tag resources are available for a variety of other symbiotic corals and anemones. These resources make it possible to profile gene expression, protein abundance, and protein localization associated with the symbiotic state. Here we review the history of "omics" studies of cnidarian-algal symbiosis and the current availability of sequence resources for corals and anemones, identifying genes putatively involved in symbiosis across 10 anthozoan species. The public availability of candidate symbiosis-associated genes leaves the field of cnidarian-algal symbiosis poised for in-depth comparative studies of sequence diversity and gene expression and for targeted functional studies of genes associated with symbiosis. Reviewing the progress to date suggests directions for future investigations of cnidarian-algal symbiosis that include (i) sequencing of Symbiodinium, (ii) proteomic analysis of the symbiosome membrane complex, (iii) glycomic analysis of Symbiodinium cell surfaces, and (iv) expression profiling of the gastrodermal cells hosting Symbiodinium.

  9. NIN Is Involved in the Regulation of Arbuscular Mycorrhizal Symbiosis.

    PubMed

    Guillotin, Bruno; Couzigou, Jean-Malo; Combier, Jean-Philippe

    2016-01-01

    Arbuscular mycorrhizal (AM) symbiosis is an intimate and ancient symbiosis found between most of terrestrial plants and fungi from the Glomeromycota family. Later during evolution, the establishment of the nodulation between legume plants and soil bacteria known as rhizobia, involved several genes of the signaling pathway previously implicated for AM symbiosis. For the past years, the identification of the genes belonging to this Common Symbiotic Signaling Pathway have been mostly done on nodulation. Among the different genes already well identified as required for nodulation, we focused our attention on the involvement of Nodule Inception (NIN) in AM symbiosis. We show here that NIN expression is induced during AM symbiosis, and that the Medicago truncatula nin mutant is less colonized than the wild-type M. truncatula strain. Moreover, nin mutant displays a defect in the ability to be infected by the fungus Rhizophagus irregularis. This work brings a new evidence of the common genes involved in overlapping signaling pathways of both nodulation and in AM symbiosis.

  10. NIN Is Involved in the Regulation of Arbuscular Mycorrhizal Symbiosis

    PubMed Central

    Guillotin, Bruno; Couzigou, Jean-Malo; Combier, Jean-Philippe

    2016-01-01

    Arbuscular mycorrhizal (AM) symbiosis is an intimate and ancient symbiosis found between most of terrestrial plants and fungi from the Glomeromycota family. Later during evolution, the establishment of the nodulation between legume plants and soil bacteria known as rhizobia, involved several genes of the signaling pathway previously implicated for AM symbiosis. For the past years, the identification of the genes belonging to this Common Symbiotic Signaling Pathway have been mostly done on nodulation. Among the different genes already well identified as required for nodulation, we focused our attention on the involvement of Nodule Inception (NIN) in AM symbiosis. We show here that NIN expression is induced during AM symbiosis, and that the Medicago truncatula nin mutant is less colonized than the wild-type M. truncatula strain. Moreover, nin mutant displays a defect in the ability to be infected by the fungus Rhizophagus irregularis. This work brings a new evidence of the common genes involved in overlapping signaling pathways of both nodulation and in AM symbiosis. PMID:27899928

  11. HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice

    SciTech Connect

    Hanna, Zaher . E-mail: Zaher.Hanna@ircm.qc.ca; Priceputu, Elena; Hu, Chunyan; Vincent, Patrick; Jolicoeur, Paul

    2006-03-01

    pathologies) in respectively Nef{sup RD35/36AA} and Nef{sup D174K} Tg mice, relative to those developing in Nef{sup Wt} Tg mice. Our data suggest that the RD35/36AA and D174K mutations affect other Nef functions, namely those involved in the development of lung and kidney diseases, in addition to their known role in CD4 downregulation. Similarly, in HIV-1-infected individuals, loss of CD4 downregulation by Nef alleles may reflect their lower intrinsic pathogenicity, independently of their effects on virus replication.

  12. HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice.

    PubMed

    Hanna, Zaher; Priceputu, Elena; Hu, Chunyan; Vincent, Patrick; Jolicoeur, Paul

    2006-03-01

    mice. Our data suggest that the RD35/36AA and D174K mutations affect other Nef functions, namely those involved in the development of lung and kidney diseases, in addition to their known role in CD4 downregulation. Similarly, in HIV-1-infected individuals, loss of CD4 downregulation by Nef alleles may reflect their lower intrinsic pathogenicity, independently of their effects on virus replication.

  13. Spinocerebellar ataxia-13 Kv3.3 potassium channels: arginine-to-histidine mutations affect both functional and protein expression on the cell surface.

    PubMed

    Zhao, Jian; Zhu, Jing; Thornhill, William B

    2013-09-01

    The voltage-gated potassium channel Kv3.3 is the causative gene of SCA13 (spinocerebellar ataxia type 13), an autosomal dominant neurological disorder. The four dominant mutations identified to date cause Kv3.3 channels to be non-functional or have altered gating properties in Xenopus oocytes. In the present paper, we report that SCA13 mutations affect functional as well as protein expression of Kv3.3 channels in a mammalian cell line. The reduced protein level of SCA13 mutants is caused by a shorter protein half-life, and blocking the ubiquitin-proteasome pathway increases the total protein of SCA13 mutants more than wild-type. SCA13 mutated amino acids are highly conserved, and the side chains of these residues play a critical role in the stable expression of Kv3.3 proteins. In addition, we show that mutant Kv3.3 protein levels could be partially rescued by treatment with the chemical chaperone TMAO (trimethylamine N-oxide) and to a lesser extent with co-expression of Kv3.1b. Thus our results suggest that amino acid side chains of SCA13 positions affect the protein half-life and/or function of Kv3.3, and the adverse effect on protein expression cannot be fully rescued.

  14. Detection of a novel mutation in the ryanodine receptor gene in an Irish malignant hyperthermia pedigree: correlation of the IVCT response with the affected and unaffected haplotypes.

    PubMed Central

    Keating, K E; Giblin, L; Lynch, P J; Quane, K A; Lehane, M; Heffron, J J; McCarthy, T V

    1997-01-01

    Defects in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia (MH), an autosomal dominant disorder of skeletal muscle and one of the main causes of death resulting from anaesthesia. Susceptibility to MH (MHS) is determined by the level of tension generated in an in vitro muscle contracture test (IVCT) in response to caffeine and halothane. To date, mutation screening of the RYR1 gene in MH families has led to the identification of eight mutations. We describe here the identification of a novel mutation, Arg552Trp, in the RYR1 gene, which is clearly linked to the MHS phenotype in a large, well characterised Irish pedigree. Considering that the RYR1 protein functions as a tetramer, correlation of the IVCT with the affected and unaffected haplotypes was performed on the pedigree to investigate if the normal RYR1 allele in affected subjects contributes to the variation in the IVCT. The results show that the normal RYR1 allele is unlikely to play a role in IVCT variation. Images PMID:9138151

  15. MECP2e1 isoform mutation affects the form and function of neurons derived from Rett syndrome patient iPS cells.

    PubMed

    Djuric, Ugljesa; Cheung, Aaron Y L; Zhang, Wenbo; Mok, Rebecca S; Lai, Wesley; Piekna, Alina; Hendry, Jason A; Ross, P Joel; Pasceri, Peter; Kim, Dae-Sung; Salter, Michael W; Ellis, James

    2015-04-01

    MECP2 mutations cause the X-linked neurodevelopmental disorder Rett Syndrome (RTT) by consistently altering the protein encoded by the MECP2e1 alternative transcript. While mutations that simultaneously affect both MECP2e1 and MECP2e2 isoforms have been widely studied, the consequence of MECP2e1 deficiency on human neurons remains unknown. Here we report the first isoform-specific patient induced pluripotent stem cell (iPSC) model of RTT. RTTe1 patient iPS cell-derived neurons retain an inactive X-chromosome and express only the mutant allele. Single-cell mRNA analysis demonstrated they have a molecular signature of cortical neurons. Mutant neurons exhibited a decrease in soma size, reduced dendritic complexity and decreased cell capacitance, consistent with impaired neuronal maturation. The soma size phenotype was rescued cell-autonomously by MECP2e1 transduction in a level-dependent manner but not by MECP2e2 gene transfer. Importantly, MECP2e1 mutant neurons showed a dysfunction in action potential generation, voltage-gated Na(+) currents, and miniature excitatory synaptic current frequency and amplitude. We conclude that MECP2e1 mutation affects soma size, information encoding properties and synaptic connectivity in human neurons that are defective in RTT.

  16. Iron: an essential micronutrient for the legume-rhizobium symbiosis

    PubMed Central

    Brear, Ella M.; Day, David A.; Smith, Penelope M. C.

    2013-01-01

    Legumes, which develop a symbiosis with nitrogen-fixing bacteria, have an increased demand for iron. Iron is required for the synthesis of iron-containing proteins in the host, including the highly abundant leghemoglobin, and in bacteroids for nitrogenase and cytochromes of the electron transport chain. Deficiencies in iron can affect initiation and development of the nodule. Within root cells, iron is chelated with organic acids such as citrate and nicotianamine and distributed to other parts of the plant. Transport to the nitrogen-fixing bacteroids in infected cells of nodules is more complicated. Formation of the symbiosis results in bacteroids internalized within root cortical cells of the legume where they are surrounded by a plant-derived membrane termed the symbiosome membrane (SM). This membrane forms an interface that regulates nutrient supply to the bacteroid. Consequently, iron must cross this membrane before being supplied to the bacteroid. Iron is transported across the SM as both ferric and ferrous iron. However, uptake of Fe(II) by both the symbiosome and bacteroid is faster than Fe(III) uptake. Members of more than one protein family may be responsible for Fe(II) transport across the SM. The only Fe(II) transporter in nodules characterized to date is GmDMT1 (Glycine max divalent metal transporter 1), which is located on the SM in soybean. Like the root plasma membrane, the SM has ferric iron reductase activity. The protein responsible has not been identified but is predicted to reduce ferric iron accumulated in the symbiosome space prior to uptake by the bacteroid. With the recent publication of a number of legume genomes including Medicago truncatula and G. max, a large number of additional candidate transport proteins have been identified. Members of the NRAMP (natural resistance-associated macrophage protein), YSL (yellow stripe-like), VIT (vacuolar iron transporter), and ZIP (Zrt-, Irt-like protein) transport families show enhanced expression in

  17. Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

    PubMed Central

    Twigg, Stephen R.F.; Babbs, Christian; van den Elzen, Marijke E.P.; Goriely, Anne; Taylor, Stephen; McGowan, Simon J.; Giannoulatou, Eleni; Lonie, Lorne; Ragoussis, Jiannis; Akha, Elham Sadighi; Knight, Samantha J.L.; Zechi-Ceide, Roseli M.; Hoogeboom, Jeannette A.M.; Pober, Barbara R.; Toriello, Helga V.; Wall, Steven A.; Rita Passos-Bueno, M.; Brunner, Han G.; Mathijssen, Irene M.J.; Wilkie, Andrew O.M.

    2013-01-01

    Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries—a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5′ untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5′ UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females. PMID:23335590

  18. Mutations associated with Dent's disease affect gating and voltage dependence of the human anion/proton exchanger ClC-5.

    PubMed

    Alekov, Alexi K

    2015-01-01

    Dent's disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5; however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent's disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 toward more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function.

  19. Characterization of a Spontaneous Novel Mutation in the NPC2 Gene in a Cat Affected by Niemann Pick Type C Disease

    PubMed Central

    Zampieri, Stefania; Bianchi, Ezio; Cantile, Carlo; Saleri, Roberta; Bembi, Bruno; Dardis, Andrea

    2014-01-01

    Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids within the lysosomes due to mutation in NPC1 or NPC2 genes. A feline model of NPC carrying a mutation in NPC1 gene has been previously described. We have identified two kittens affected by NPC disease due to a mutation in NPC2 gene. They manifested with tremors at the age of 3 months, which progressed to dystonia and severe ataxia. At 6 months of age cat 2 was unable to stand without assistance and had bilaterally reduced menace response. It died at the age of 10 months. Post-mortem histological analysis of the brain showed the presence of neurons with cytoplasmic swelling and vacuoles, gliosis of the substantia nigra and degeneration of the white matter. Spheroids with accumulation of ubiquitinated aggregates were prominent in the cerebellar cortex. Purkinje cells were markedly reduced in number and they showed prominent intracytoplasmic storage. Scattered perivascular aggregates of lymphocytes and microglial cells proliferation were present in the thalamus and midbrain. Proliferation of Bergmann glia was also observed. In the liver, hepatocytes were swollen because of accumulation of small vacuoles and foamy Kupffer cells were also detected. Foamy macrophages were observed within the pulmonary interstitium and alveoli as well. At 9 months cat 1 was unable to walk, developed seizures and it was euthanized at 21 months. Filipin staining of cultured fibroblasts showed massive storage of unesterified cholesterol. Molecular analysis of NPC1 and NPC2 genes showed the presence of a homozygous intronic mutation (c.82+5G>A) in the NPC2 gene. The subsequent analysis of the mRNA showed that the mutation causes the retention of 105 bp in the mature mRNA, which leads to the in frame insertion of 35 amino acids between residues 28 and 29 of NPC2 protein (p.G28_S29ins35). PMID:25396745

  20. Cyclophilin and the regulation of symbiosis in Aiptasia pallida.

    PubMed

    Perez, S; Weis, V

    2008-08-01

    The sea anemone Aiptasia pallida, symbiotic with intracellular dinoflagellates, expresses a peptydyl-prolyl cis-trans isomerase (PPIase) belonging to the conserved family of cytosolic cyclophilins (ApCypA). Protein extracts from A. pallida exhibited PPIase activity. Given the high degree of conservation of ApCypA and its known function in the cellular stress response, we hypothesized that it plays a similar role in the cnidarian-dinoflagellate symbiosis. To explore its role, we inhibited the activity of cyclophilin with cyclosporin A (CsA). CsA effectively inhibited the PPIase activity of protein extracts from symbiotic A. pallida. CsA also induced the dose-dependent release of symbiotic algae from host tissues (bleaching). Laser scanning confocal microscopy using superoxide and nitric oxide-sensitive fluorescent dyes on live specimens of A. pallida revealed that CsA strongly induced the production of these known mediators of bleaching. We tested whether the CsA-sensitive isomerase activity is important for maintaining the activity of the antioxidant enzyme superoxide dismutase (SOD). SOD activity of protein extracts was not affected by pre-incubation with CsA in vitro.

  1. Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant

    PubMed Central

    Durand, Adeline

    2016-01-01

    Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo. PMID:27280472

  2. Stars and symbiosis: microRNA- and microRNA*-mediated transcript cleavage involved in arbuscular mycorrhizal symbiosis.

    PubMed

    Devers, Emanuel A; Branscheid, Anja; May, Patrick; Krajinski, Franziska

    2011-08-01

    The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out high-throughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development.

  3. The diageotropica mutation and synthetic auxins differentially affect the expression of auxin-regulated genes in tomato.

    PubMed Central

    Mito, N; Bennett, A B

    1995-01-01

    The effect of a tomato (Lycopersicon esculentum) mutation, diageotropica (dgt), on the accumulation of mRNA corresponding to tomato homologs of three auxin-regulated genes, LeAux, LeSAUR, and Lepar, was examined. The dgt mutation inhibited the induction of LeAux and LeSAUR mRNA accumulation by naphthalene acetic acid (NAA) but had no effect on NAA-induced Lepar mRNA accumulation. The effect of two synthetic auxins, NAA and 3,7-dichloro-8-quinoline carboxylic acid (quinclorac), on the accumulation of LeAux, LeSAUR, and Lepar mRNA was also examined. Quinclorac induced the expression of each of the auxin-regulated genes, confirming its proposed mode of herbicidal action as an auxin-type herbicide. Concentrations of quinclorac at least 100-fold higher than NAA were required to induce LeAux and LeSAUR mRNA accumulation to similar levels, whereas Lepar mRNA accumulation was induced by similar concentrations of NAA and quinclorac. Collectively, these data suggest the presence of two auxin-dependent signal transduction pathways: one that regulates LeSAUR and LeAux mRNA accumulation and is interrupted by the dgt mutation and a second that regulates Lepar mRNA accumulation and is not defective in dgt tomato hypocotyls. These two auxin-regulated signal transduction pathways can be further discriminated by the action of two synthetic auxins, NAA and quinclorac. PMID:7480327

  4. CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells.

    PubMed

    Tikka, Saara; Ng, Yan Peng; Di Maio, Giuseppe; Mykkänen, Kati; Siitonen, Maija; Lepikhova, Tatiana; Pöyhönen, Minna; Viitanen, Matti; Virtanen, Ismo; Kalimo, Hannu; Baumann, Marc

    2012-12-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia caused by mutations in NOTCH3 gene. Pathology is manifested in small- and middle-sized arteries throughout the body, though primarily in cerebral white matter. Hemodynamics is altered in CADASIL and NOTCH3 is suggested to regulate actin filament polymerization and thereby vascular tone. We analyzed NOTCH3 expression and morphology of actin cytoskeleton in genetically genuine cultured human CADASIL vascular smooth muscle cells (VSMCs) (including a cell line homozygous for p.Arg133Cys mutation) derived from different organs, and in control VSMCs with short hairpin RNA (shRNA)-silenced NOTCH3. NOTCH3 protein level was higher in VSMCs derived from adult than newborn arteries in both CADASIL and control VSMCs. CADASIL VSMCs showed altered actin cytoskeleton including increased branching and node formation, and more numerous and smaller adhesion sites than control VSMCs. Alterations in actin cytoskeleton in shRNA-silenced VSMCs were similar as in CADASIL VSMCs. Severity of the alterations in actin filaments corresponded to NOTCH3 expression level being most severe in VSMCs derived from adult cerebral arteries. These observations suggest that hypomorphic NOTCH3 activity causes alterations in actin organization in CADASIL. Furthermore, arteries from different organs have specific characteristics, which modify the effects of the NOTCH3 mutation and which is one explanation for the exceptional susceptibility of cerebral white matter arteries.

  5. Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila

    PubMed Central

    1992-01-01

    We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2. PMID:1469046

  6. A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis

    PubMed Central

    Gillmor, C. Stewart; Roeder, Adrienne H. K.; Sieber, Patrick; Somerville, Chris; Lukowitz, Wolfgang

    2016-01-01

    Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms. PMID:26745275

  7. The ataxia related G1107D mutation of the plasma membrane Ca(2+) ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process.

    PubMed

    Calì, Tito; Frizzarin, Martina; Luoni, Laura; Zonta, Francesco; Pantano, Sergio; Cruz, Carlos; Bonza, Maria Cristina; Bertipaglia, Ilenia; Ruzzene, Maria; De Michelis, Maria Ida; Damiano, Nunzio; Marin, Oriano; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Lopreiato, Raffaele; Carafoli, Ernesto

    2017-01-01

    The plasma membrane Ca(2+) ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca(2+)), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca(2+)), Ca(2+)-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca(2+) in the activated state, and the autoinhibition mechanism in its resting state.

  8. Academia-industry symbiosis in organic chemistry.

    PubMed

    Michaudel, Quentin; Ishihara, Yoshihiro; Baran, Phil S

    2015-03-17

    Collaboration between academia and industry is a growing phenomenon within the chemistry community. These sectors have long held strong ties since academia traditionally trains the future scientists of the corporate world, but the recent drastic decrease of public funding is motivating the academic world to seek more private grants. This concept of industrial "sponsoring" is not new, and in the past, some companies granted substantial amounts of money per annum to various academic institutions in exchange for prime access to all their scientific discoveries and inventions. However, academic and industrial interests were not always aligned, and therefore the investment has become increasingly difficult to justify from industry's point of view. With fluctuating macroeconomic factors, this type of unrestricted grant has become more rare and has been largely replaced by smaller and more focused partnerships. In our view, forging a partnership with industry can be a golden opportunity for both parties and can represent a true symbiosis. This type of project-specific collaboration is engendered by industry's desire to access very specific academic expertise that is required for the development of new technologies at the forefront of science. Since financial pressures do not allow companies to spend the time to acquire this expertise and even less to explore fundamental research, partnering with an academic laboratory whose research is related to the problem gives them a viable alternative. From an academic standpoint, it represents the perfect occasion to apply "pure science" research concepts to solve problems that benefit humanity. Moreover, it offers a unique opportunity for students to face challenges from the "real world" at an early stage of their career. Although not every problem in industry can be solved by research developments in academia, we argue that there is significant scientific overlap between these two seemingly disparate groups, thereby presenting an

  9. Academia–Industry Symbiosis in Organic Chemistry

    PubMed Central

    2015-01-01

    Conspectus Collaboration between academia and industry is a growing phenomenon within the chemistry community. These sectors have long held strong ties since academia traditionally trains the future scientists of the corporate world, but the recent drastic decrease of public funding is motivating the academic world to seek more private grants. This concept of industrial “sponsoring” is not new, and in the past, some companies granted substantial amounts of money per annum to various academic institutions in exchange for prime access to all their scientific discoveries and inventions. However, academic and industrial interests were not always aligned, and therefore the investment has become increasingly difficult to justify from industry’s point of view. With fluctuating macroeconomic factors, this type of unrestricted grant has become more rare and has been largely replaced by smaller and more focused partnerships. In our view, forging a partnership with industry can be a golden opportunity for both parties and can represent a true symbiosis. This type of project-specific collaboration is engendered by industry’s desire to access very specific academic expertise that is required for the development of new technologies at the forefront of science. Since financial pressures do not allow companies to spend the time to acquire this expertise and even less to explore fundamental research, partnering with an academic laboratory whose research is related to the problem gives them a viable alternative. From an academic standpoint, it represents the perfect occasion to apply “pure science” research concepts to solve problems that benefit humanity. Moreover, it offers a unique opportunity for students to face challenges from the “real world” at an early stage of their career. Although not every problem in industry can be solved by research developments in academia, we argue that there is significant scientific overlap between these two seemingly disparate

  10. Identification of Genetic Loci Affecting the Severity of Symptoms of Hirschsprung Disease in Rats Carrying Ednrbsl Mutations by Quantitative Trait Locus Analysis

    PubMed Central

    Torigoe, Daisuke; Lei, Chuzhao; Lan, Xianyong; Chen, Hong; Sasaki, Nobuya; Wang, Jinxi; Agui, Takashi

    2015-01-01

    Hirschsprung’s disease (HSCR) is a congenital disease in neonates characterized by the absence of the enteric ganglia in a variable length of the distal colon. This disease results from multiple genetic interactions that modulate the ability of enteric neural crest cells to populate developing gut. We previously reported that three rat strains with different backgrounds (susceptible AGH-Ednrbsl/sl, resistant F344-Ednrbsl/sl, and LEH-Ednrbsl/sl) but the same null mutation of Ednrb show varying severity degrees of aganglionosis. This finding suggests that strain-specific genetic factors affect the severity of HSCR. Consistent with this finding, a quantitative trait locus (QTL) for the severity of HSCR on chromosome (Chr) 2 was identified using an F2 intercross between AGH and F344 strains. In the present study, we performed QTL analysis using an F2 intercross between the susceptible AGH and resistant LEH strains to identify the modifier/resistant loci for HSCR in Ednrb-deficient rats. A significant locus affecting the severity of HSCR was also detected within the Chr 2 region. These findings strongly suggest that a modifier gene of aganglionosis exists on Chr 2. In addition, two potentially causative SNPs (or mutations) were detected upstream of a known HSCR susceptibility gene, Gdnf. These SNPs were possibly responsible for the varied length of gut affected by aganglionosis. PMID:25790447

  11. Immunosuppression during Rhizobium-legume symbiosis.

    PubMed

    Luo, Li; Lu, Dawei

    2014-01-01

    Rhizobium infects host legumes to elicit new plant organs, nodules where dinitrogen is fixed as ammonia that can be directly utilized by plants. The nodulation factor (NF) produced by Rhizobium is one of the determinant signals for rhizobial infection and nodule development. Recently, it was found to suppress the innate immunity on host and nonhost plants as well as its analogs, chitins. Therefore, NF can be recognized as a microbe/pathogen-associated molecular pattern (M/PAMP) like chitin to induce the M/PAMP triggered susceptibility (M/PTS) of host plants to rhizobia. Whether the NF signaling pathway is directly associated with the innate immunity is not clear till now. In fact, other MAMPs such as lipopolysaccharide (LPS), exopolysaccharide (EPS) and cyclic-β-glucan, together with type III secretion system (T3SS) effectors are also required for rhizobial infection or survival in leguminous nodule cells. Interestingly, most of them play similarly negative roles in the innate immunity of host plants, though their signaling is not completely elucidated. Taken together, we believe that the local immunosuppression on host plants induced by Rhizobium is essential for the establishment of their symbiosis.

  12. Phylogeny, genomics, and symbiosis of Photobacterium.

    PubMed

    Urbanczyk, Henryk; Ast, Jennifer C; Dunlap, Paul V

    2011-03-01

    Photobacterium comprises several species in Vibrionaceae, a large family of Gram-negative, facultatively aerobic, bacteria that commonly associate with marine animals. Members of the genus are widely distributed in the marine environment and occur in seawater, surfaces, and intestines of marine animals, marine sediments and saline lake water, and light organs of fish. Seven Photobacterium species are luminous via the activity of the lux genes, luxCDABEG. Much recent progress has been made on the phylogeny, genomics, and symbiosis of Photobacterium. Phylogenetic analysis demonstrates a robust separation between Photobacterium and its close relatives, Aliivibrio and Vibrio, and reveals the presence of two well-supported clades. Clade 1 contains luminous and symbiotic species and one species with no luminous members, and Clade 2 contains mostly nonluminous species. The genomes of Photobacterium are similar in size, structure, and organization to other members of Vibrionaceae, with two chromosomes of unequal size and multiple rrn operons. Many species of marine fish form bioluminescent symbioses with three Photobacterium species: Photobacterium kishitanii, Photobacterium leiognathi, and Photobacterium mandapamensis. These associations are highly, but not strictly species specific, and they do not exhibit symbiont-host codivergence. Environmental congruence instead of host selection might explain the patterns of symbiont-host affiliation observed from nature.

  13. Bacterial communities associated with the lichen symbiosis.

    PubMed

    Bates, Scott T; Cropsey, Garrett W G; Caporaso, J Gregory; Knight, Rob; Fierer, Noah

    2011-02-01

    Lichens are commonly described as a mutualistic symbiosis between fungi and "algae" (Chlorophyta or Cyanobacteria); however, they also have internal bacterial communities. Recent research suggests that lichen-associated microbes are an integral component of lichen thalli and that the classical view of this symbiotic relationship should be expanded to include bacteria. However, we still have a limited understanding of the phylogenetic structure of these communities and their variability across lichen species. To address these knowledge gaps, we used bar-coded pyrosequencing to survey the bacterial communities associated with lichens. Bacterial sequences obtained from four lichen species at multiple locations on rock outcrops suggested that each lichen species harbored a distinct community and that all communities were dominated by Alphaproteobacteria. Across all samples, we recovered numerous bacterial phylotypes that were closely related to sequences isolated from lichens in prior investigations, including those from a lichen-associated Rhizobiales lineage (LAR1; putative N(2) fixers). LAR1-related phylotypes were relatively abundant and were found in all four lichen species, and many sequences closely related to other known N(2) fixers (e.g., Azospirillum, Bradyrhizobium, and Frankia) were recovered. Our findings confirm the presence of highly structured bacterial communities within lichens and provide additional evidence that these bacteria may serve distinct functional roles within lichen symbioses.

  14. Mutations affecting the development of the peripheral nervous system in Drosophila: a molecular screen for novel proteins.

    PubMed Central

    Prokopenko, S N; He, Y; Lu, Y; Bellen, H J

    2000-01-01

    In our quest for novel genes required for the development of the embryonic peripheral nervous system (PNS), we have performed three genetic screens using MAb 22C10 as a marker of terminally differentiated neurons. A total of 66 essential genes required for normal PNS development were identified, including 49 novel genes. To obtain information about the molecular nature of these genes, we decided to complement our genetic screens with a molecular screen. From transposon-tagged mutations identified on the basis of their phenotype in the PNS we selected 31 P-element strains representing 26 complementation groups on the second and third chromosomes to clone and sequence the corresponding genes. We used plasmid rescue to isolate and sequence 51 genomic fragments flanking the sites of these P-element insertions. Database searches using sequences derived from the ends of plasmid rescues allowed us to assign genes to one of four classes: (1) previously characterized genes (11), (2) first mutations in cloned genes (1), (3) P-element insertions in genes that were identified, but not characterized molecularly (1), and (4) novel genes (13). Here, we report the cloning, sequence, Northern analysis, and the embryonic expression pattern of candidate cDNAs for 10 genes: astray, chrowded, dalmatian, gluon, hoi-polloi, melted, pebble, skittles, sticky ch1, and vegetable. This study allows us to draw conclusions about the identity of proteins required for the development of the nervous system in Drosophila and provides an example of a molecular approach to characterize en masse transposon-tagged mutations identified in genetic screens. PMID:11102367

  15. Sweat chloride and immunoreactive trypsinogen in infants carrying two CFTR mutations and not affected by cystic fibrosis.

    PubMed

    Castellani, Carlo; Tridello, Gloria; Tamanini, Anna; Assael, Baroukh M

    2016-01-11

    Newborns with raised immunotrypsinogen levels who have non-pathological sweat chloride values and carry two cystic fibrosis transmembrane regulator (CFTR) mutations of which at least one is not acknowledged to be cystic fibrosis (CF)-causing are at risk of developing clinical manifestations consistent with CFTR-related disorders or even CF. It is not known whether newborns with similar genotypes and normal immunoreactive trypsinogen (IRT) may share the same risk. This study found that newborns with these characteristics and normal IRT have lower sweat chloride values than those with raised IRT (p=0.007).

  16. Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions

    PubMed Central

    Sohn, Mira; Ivanova, Pavlina; Brown, H. Alex; Varnai, Peter; Kim, Yeun Ju; Balla, Tamas

    2016-01-01

    Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in the PTDSS1 gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein–related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism. PMID:27044099

  17. Parathyroid Hormone Receptor Type 1/Indian Hedgehog Expression Is Preserved in the Growth Plate of Human Fetuses Affected with Fibroblast Growth Factor Receptor Type 3 Activating Mutations

    PubMed Central

    Cormier, Sarah; Delezoide, Anne-Lise; Benoist-Lasselin, Catherine; Legeai-Mallet, Laurence; Bonaventure, Jacky; Silve, Caroline

    2002-01-01

    The fibroblast growth factor receptor type 3 (FGFR3) and Indian hedgehog (IHH)/parathyroid hormone (PTH)/PTH-related peptide receptor type 1 (PTHR1) systems are both essential regulators of endochondral ossification. Based on mouse models, activation of the FGFR3 system is suggested to regulate the IHH/PTHR1 pathway. To challenge this possible interaction in humans, we analyzed the femoral growth plates from fetuses carrying activating FGFR3 mutations (9 achondroplasia, 21 and 8 thanatophoric dysplasia types 1 and 2, respectively) and 14 age-matched controls by histological techniques and in situ hybridization using riboprobes for human IHH, PTHR1, type 10 and type 1 collagen transcripts. We show that bone-perichondrial ring enlargement and growth plate increased vascularization in FGFR3-mutated fetuses correlate with the phenotypic severity of the disease. PTHR1 and IHH expression in growth plates, bone-perichondrial rings and vascular canals is not affected by FGFR3 mutations, irrespective of the mutant genotype and age, and is in keeping with cell phenotypes. These results indicate that in humans, FGFR3 signaling does not down-regulate the main players of the IHH/PTHR1 pathway. Furthermore, we show that cells within the bone-perichondrial ring in controls and patients express IHH, PTHR1, and type 10 and type 1 collagen transcripts, suggesting that bone-perichondrial ring formation involves cells of both chondrocytic and osteoblastic phenotypes. PMID:12368206

  18. A mutation in the RET proto-oncogene in Hirschsprung's disease affects the tyrosine kinase activity associated with multiple endocrine neoplasia type 2A and 2B.

    PubMed Central

    Cosma, M P; Panariello, L; Quadro, L; Dathan, N A; Fattoruso, O; Colantuoni, V

    1996-01-01

    We demonstrate that a Hirschsprung (HSCR) mutation in the tyrosine kinase domain of the RET proto-oncogene abolishes in cis the tyrosine-phosphorylation associated with the activating mutation in multiple endocrine neoplasia type 2A (MEN2A) in transiently transfected Cos cells. Yet the double mutant RET2AHS retains the ability to form stable dimers, thus dissociating the dimerization from the phosphorylation potential. Co-transfection experiments with single and double mutants carrying plasmids RET2A and RET2AHS in different ratios drastically reduced the phosphorylation levels of the RET2A protein, suggesting a dominant-negative effect of the HSCR mutation. Also, the phosphorylation associated with the multiple endocrine neoplasia type 2B (MEN2B) allele was affected in experiments with single and double mutants carrying plasmids co-transfected under the same conditions. Finally, analysis of the enzymic activity of MEN2A and MEN2B tumours confirmed the relative levels of tyrosine phosphorylation observed in Cos cells, indicating that this condition, in vivo, may account for the RET transforming potential. PMID:8670046

  19. The RetC620R Mutation Affects Renal and Enteric Development in a Mouse Model of Hirschsprung’s Disease

    PubMed Central

    Carniti, Cristiana; Belluco, Sara; Riccardi, Elena; Cranston, Aaron N.; Mondellini, Piera; Ponder, Bruce A.J.; Scanziani, Eugenio; Pierotti, Marco A.; Bongarzone, Italia

    2006-01-01

    In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung’s disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RETC620R substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although RetC620R homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. RetC620R heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The RetC620R allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease. PMID:16565500

  20. Mutations in Arabidopsis thaliana genes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces

    NASA Technical Reports Server (NTRS)

    Rutherford, R.; Gallois, P.; Masson, P. H.

    1998-01-01

    Arabidopsis thaliana roots grow in a wavy pattern upon a slanted surface. A novel mutation in the anthranilate synthase alpha 1 (ASA1) gene, named trp5-2wvc1, and mutations in the tryptophan synthase alpha and beta 1 genes (trp3-1 and trp2-1, respectively) confer a compressed root wave phenotype on tilted agar surfaces. When trp5-2wvc1 seedlings are grown on media supplemented with anthranilate metabolites, their roots wave like wild type. Genetic and pharmacological experiments argue that the compressed root wave phenotypes of trp5-2wvc1, trp2-1 and trp3-1 seedlings are not due to reduced IAA biosynthetic potential, but rather to a deficiency in L-tryptophan (L-Trp), or in a L-Trp derivative. Although the roots of 7-day-old seedlings possess higher concentrations of free L-Trp than the shoot as a whole, trp5-2wvc1 mutants show no detectable alteration in L-Trp levels in either tissue type, suggesting that a very localized shortage of L-Trp, or of a L-Trp-derived compound, is responsible for the observed phenotype.

  1. Mutations in the hormone regulatory element of mouse mammary tumor virus differentially affect the response to progestins, androgens, and glucocorticoids.

    PubMed Central

    Gowland, P L; Buetti, E

    1989-01-01

    Transcription of the mouse mammary tumor virus DNA is known to be induced by several steroid hormones. Using chimeric MMTV plasmids containing mutations within the hormone regulatory element, we have previously studied the regions required for the glucocorticoid response in mouse fibroblasts. Here we report the characterization of elements essential for the stimulation by progestins and androgens as compared with glucocorticoids. The same set of mutant plasmids was transfected into the human mammary tumor cell line T47D, and the specific transcripts were analyzed by an S1 nuclease protection assay. Androgen-mediated stimulation, although weak, showed an extended sensitivity to mutations, with a slight preference for the proximal region. The results with progestin suggest that sequences within all the described sites protected by the receptor in vitro are required and that the promoter-proximal region (-128 to -78 from the RNA start site) is more important than the distal one (-190 to -160). Moreover, a binding site for nuclear factor I was not required for the progestin response, whereas it was required for glucocorticoids. Thus, the various steroid receptors play a role in the differential regulation of mouse mammary tumor virus transcription by recognizing distinct sequence differences in the hormone regulatory element and interacting with different factors bound to the promoter. Images PMID:2550809

  2. The cancer-associated K351N mutation affects the ubiquitination and the translocation to mitochondria of p53 protein.

    PubMed

    Muscolini, Michela; Montagni, Elisa; Palermo, Vanessa; Di Agostino, Silvia; Gu, Wei; Abdelmoula-Souissi, Salma; Mazzoni, Cristina; Blandino, Giovanni; Tuosto, Loretta

    2011-11-18

    Stress-induced monoubiquitination of p53 is a crucial event for the nuclear-cytoplasm-mitochondria trafficking and transcription-independent pro-apoptotic functions of p53. Although an intact ubiquitination pathway and a functional nuclear export sequence are required for p53 nuclear export, the role of specific residues within this region in regulating both processes remains largely unknown. Here we characterize the mechanisms accounting for the nuclear accumulation of a new point mutation (Lys-351 to Asn) in the nuclear export sequence of p53 identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution abrogates the monoubiquitination of p53 induced by both Mdm2 and MSL2 E3-ligases. As a consequence, cells expressing p53 K351N mutant showed defects in cisplatin-induced translocation of p53 to mitochondria, Bax oligomerization, and mitochondrial membrane depolarization. These data identify K351N as a critical mutation of p53 that contributes to the development and maintenance of resistance to cisplatin.

  3. The A1555G mutation in the 12S rRNA gene of human mtDNA: recurrent origins and founder events in families affected by sensorineural deafness.

    PubMed

    Torroni, A; Cruciani, F; Rengo, C; Sellitto, D; López-Bigas, N; Rabionet, R; Govea, N; López De Munain, A; Sarduy, M; Romero, L; Villamar, M; del Castillo, I; Moreno, F; Estivill, X; Scozzari, R

    1999-11-01

    The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to >/=30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments.

  4. The A1555G Mutation in the 12S rRNA Gene of Human mtDNA: Recurrent Origins and Founder Events in Families Affected by Sensorineural Deafness

    PubMed Central

    Torroni, Antonio; Cruciani, Fulvio; Rengo, Chiara; Sellitto, Daniele; López-Bigas, Núria; Rabionet, Raquel; Govea, Nancy; López de Munain, Adolfo; Sarduy, Maritza; Romero, Lourdes; Villamar, Manuela; del Castillo, Ignacio; Moreno, Felipe; Estivill, Xavier; Scozzari, Rosaria

    1999-01-01

    Summary The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to ⩾30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments. PMID:10521300

  5. Limited proteolysis differentially modulates the stability and subcellular localization of domains of RPGRIP1 that are distinctly affected by mutations in Leber's congenital amaurosis.

    PubMed

    Lu, Xinrong; Guruju, Mallikarjuna; Oswald, John; Ferreira, Paulo A

    2005-05-15

    The retinitis pigmentosa GTPase regulator (RPGR) protein interacts with the retinitis pigmentosa GTPase regulator interacting protein-1 (RPGRIP1). Genetic lesions in the cognate genes lead to distinct and severe human retinal dystrophies. The biological role of these proteins in retinal function and pathogenesis of retinal diseases is elusive. Here, we present the first physiological assay of the role of RPGRIP1 and mutations therein. We found that the monoallelic and homozygous mutations, DeltaE1279 and D1114G, in the RPGR-interacting domain (RID) of RPGRIP1, enhance and abolish, respectively, its interaction in vivo with RPGR without affecting the stability of RID. In contrast to RID(WT) and RID(D1114G), chemical genetics shows that the interaction of RID(DeltaE1279) with RPGR is resistant to various stress treatments such as osmotic, pH and heat-shock stimuli. Hence, RID(D1114G) and RID(DeltaE1279) constitute loss- and gain-of-function mutations. Moreover, we find that the isoforms, bRPGRIP1 and bRPGRIP1b, undergo limited proteolysis constitutively in vivo in the cytoplasm compartment. This leads to the relocation and accumulation of a small and stable N-terminal domain of approximately 7 kDa to the nucleus, whereas the cytosolic C-terminal domain of RPGRIP1 is degraded and short-lived. The RID(D1114G) and RID(DeltaE1279) mutations exhibit strong cis-acting and antagonistic biological effects on the nuclear relocation, subcellular distribution and proteolytic cleavage of RPGRIP1 and/or domains thereof. These data support distinct and spatiotemporal subcellular-specific roles to RPGRIP1. A novel RPGRIP1-mediated nucleocytoplasmic crosstalk and transport pathway regulated by RID, and hence by RPGR, emerges with implications in the molecular pathogenesis of retinopathies, and a model to other diseases.

  6. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

    PubMed

    Floss, Daniela S; Levy, Julien G; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J

    2013-12-17

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

  7. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis

    PubMed Central

    Floss, Daniela S.; Levy, Julien G.; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J.

    2013-01-01

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis. PMID:24297892

  8. Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis.

    PubMed

    Penterman, Jon; Abo, Ryan P; De Nisco, Nicole J; Arnold, Markus F F; Longhi, Renato; Zanda, Matteo; Walker, Graham C

    2014-03-04

    The α-proteobacterium Sinorhizobium meliloti establishes a chronic intracellular infection during the symbiosis with its legume hosts. Within specialized host cells, S. meliloti differentiates into highly polyploid, enlarged nitrogen-fixing bacteroids. This differentiation is driven by host cells through the production of defensin-like peptides called "nodule-specific cysteine-rich" (NCR) peptides. Recent research has shown that synthesized NCR peptides exhibit antimicrobial activity at high concentrations but cause bacterial endoreduplication at sublethal concentrations. We leveraged synchronized S. meliloti populations to determine how treatment with a sublethal NCR peptide affects the cell cycle and physiology of bacteria at the molecular level. We found that at sublethal levels a representative NCR peptide specifically blocks cell division and antagonizes Z-ring function. Gene-expression profiling revealed that the cell division block was produced, in part, through the substantial transcriptional response elicited by sublethal NCR treatment that affected ∼15% of the genome. Expression of critical cell-cycle regulators, including ctrA, and cell division genes, including genes required for Z-ring function, were greatly attenuated in NCR-treated cells. In addition, our experiments identified important symbiosis functions and stress responses that are induced by sublethal levels of NCR peptides and other antimicrobial peptides. Several of these stress-response pathways also are found in related α-proteobacterial pathogens and might be used by S. meliloti to sense host cues during infection. Our data suggest a model in which, in addition to provoking stress responses, NCR peptides target intracellular regulatory pathways to drive S. meliloti endoreduplication and differentiation during symbiosis.

  9. Mutations affecting transport of the hexitols D-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping.

    PubMed Central

    Lengeler, J

    1975-01-01

    Mutants of Escherichia coli K-12 unable to grow on any of the three naturally occurring hexitols D-manitol, D-glucitol, and galactitol and, among these specifically, mutants with altered transport and phosphorylating activity have been isolated. Different isolation procedures have been utilized, including suicide by D-[3H]mannitol, chemotaxis, and resistance to the toxic hexitol analogue 2-deoxy-arabino-hexitol. Mutations thus obtained have been mapped in four distinct operons. (i) Mutations affecting an enzyme II-complexmt1 activity of the phosphoenolpyruvate-dependent phosphotransferase system all map in gene mtlA. This gene has previously been shown (Solomon and Lin, 1972) to be part of an operon, mtl, located at 71 min on the E. coli linkage map containing, in addition to mtlA, the cis-dominant regulatory gene mtlC and mtlD, the structural gene for the enzyme D-mannitol-1-phosphate dehydrogenase. The gene order in this operon, induced by D-mannitol, is mtlC A D. (ii) Mutations in gene gutA affecting a second enzyme II-complexgut of the phosphotransferase system map at 51 min, clustered in operon gutC A D together with the cis-dominant regulatory gene gutC and the structural gene gutD for the enzyme D-glucitol-6-phosphate dehydrogenase. The gut operon, previously called sbl or srl, is induced by D-glucitol. (iii) Mutations affecting the transport and catabolism of galactitol are clustered in a third operon, gatC A D, located at 40.5 min. This operon again contains a cis-dominant regulatory gene, gatC, the structural gene gatD for galactitol-1-phosphate dehydrogenase, and gene gatA coding for a thrid hexitol-specific enzyme II-complexgat. Other genes coding for two additional enzymes involved in galactitol catabolism apparently are not linked to gatC A D. (iv) A fourth class of mutants pleiotropically negative for hexitol growth and transport maps in the pts operon. Triple-negative mutants (mtlA gutA gatA) do not have further transport or phosphorylating activity

  10. Effects of nano-ZnO on the agronomically relevant Rhizobium-legume symbiosis.

    PubMed

    Huang, Yu Chu; Fan, Ruimei; Grusak, Michael A; Sherrier, Janine D; Huang, C P

    2014-11-01

    The impact of nano-ZnO (nZnO) on Rhizobium-legume symbiosis was studied with garden pea and its compatible bacterial partner Rhizobium leguminosarum bv. viciae 3841. Exposure of peas to nZnO had no impact on germination, but significantly affected root length. Chronic exposure of plant to nZnO impacted its development by decreasing the number of the first- and the second-order lateral roots, stem length, leaf surface area, and transpiration. The effect of nZnO dissolution on phytotoxicity was also examined. Results showed that Zn(2+) had negative impact on plant development. Exposure of R. leguminosarum bv. viciae 3841 to nZnO brought about morphological changes by rendering the microbial cells toward round shape and damaging the bacterial surface. Furthermore, the presence of nZnO in the rhizosphere affected root nodulation, delayed the onset of nitrogen fixation, and caused early senescence of nodules. Attachment of nanoparticles on the root surface and dissolution of Zn(2+) are important factors affecting the phytotocity of nZnO. Hence, the presence of nZnO in the environment is potentially hazardous to the Rhizobium-legume symbiosis system.

  11. Point mutations in Staphylococcus aureus PBP 2 gene affect penicillin-binding kinetics and are associated with resistance.

    PubMed Central

    Hackbarth, C J; Kocagoz, T; Kocagoz, S; Chambers, H F

    1995-01-01

    In Staphylococcus aureus, penicillin-binding protein 2 (PBP 2) has been implicated in non-PBP 2a-mediated methicillin resistance. The PBP 2 gene (pbpB) was cloned from an expression library of a methicillin-susceptible strain of S. aureus (209P), and its entire sequence was compared with that of the pbpB gene from strains BB255, BB255R, and CDC6. Point mutations that resulted in amino acid substitutions near the conserved penicillin-binding motifs were detected in BB255R and CDC6, two low-level methicillin-resistant strains. Penicillin binding to PBP 2 in both BB255R and CDC6 is altered, and kinetic analysis indicated that altered binding of PBP 2 by penicillin was due to both lower binding affinity and more rapid release of bound drug. These structural and biochemical changes may contribute to the strains' resistance to beta-lactam antibiotics. PMID:7695289

  12. Mutations in ampG and Lytic Transglycosylase Genes Affect the Net Release of Peptidoglycan Monomers from Vibrio fischeri▿ †

    PubMed Central

    Adin, Dawn M.; Engle, Jacquelyn T.; Goldman, William E.; McFall-Ngai, Margaret J.; Stabb, Eric V.

    2009-01-01

    The light-organ symbiont Vibrio fischeri releases N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramylalanyl-γ-glutamyldiaminopimelylalanine, a disaccharide-tetrapeptide component of peptidoglycan that is referred to here as “PG monomer.” In contrast, most gram-negative bacteria recycle PG monomer efficiently, and it does not accumulate extracellularly. PG monomer can stimulate normal light-organ morphogenesis in the host squid Euprymna scolopes, resulting in regression of ciliated appendages similar to that triggered by infection with V. fischeri. We examined whether the net release of PG monomers by V. fischeri resulted from lytic transglycosylase activity or from defects in AmpG, the permease through which PG monomers enter the cytoplasm for recycling. An ampG mutant displayed a 100-fold increase in net PG monomer release, indicating that AmpG is functional. The ampG mutation also conferred the uncharacteristic ability to induce light-organ morphogenesis even when placed in a nonmotile flaJ mutant that cannot infect the light-organ crypts. We targeted five potential lytic transglycosylase genes singly and in specific combinations to assess their role in PG monomer release. Combinations of mutations in ltgA, ltgD, and ltgY decreased net PG monomer release, and a triple mutant lacking all three of these genes had little to no accumulation of PG monomers in culture supernatants. This mutant colonized the host as well as the wild type did; however, the mutant-infected squid were more prone to later superinfection by a second V. fischeri strain. We propose that the lack of PG monomer release by this mutant results in less regression of the infection-promoting ciliated appendages, leading to this propensity for superinfection. PMID:19074387

  13. Temperature Sensitivity Caused by Mutant Release Factor 1 Is Suppressed by Mutations That Affect 16S rRNA Maturation

    PubMed Central

    Kaczanowska, Magdalena; Rydén-Aulin, Monica

    2004-01-01

    To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the rplM-rpsI operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the yrdC open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1. PMID:15126466

  14. Arbuscular mycorrhizal symbiosis decreases strigolactone production in tomato.

    PubMed

    López-Ráez, Juan A; Charnikhova, Tatsiana; Fernández, Ivan; Bouwmeester, Harro; Pozo, Maria J

    2011-02-15

    Strigolactones are a new class of plant hormones emerging as important signals in the control of plant architecture. In addition, they are key elements in plant communication with several rhizosphere organisms. Strigolactones are exuded into the soil, where they act as host detection signals for arbuscular mycorrhizal (AM) fungi, but also as germination stimulants for root parasitic plant seeds. Under phosphate limiting conditions, plants up-regulate the secretion of strigolactones into the rhizosphere to promote the formation of AM symbiosis. Using tomato as a model plant, we have recently shown that AM symbiosis induces changes in transcriptional and hormonal profiles. Using the same model system, here we analytically demonstrate, using liquid chromatography-tandem mass spectrometry, that strigolactone production is also significantly reduced upon AM symbiosis. Considering the dual role of the strigolactones in the rhizosphere as signals for AM fungi and parasitic plants, we discuss the potential implications of these changes in the plant interaction with both organisms.

  15. Symbiosis-promoting and deleterious effects of NopT, a novel type 3 effector of Rhizobium sp. strain NGR234.

    PubMed

    Dai, Wei-Jun; Zeng, Yong; Xie, Zhi-Ping; Staehelin, Christian

    2008-07-01

    Establishment of symbiosis between certain host plants and nitrogen-fixing bacteria ("rhizobia") depends on type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS). Here, we report that the open reading frame y4zC of strain NGR234 encodes a novel rhizobial type 3 effector, termed NopT (for nodulation outer protein T). Analysis of secreted proteins from NGR234 and T3SS mutants revealed that NopT is secreted via the T3SS. NopT possessed autoproteolytic activity when expressed in Escherichia coli or human HEK 293T cells. The processed NopT exposed a glycine (G50) to the N terminus, which is predicted to be myristoylated in eukaryotic cells. NopT with a point mutation at position C93, H205, or D220 (catalytic triad) showed strongly reduced autoproteolytic activity, indicating that NopT is a functional protease of the YopT-AvrPphB effector family. When transiently expressed in tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. Arabidopsis plants transformed with nopT showed chlorotic and necrotic symptoms, indicating a cytotoxic effect. Inoculation experiments with mutant derivatives of NGR234 indicated that NopT affected nodulation either positively (Phaseolus vulgaris cv. Yudou No. 1; Tephrosia vogelii) or negatively (Crotalaria juncea). We suggest that NopT-related polymorphism may be involved in evolutionary adaptation of NGR234 to particular host legumes.

  16. [LEGUME-RHIZOBIUM SYMBIOSIS PROTEOMICS: ACHIEVEMENTS AND PERSPECTIVES].

    PubMed

    Kondratiuk, Iu Iu; Mamenko, P M; Kots, S Ya

    2015-01-01

    The present review contains results of proteomic researches of legume-rhizobium symbiosis. The technical difficulties associated with the methods of obtaining protein extracts from symbiotic structures and ways of overcoming them were discussed. The changes of protein synthesis under formation and functioning of symbiotic structures were shown. Special attention has been given to the importance of proteomic studies of plant-microbe structures in the formation of adaptation strategies under adverse environmental conditions. The technical and conceptual perspectives of legume-rhizobium symbiosis proteomics were shown.

  17. Roles of poly-3-hydroxybutyrate (PHB) and glycogen in symbiosis of Sinorhizobium meliloti with Medicago sp.

    PubMed

    Wang, Chunxia; Saldanha, Marsha; Sheng, Xiaoyan; Shelswell, Kristopher J; Walsh, Keith T; Sobral, Bruno W S; Charles, Trevor C

    2007-02-01

    Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia-legume symbiosis are not fully understood. Glycogen synthase mutations were constructed by in-frame deletion (glgA1) or insertion (glgA2). These mutations were combined with a phbC mutation to make all combinations of double and triple mutants. PHB was not detectable in any of the mutants containing the phbC mutation; glycogen was not detectable in any of the mutants containing the glgA1 mutation. PHB levels were significantly lower in the glgA1 mutant, while glycogen levels were increased in the phbC mutant. Exopolysaccharide (EPS) was not detected in any of the phbC mutants, while the glgA1 and glgA2 mutants produced levels of EPS similar to the wild-type. Symbiotic properties of these strains were investigated on Medicago truncatula and Medicago sativa. The results indicated that the strains unable to synthesize PHB, or glycogen, were still able to form nodules and fix nitrogen. However, phbC mutations caused greater nodule formation delay on M. truncatula than on M. sativa. Time-course studies showed that (1) the ability to synthesize PHB is important for N(2) fixation in M. truncatula nodules and younger M. sativa nodules, and (2) the blocking of glycogen synthesis resulted in lower levels of N(2) fixation on M. truncatula and older nodules on M. sativa. These data have important implications for understanding how PHB and glycogen function in the interactions of S. meliloti with Medicago spp.

  18. LTBP2 mutations cause Weill-Marchesani and Weill-Marchesani-like syndrome and affect disruptions in the extracellular matrix.

    PubMed

    Haji-Seyed-Javadi, Ramona; Jelodari-Mamaghani, Sahar; Paylakhi, Seyed Hassan; Yazdani, Shahin; Nilforushan, Naveed; Fan, Jian-Bing; Klotzle, Brandy; Mahmoudi, Mohammad Jafar; Ebrahimian, Mohammad Jafar; Chelich, Noori; Taghiabadi, Ehsan; Kamyab, Kambiz; Boileau, Catherine; Paisan-Ruiz, Coro; Ronaghi, Mostafa; Elahi, Elahe

    2012-08-01

    Latent transforming growth factor (TGF) beta-binding protein 2 (LTBP2) is an extracellular matrix (ECM) protein that associates with fibrillin-1 containing microfibrils. Various factors prompted considering LTBP2 in the etiology of isolated ectopia lentis and associated conditions such as Weill-Marchesani syndrome (WMS) and Marfan syndrome (MFS). LTBP2 was screened in 30 unrelated Iranian patients. Mutations were found only in one WMS proband and one MFS proband. Homozygous c.3529G>A (p.Val1177Met) was shown to cause autosomal recessive WMS or WM-like syndrome by several approaches, including homozygosity mapping. Light, fluorescent, and electron microscopy evidenced disruptions of the microfibrillar network in the ECM of the proband's skin. In conjunction with recent findings regarding other ECM proteins, the results presented strongly support the contention that anomalies in WMS patients are due to disruptions in the ECM. Heterozygous c.1642C >T (p.Arg548*) possibly contributed to MFS-related phenotypes, including ocular manifestations, mitral valve prolapse, and pectus excavatum, but was not cause of MFS.

  19. Mutation in the xpsD gene of Xanthomonas axonopodis pv. citri affects cellulose degradation and virulence.

    PubMed

    Baptista, Juliana Cristina; Machado, Marcos Antonio; Homem, Rafael Augusto; Torres, Pablo Sebastián; Vojnov, Adrian Alberto; do Amaral, Alexandre Morais

    2010-01-01

    The Gram-negative bacterium Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, is a major threat to the citrus industry worldwide. Although this is a leaf spot pathogen, it bears genes highly related to degradation of plant cell walls, which are typically found in plant pathogens that cause symptoms of tissue maceration. Little is known on Xac capacity to cause disease and hydrolyze cellulose. We investigated the contribution of various open reading frames on degradation of a cellulose compound by means of a global mutational assay to selectively screen for a defect in carboxymethyl cellulase (CMCase) secretion in X. axonopodis pv. citri. Screening on CMC agar revealed one mutant clone defective in extracellular glycanase activity, out of nearly 3,000 clones. The insertion was located in the xpsD gene, a component of the type II secretion system (T2SS) showing an influence in the ability of Xac to colonize tissues and hydrolyze cellulose. In summary, these data show for the first time, that X. axonopodis pv. citri is capable of hydrolyzing cellulose in a T2SS-dependent process. Furthermore, it was demonstrated that the ability to degrade cellulose contributes to the infection process as a whole.

  20. A Protein Kinase C phosphorylation motif in GLUT1 affects glucose transport and is mutated in GLUT1 deficiency syndrome

    PubMed Central

    Lee, Eunice E.; Ma, Jing; Sacharidou, Anastasia; Mi, Wentao; Salato, Valerie K.; Nguyen, Nam; Jiang, Youxing; Pascual, Juan M.; North, Paula E.; Shaul, Philip W.; Mettlen, Marcel; Wang, Richard C.

    2015-01-01

    Summary Protein Kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in-vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify Serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally-occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport. PMID:25982116

  1. A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

    PubMed

    Lee, Eunice E; Ma, Jing; Sacharidou, Anastasia; Mi, Wentao; Salato, Valerie K; Nguyen, Nam; Jiang, Youxing; Pascual, Juan M; North, Paula E; Shaul, Philip W; Mettlen, Marcel; Wang, Richard C

    2015-06-04

    Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

  2. Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

    SciTech Connect

    Wu Liguo; Hutt-Fletcher, Lindsey M. . E-mail: lhuttf@lsuhsc.edu

    2007-06-20

    Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.

  3. Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity.

    PubMed Central

    Katavic, V; Reed, D W; Taylor, D C; Giblin, E M; Barton, D L; Zou, J; Mackenzie, S L; Covello, P S; Kunst, L

    1995-01-01

    In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids. PMID:7784510

  4. Mutation in the xpsD gene of Xanthomonas axonopodis pv. citri affects cellulose degradation and virulence

    PubMed Central

    2010-01-01

    The Gram-negative bacterium Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, is a major threat to the citrus industry worldwide. Although this is a leaf spot pathogen, it bears genes highly related to degradation of plant cell walls, which are typically found in plant pathogens that cause symptoms of tissue maceration. Little is known on Xac capacity to cause disease and hydrolyze cellulose. We investigated the contribution of various open reading frames on degradation of a cellulose compound by means of a global mutational assay to selectively screen for a defect in carboxymethyl cellulase (CMCase) secretion in X. axonopodis pv. citri. Screening on CMC agar revealed one mutant clone defective in extracellular glycanase activity, out of nearly 3,000 clones. The insertion was located in the xpsD gene, a component of the type II secretion system (T2SS) showing an influence in the ability of Xac to colonize tissues and hydrolyze cellulose. In summary, these data show for the first time, that X. axonopodis pv. citri is capable of hydrolyzing cellulose in a T2SS-dependent process. Furthermore, it was demonstrated that the ability to degrade cellulose contributes to the infection process as a whole. PMID:21637619

  5. Novel and recurrent mutations in the TAT gene in Tunisian families affected with Richner-Hanhart syndrome.

    PubMed

    Bouyacoub, Yosra; Zribi, Hela; Azzouz, Hatem; Nasrallah, Fehmi; Abdelaziz, Rim Ben; Kacem, Monia; Rekaya, Ben; Messaoud, Olfa; Romdhane, Lilia; Charfeddine, Cherine; Bouziri, Mustapha; Bouziri, Sonia; Tebib, Neji; Mokni, Mourad; Kaabachi, Naziha; Boubaker, Samir; Abdelhak, Sonia

    2013-10-15

    Tyrosinemia type II, also designated as oculocutaneous tyrosinemia or Richner-Hanhart syndrome (RHS), is a very rare autosomal recessive disorder. In the present study, we report clinical features and molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in two young patients, both born to consanguineous unions between first-degree cousins. These two unrelated families originated from Northern and Southern Tunisia. The clinical diagnosis was based on the observation of several complications related to Richner-Hanhart syndrome: recurrent eye redness, tearing and burning pain, photophobia, bilateral pseudodendritic keratitis, an erythematous and painful focal palmo-plantar hyperkeratosis and a mild delay of mental development. The diagnosis was confirmed by biochemical analysis. Sequencing of the TAT gene revealed the presence of a previously reported missense mutation (c.452G>A, p.Cys151Tyr) in a Tunisian family, and a novel G duplication (c.869dupG, p.Trp291Leufs 6). Early diagnosis of RHS and protein-restricted diet are crucial to reduce the risk and the severity of long-term complications of hypertyrosinemia such as intellectual disability.

  6. Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm.

    PubMed

    Satoh, Hikaru; Nishi, Aiko; Yamashita, Kazuhiro; Takemoto, Yoko; Tanaka, Yasumasa; Hosaka, Yuko; Sakurai, Aya; Fujita, Naoko; Nakamura, Yasunori

    2003-11-01

    We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.

  7. Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis.

    PubMed

    El Khalloufi, Fatima; Oufdou, Khalid; Lahrouni, Majida; El Ghazali, Issam; Saqrane, Sanaa; Vasconcelos, Vitor; Oudra, Brahim

    2011-03-01

    The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.

  8. NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses.

    PubMed

    DeDiego, Marta L; Nogales, Aitor; Lambert-Emo, Kris; Martinez-Sobrido, Luis; Topham, David J

    2016-11-01

    Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus.

  9. A novel mutation in the N-terminal domain of Drosophila BubR1 affects the spindle assembly checkpoint function of BubR1

    PubMed Central

    Duranteau, Marie; Montagne, Jean-Jacques

    2016-01-01

    ABSTRACT The spindle assembly checkpoint (SAC) is a surveillance mechanism that ensures accurate segregation of chromosomes into two daughter cells. BubR1, a key component of the SAC, also plays a role in the mitotic timing since depletion of BubR1 leads to accelerated mitosis. We previously found that mutation of the KEN1-box domain of Drosophila BubR1 (bubR1-KEN1 mutant) affects the binding of BubR1 to Cdc20, the activating co-factor of the APC/C, and does not accelerate the mitotic timing despite resulting in a defective SAC, which was unlike what was reported in mammalian cells. Here, we show that a mutation in a novel Drosophila short sequence (bubR1-KAN mutant) leads to an accelerated mitotic timing as well as SAC failure. Moreover, our data indicate that the level of Fzy, the Drosophila homolog of Cdc20, recruited to kinetochores is diminished in bubR1-KEN1 mutant cells and further diminished in bubR1-KAN mutant cells. Altogether, our data show that this newly identified Drosophila BubR1 KAN motif is required for a functional SAC and suggest that it may play an important role on Cdc20/Fzy kinetochore recruitment. PMID:27742609

  10. Mutations within the LINC-HELLP non-coding RNA differentially bind ribosomal and RNA splicing complexes and negatively affect trophoblast differentiation.

    PubMed

    van Dijk, Marie; Visser, Allerdien; Buabeng, Kwadwo M L; Poutsma, Ankie; van der Schors, Roel C; Oudejans, Cees B M

    2015-10-01

    LINC-HELLP, showing chromosomal linkage with the pregnancy-specific HELLP syndrome in Dutch families, reduces differentiation from a proliferative to an invasive phenotype of first-trimester extravillous trophoblasts. Here we show that mutations in LINC-HELLP identified in HELLP families negatively affect this trophoblast differentiation either by inducing proliferation rate or by causing cell cycle exit as shown by a decrease in both proliferation and invasion. As LincRNAs predominantly function through interactions with proteins, we identified the directly interacting proteins using chromatin isolation by RNA purification followed by protein mass spectrometry. We found 22 proteins predominantly clustering in two functional networks, i.e. RNA splicing and the ribosome. YBX1, PCBP1, PCBP2, RPS6 and RPL7 were validated, and binding to these proteins was influenced by the HELLP mutations carried. Finally, we show that the LINC-HELLP transcript levels are significantly upregulated in plasma of women in their first trimester of pregnancy compared with non-pregnant women, whereas this upregulation seems absent in a pilot set of patients later developing pregnancy complications, indicative of its functional significance in vivo.

  11. A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi

    PubMed Central

    Stoetzel, Corinne; Bär, Séverine; De Craene, Johan-Owen; Scheidecker, Sophie; Etard, Christelle; Chicher, Johana; Reck, Jennifer R.; Perrault, Isabelle; Geoffroy, Véronique; Chennen, Kirsley; Strähle, Uwe; Hammann, Philippe; Friant, Sylvie; Dollfus, Hélène

    2016-01-01

    Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium. PMID:27882921

  12. Signaling events during initiation of arbuscular mycorrhizal symbiosis.

    PubMed

    Schmitz, Alexa M; Harrison, Maria J

    2014-03-01

    Under nutrient-limiting conditions, plants will enter into symbiosis with arbuscular mycorrhizal (AM) fungi for the enhancement of mineral nutrient acquisition from the surrounding soil. AM fungi live in close, intracellular association with plant roots where they transfer phosphate and nitrogen to the plant in exchange for carbon. They are obligate fungi, relying on their host as their only carbon source. Much has been discovered in the last decade concerning the signaling events during initiation of the AM symbiosis, including the identification of signaling molecules generated by both partners. This signaling occurs through symbiosis-specific gene products in the host plant, which are indispensable for normal AM development. At the same time, plants have adapted complex mechanisms for avoiding infection by pathogenic fungi, including an innate immune response to general microbial molecules, such as chitin present in fungal cell walls. How it is that AM fungal colonization is maintained without eliciting a defensive response from the host is still uncertain. In this review, we present a summary of the molecular signals and their elicited responses during initiation of the AM symbiosis, including plant immune responses and their suppression.

  13. Quorum sensing in the squid-Vibrio symbiosis.

    PubMed

    Verma, Subhash C; Miyashiro, Tim

    2013-08-07

    Quorum sensing is an intercellular form of communication that bacteria use to coordinate group behaviors such as biofilm formation and the production of antibiotics and virulence factors. The term quorum sensing was originally coined to describe the mechanism underlying the onset of luminescence production in cultures of the marine bacterium Vibrio fischeri. Luminescence and, more generally, quorum sensing are important for V. fischeri to form a mutualistic symbiosis with the Hawaiian bobtail squid, Euprymna scolopes. The symbiosis is established when V. fischeri cells migrate via flagella-based motility from the surrounding seawater into a specialized structure injuvenile squid called the light organ. The cells grow to high cell densities within the light organ where the infection persists over the lifetime of the animal. A hallmark of a successful symbiosis is the luminescence produced by V. fischeri that camouflages the squid at night by eliminating its shadow within the water column. While the regulatory networks governing quorum sensing are critical for properly regulating V. fischeri luminescence within the squid light organ, they also regulate luminescence-independent processes during symbiosis. In this review, we discuss the quorum-sensing network of V. fischeri and highlight its impact at various stages during host colonization.

  14. Loss of Msp1p in Schizosaccharomyces pombe induces a ROS-dependent nuclear mutator phenotype that affects mitochondrial fission genes.

    PubMed

    Delerue, Thomas; Khosrobakhsh, Farnoosh; Daloyau, Marlène; Emorine, Laurent Jean; Dedieu, Adrien; Herbert, Christopher J; Bonnefoy, Nathalie; Arnauné-Pelloquin, Laetitia; Belenguer, Pascale

    2016-10-01

    Mitochondria continually fuse and divide to dynamically adapt to changes in metabolism and stress. Mitochondrial dynamics are also required for mitochondrial DNA (mtDNA) integrity; however, the underlying reason is not known. In this study, we examined the link between mitochondrial fusion and mtDNA maintenance in Schizosaccharomyces pombe, which cannot survive without mtDNA, by screening for suppressors of the lethality induced by loss of the dynamin-related large GTPase Msp1p. Our findings reveal that inactivation of Msp1p induces a ROS-dependent nuclear mutator phenotype that affects mitochondrial fission genes involved in suppressing mitochondrial fragmentation and mtDNA depletion. This indicates that mitochondrial fusion is crucial for maintaining the integrity of both mitochondrial and nuclear genetic information. Furthermore, our study suggests that the primary roles of Msp1p are to organize mitochondrial membranes, thus making them competent for fusion, and maintain the integrity of mtDNA.

  15. [Effects of monorecessive and double recessive mutations affecting coat color on the monoamine content of the brain of the American mink (Mustela vison Schreber, 1777)].

    PubMed

    Trapezov, O V; Trapezova, L I; alekhina, T A; Klochkov, D V; Ivanov, Iu N

    2009-12-01

    The effects of mutations affecting the coat color on the dopamine, noradrenaline, and serotonin contents of the hypothalamus and brainstem of the American mink have been studied. The sample comprised standard (+/+) and mutant minks, including the monorecessive pastel (b/b), silver-blue (p/p), and white hedlund (h/h) and the combination double recessive sapphire (a/a p/p) and pearl (k/k p/p) ones. The dopamine content of the brainstem of the monorecessive pastel (b/b) and silver-blue (p/p) minks has been found to be higher than in standard (+/+) minks. Conversely, the homozigosity for two coat color loci in double recessive pearl minks (k/k p/p) significantly decreases the noradrenaline and serotonin contents of the hypothalamus. In addition, monorecessive and double recessive minks differ from each other in the serotonin contents of the midbrain and medulla.

  16. A Sinorhizobium meliloti RpoH-Regulated Gene Is Involved in Iron-Sulfur Protein Metabolism and Effective Plant Symbiosis under Intrinsic Iron Limitation

    PubMed Central

    Sasaki, Shohei; Minamisawa, Kiwamu

    2016-01-01

    ABSTRACT In Sinorhizobium meliloti, RpoH-type sigma factors have a global impact on gene expression during heat shock and play an essential role in symbiosis with leguminous plants. Using mutational analysis of a set of genes showing highly RpoH-dependent expression during heat shock, we identified a gene indispensable for effective symbiosis. This gene, designated sufT, was located downstream of the sufBCDS homologs that specify the iron-sulfur (Fe/S) cluster assembly pathway. The identified transcription start site was preceded by an RpoH-dependent promoter consensus sequence. SufT was related to a conserved protein family of unknown molecular function, of which some members are involved in Fe/S cluster metabolism in diverse organisms. A sufT mutation decreased bacterial growth in both rich and minimal media, tolerance to stresses such as iron starvation, and activities of some Fe/S cluster-dependent enzymes. These results support the involvement of SufT in SUF (sulfur mobilization) system-mediated Fe/S protein metabolism. Furthermore, we isolated spontaneous pseudorevertants of the sufT mutant with partially recovered growth; each of them had a mutation in rirA. This gene encodes a global iron regulator whose loss increases the intracellular iron content. Deletion of rirA in the original sufT mutant improved growth and restored Fe/S enzyme activities and effective symbiosis. These results suggest that enhanced iron availability compensates for the lack of SufT in the maintenance of Fe/S proteins. IMPORTANCE Although RpoH-type sigma factors of the RNA polymerase are present in diverse proteobacteria, their role as global regulators of protein homeostasis has been studied mainly in the enteric gammaproteobacterium Escherichia coli. In the soil alphaproteobacterium Sinorhizobium meliloti, the rpoH mutations have a strong impact on symbiosis with leguminous plants. We found that sufT is a unique member of the S. meliloti RpoH regulon; sufT contributes to Fe

  17. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

    PubMed Central

    González-Alonso, Paula; Chamizo, Cristina; Moreno, Víctor; Madoz-Gúrpide, Juan; Carvajal, Nerea; Daoud, Lina; Zazo, Sandra; Martín-Aparicio, Ester; Cristóbal, Ion; Rincón, Raúl; García-Foncillas, Jesús; Rojo, Federico

    2015-01-01

    Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches. PMID:26287187

  18. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma.

    PubMed

    González-Alonso, Paula; Chamizo, Cristina; Moreno, Víctor; Madoz-Gúrpide, Juan; Carvajal, Nerea; Daoud, Lina; Zazo, Sandra; Martín-Aparicio, Ester; Cristóbal, Ion; Rincón, Raúl; García-Foncillas, Jesús; Rojo, Federico

    2015-08-17

    Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  19. A 2-component system is involved in the early stages of the Pisolithus tinctorius-Pinus greggii symbiosis.

    PubMed

    Herrera-Martínez, Aseneth; Ruiz-Medrano, Roberto; Galván-Gordillo, Santiago Valentín; Toscano Morales, Roberto; Gómez-Silva, Lidia; Valdés, María; Hinojosa-Moya, Jesús; Xoconostle-Cázares, Beatriz

    2014-01-01

    Ectomycorrhizal symbiosis results in profound morphological and physiological modifications in both plant and fungus. This in turn is the product of differential gene expression in both co-symbionts, giving rise to specialized cell types capable of performing novel functions. During the precolonization stage, chemical signals from root exudates are sensed by the ectomycorrizal fungus, and vice versa, which are in principle responsible for the observed change in the developmental symbionts program. Little is known about the molecular mechanisms involved in the signaling and recognition between ectomycorrhizal fungi and their host plants. In the present work, we characterized a novel lactone, termed pinelactone, and identified a gene encoding for a histidine kinase in Pisolithus tictorius, which function is proposed to be the perception of the aforementioned metabolites. In this study, the use of closantel, a specific inhibitor of histidine kinase phosphorylation, affected the capacity for fungal colonization in the symbiosis between Pisolithus tinctorius and Pinus greggii, indicating that a 2-component system (TCS) may operate in the early events of plant-fungus interaction. Indeed, the metabolites induced the accumulation of Pisolithus tinctorius mRNA for a putative histidine kinase (termed Pthik1). Of note, Pthik1 was able to partially complement a S. cerevisiae histidine kinase mutant, demonstrating its role in the response to the presence of the aforementioned metabolites. Our results indicate a role of a 2-component pathway in the early stages of ectomycorrhizal symbiosis before colonization. Furthermore, a novel lactone from Pinus greggii root exudates may activate a signal transduction pathway that contributes to the establishment of the ectomycorrhizal symbiosis.

  20. Mutations in domain a′ of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

    PubMed Central

    Ruoppolo, Margherita; Orrù, Stefania; Talamo, Fabio; Ljung, Johanna; Pirneskoski, Annamari; Kivirikko, Kari I.; Marino, Gennaro; Koivunen, Peppi

    2003-01-01

    Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a′, and two inactive ones, b and b′, all four domains having the thioredoxin fold. Domain b′ contains the primary peptide binding site, but a′ is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Δ455–457, and abb′, and the individual domains a and a′. The first two mutants contained alterations in the last α helix of domain a′, while the third lacked the entire domain a′. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Δ455–457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb′, and over 15-fold in the cases of the individual domains a and a′. In addition, PDI F449R and PDI abb′ affected the distribution of folding intermediates. Domains a and a′ catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process. PMID:12717017

  1. A point mutation in the EGF-4 domain of β(3) integrin is responsible for the formation of the Sec(a) platelet alloantigen and affects receptor function.

    PubMed

    Sachs, Ulrich J; Bakchoul, Tamam; Eva, Olga; Giptner, Astrid; Bein, Gregor; Aster, Richard H; Gitter, Maria; Peterson, Julie; Santoso, Sentot

    2012-01-01

    Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Sec(a)) residing on αIIbβ3. The location of Sec(a) on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the β3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbβ3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbβ3 receptor function.

  2. Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template.

    PubMed

    Fisher, Timothy S; Darden, Tom; Prasad, Vinayaka R

    2003-05-01

    Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.

  3. Mutations affecting the BHLHA9 DNA-binding domain cause MSSD, mesoaxial synostotic syndactyly with phalangeal reduction, Malik-Percin type.

    PubMed

    Malik, Sajid; Percin, Ferda E; Bornholdt, Dorothea; Albrecht, Beate; Percesepe, Antonio; Koch, Manuela C; Landi, Antonio; Fritz, Barbara; Khan, Rizwan; Mumtaz, Sara; Akarsu, Nurten A; Grzeschik, Karl-Heinz

    2014-12-04

    Mesoaxial synostotic syndactyly, Malik-Percin type (MSSD) (syndactyly type IX) is a rare autosomal-recessive nonsyndromic digit anomaly with only two affected families reported so far. We previously showed that the trait is genetically distinct from other syndactyly types, and through autozygosity mapping we had identified a locus on chromosome 17p13.3 for this unique limb malformation. Here, we extend the number of independent pedigrees from various geographic regions segregating MSSD to a total of six. We demonstrate that three neighboring missense mutations affecting the highly conserved DNA-binding region of the basic helix-loop-helix A9 transcription factor (BHLHA9) are associated with this phenotype. Recombinant BHLHA9 generated by transient gene expression is shown to be located in the cytoplasm and the cell nucleus. Transcription factors 3, 4, and 12, members of the E protein (class I) family of helix-loop-helix transcription factors, are highlighted in yeast two-hybrid analysis as potential dimerization partners for BHLHA9. In the presence of BHLHA9, the potential of these three proteins to activate expression of an E-box-regulated target gene is reduced considerably. BHLHA9 harboring one of the three substitutions detected in MSSD-affected individuals eliminates entirely the transcription activation by these class I bHLH proteins. We conclude that by dimerizing with other bHLH protein monomers, BHLHA9 could fine tune the expression of regulatory factors governing determination of central limb mesenchyme cells, a function made impossible by altering critical amino acids in the DNA binding domain. These findings identify BHLHA9 as an essential player in the regulatory network governing limb morphogenesis in humans.

  4. Genes affected by mouse mammary tumor virus (MMTV) proviral insertions in mouse mammary tumors are deregulated or mutated in primary human mammary tumors

    PubMed Central

    Callahan, Robert; Mudunuri, Uma; Bargo, Sharon; Raafat, Ahmed; McCurdy, David; Boulanger, Corinne; Lowther, William; Stephens, Robert; Luke, Brian T.; Stewart, Claudia; Wu, Xiaolin; Munroe, David; Smith, Gilbert H.

    2012-01-01

    The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene

  5. Personality psychology's comeback and its emerging symbiosis with social psychology.

    PubMed

    Swann, William B; Seyle, Conor

    2005-02-01

    Psychology's early allegiance to behaviorism and experimental methods led many to disparage personality approaches throughout much of last century. Doubts about personality psychology's viability culminated in Mischel's assertion that measures of personality account for modest amounts of variance in behavior. In the years immediately following this critique, interest in personality research waned and many psychology departments dropped their training programs in personality. Throughout the past two decades, however, personality psychology has enjoyed a resurgence. The authors discuss several possible explanations for personality's comeback and then describe the emergence of a promising symbiosis between personality psychology and its sister discipline, social psychology. The article concludes by noting that although this emerging symbiosis is likely to continue bearing considerable theoretical fruit, the traditional distinction between personal, situational, and interactional determinants of behavior continues to be useful within appropriate contexts.

  6. Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation.

    PubMed

    Göhre, Vera; Paszkowski, Uta

    2006-05-01

    High concentrations of heavy metals (HM) in the soil have detrimental effects on ecosystems and are a risk to human health as they can enter the food chain via agricultural products or contaminated drinking water. Phytoremediation, a sustainable and inexpensive technology based on the removal of pollutants from the environment by plants, is becoming an increasingly important objective in plant research. However, as phytoremediation is a slow process, improvement of efficiency and thus increased stabilization or removal of HMs from soils is an important goal. Arbuscular mycorrhizal (AM) fungi provide an attractive system to advance plant-based environmental clean-up. During symbiotic interaction the hyphal network functionally extends the root system of their hosts. Thus, plants in symbiosis with AM fungi have the potential to take up HM from an enlarged soil volume. In this review, we summarize current knowledge about the contribution of the AM symbiosis to phytoremediation of heavy metals.

  7. Tumor Infiltrating Lymphocytes Affect the Outcome of Patients with Operable Triple-Negative Breast Cancer in Combination with Mutated Amino Acid Classes

    PubMed Central

    Kotoula, Vassiliki; Lakis, Sotiris; Vlachos, Ioannis S.; Giannoulatou, Eleni; Zagouri, Flora; Alexopoulou, Zoi; Gogas, Helen; Pectasides, Dimitrios; Aravantinos, Gerasimos; Efstratiou, Ioannis; Pentheroudakis, George; Papadopoulou, Kyriaki; Chatzopoulos, Kyriakos; Papakostas, Pavlos; Sotiropoulou, Maria; Nicolaou, Irene; Razis, Evangelia; Psyrri, Amanda; Kosmidis, Paris; Papadimitriou, Christos; Fountzilas, George

    2016-01-01

    Background Stromal tumor infiltrating lymphocytes (TILs) density is an outcome predictor in triple-negative breast cancer (TNBC). Herein we asked whether TILs are related to coding mutation load and to the chemical class of the resulting mutated amino acids, i.e., charged, polar, and hydrophobic mutations. Methods We examined paraffin tumors from TNBC patients who had been treated with adjuvant chemotherapy mostly within clinical trials (training cohort, N = 133; validation, N = 190) for phenotype concordance; TILs density; mutation load and types. Results Concordance of TNBC phenotypes was 42.1% upon local / central, and 72% upon central / central pathology assessment. TILs were not associated with mutation load, type and class of mutated amino acids. Polar and charged mutation patterns differed between TP53 and PIK3CA (p<0.001). Hydrophobic mutations predicted for early relapse in patients with high nodal burden and <50% TILs tumors (training: HR 3.03, 95%CI 1.11–8.29, p = 0.031; validation: HR 2.90, 95%CI 0.97–8.70, p = 0.057), especially if compared to patients with >50% TILs tumors (training p = 0.003; validation p = 0.015). Conclusions TILs density is unrelated to mutation load in TNBC, which may be regarded as an unstable phenotype. If further validated, hydrophobic mutations along with TILs density may help identifying TNBC patients in higher risk for relapse. PMID:27685159

  8. Establishment of coral-algal symbiosis requires attraction and selection.

    PubMed

    Yamashita, Hiroshi; Suzuki, Go; Kai, Sayaka; Hayashibara, Takeshi; Koike, Kazuhiko

    2014-01-01

    Coral reef ecosystems are based on coral-zooxanthellae symbiosis. During the initiation of symbiosis, majority of corals acquire their own zooxanthellae (specifically from the dinoflagellate genus Symbiodinium) from surrounding environments. The mechanisms underlying the initial establishment of symbiosis have attracted much interest, and numerous field and laboratory experiments have been conducted to elucidate this establishment. However, it is still unclear whether the host corals selectively or randomly acquire their symbionts from surrounding environments. To address this issue, we initially compared genetic compositions of Symbiodinium within naturally settled about 2-week-old Acropora coral juveniles (recruits) and those in the adjacent seawater as the potential symbiont source. We then performed infection tests using several types of Symbiodinium culture strains and apo-symbiotic (does not have Symbiodinium cells yet) Acropora coral larvae. Our field observations indicated apparent preference toward specific Symbiodinium genotypes (A1 and D1-4) within the recruits, despite a rich abundance of other Symbiodinium in the environmental population pool. Laboratory experiments were in accordance with this field observation: Symbiodinium strains of type A1 and D1-4 showed higher infection rates for Acropora larvae than other genotype strains, even when supplied at lower cell densities. Subsequent attraction tests revealed that three Symbiodinium strains were attracted toward Acropora larvae, and within them, only A1 and D1-4 strains were acquired by the larvae. Another three strains did not intrinsically approach to the larvae. These findings suggest the initial establishment of corals-Symbiodinium symbiosis is not random, and the infection mechanism appeared to comprise two steps: initial attraction step and subsequent selective uptake by the coral.

  9. The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis

    SciTech Connect

    Martin, F.; Aerts, A.; Ahren, D.; Brun, A.; Danchin, E. G. J.; Duchaussoy, F.; Gibon, J.; Kohler, A.; Lindquist, E.; Peresa, V.; Salamov, A.; Shapiro, H. J.; Wuyts, J.; Blaudez, D.; Buee, M.; Brokstein, P.; Canback, B.; Cohen, D.; Courty, P. E.; Coutinho, P. M.; Delaruelle, C.; Detter, J. C.; Deveau, A.; DiFazio, S.; Duplessis, S.; Fraissinet-Tachet, L.; Lucic, E.; Frey-Klett, P.; Fourrey, C.; Feussner, I.; Gay, G.; Grimwood, J.; Hoegger, P. J.; Jain, P.; Kilaru, S.; Labbe, J.; Lin, Y. C.; Legue, V.; Le Tacon, F.; Marmeisse, R.; Melayah, D.; Montanini, B.; Muratet, M.; Nehls, U.; Niculita-Hirzel, H.; Secq, M. P. Oudot-Le; Peter, M.; Quesneville, H.; Rajashekar, B.; Reich, M.; Rouhier, N.; Schmutz, J.; Yin, T.; Chalot, M.; Henrissat, B.; Kues, U.; Lucas, S.; Van de Peer, Y.; Podila, G. K.; Polle, A.; Pukkila, P. J.; Richardson, P. M.; Rouze, P.; Sanders, I. R.; Stajich, J. E.; Tunlid, A.; Tuskan, G.; Grigoriev, I. V.

    2007-08-10

    Mycorrhizal symbioses the union of roots and soil fungi are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants 1, 2. Boreal, temperate and montane forests all depend on ectomycorrhizae1. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are

  10. Coral Reef Genomics: Developing tools for functional genomics ofcoral symbiosis

    SciTech Connect

    Schwarz, Jodi; Brokstein, Peter; Manohar, Chitra; Coffroth, MaryAlice; Szmant, Alina; Medina, Monica

    2005-03-01

    Symbioses between cnidarians and dinoflagellates in the genus Symbiodinium are widespread in the marine environment. The importance of this symbiosis to reef-building corals and reef nutrient and carbon cycles is well documented, but little is known about the mechanisms by which the partners establish and regulate the symbiosis. Because the dinoflagellate symbionts live inside the cells of their host coral, the interactions between the partners occur on cellular and molecular levels, as each partner alters the expression of genes and proteins to facilitate the partnership. These interactions can examined using high-throughput techniques that allow thousands of genes to be examined simultaneously. We are developing the groundwork so that we can use DNA microarray profiling to identify genes involved in the Montastraea faveolata and Acropora palmata symbioses. Here we report results from the initial steps in this microarray initiative, that is, the construction of cDNA libraries from 4 of 16 target stages, sequencing of 3450 cDNA clones to generate Expressed Sequenced Tags (ESTs), and annotation of the ESTs to identify candidate genes to include in the microarrays. An understanding of how the coral-dinoflagellate symbiosis is regulated will have implications for atmospheric and ocean sciences, conservation biology, the study and diagnosis of coral bleaching and disease, and comparative studies of animal-protest interactions.

  11. Phosphorus and Nitrogen Regulate Arbuscular Mycorrhizal Symbiosis in Petunia hybrida

    PubMed Central

    Nouri, Eva; Breuillin-Sessoms, Florence; Feller, Urs; Reinhardt, Didier

    2014-01-01

    Phosphorus and nitrogen are essential nutrient elements that are needed by plants in large amounts. The arbuscular mycorrhizal symbiosis between plants and soil fungi improves phosphorus and nitrogen acquisition under limiting conditions. On the other hand, these nutrients influence root colonization by mycorrhizal fungi and symbiotic functioning. This represents a feedback mechanism that allows plants to control the fungal symbiont depending on nutrient requirements and supply. Elevated phosphorus supply has previously been shown to exert strong inhibition of arbuscular mycorrhizal development. Here, we address to what extent inhibition by phosphorus is influenced by other nutritional pathways in the interaction between Petunia hybrida and R. irregularis. We show that phosphorus and nitrogen are the major nutritional determinants of the interaction. Interestingly, the symbiosis-promoting effect of nitrogen starvation dominantly overruled the suppressive effect of high phosphorus nutrition onto arbuscular mycorrhiza, suggesting that plants promote the symbiosis as long as they are limited by one of the two major nutrients. Our results also show that in a given pair of symbiotic partners (Petunia hybrida and R. irregularis), the entire range from mutually symbiotic to parasitic can be observed depending on the nutritional conditions. Taken together, these results reveal complex nutritional feedback mechanisms in the control of root colonization by arbuscular mycorrhizal fungi. PMID:24608923

  12. Phosphorus and nitrogen regulate arbuscular mycorrhizal symbiosis in Petunia hybrida.

    PubMed

    Nouri, Eva; Breuillin-Sessoms, Florence; Feller, Urs; Reinhardt, Didier

    2014-01-01

    Phosphorus and nitrogen are essential nutrient elements that are needed by plants in large amounts. The arbuscular mycorrhizal symbiosis between plants and soil fungi improves phosphorus and nitrogen acquisition under limiting conditions. On the other hand, these nutrients influence root colonization by mycorrhizal fungi and symbiotic functioning. This represents a feedback mechanism that allows plants to control the fungal symbiont depending on nutrient requirements and supply. Elevated phosphorus supply has previously been shown to exert strong inhibition of arbuscular mycorrhizal development. Here, we address to what extent inhibition by phosphorus is influenced by other nutritional pathways in the interaction between Petunia hybrida and R. irregularis. We show that phosphorus and nitrogen are the major nutritional determinants of the interaction. Interestingly, the symbiosis-promoting effect of nitrogen starvation dominantly overruled the suppressive effect of high phosphorus nutrition onto arbuscular mycorrhiza, suggesting that plants promote the symbiosis as long as they are limited by one of the two major nutrients. Our results also show that in a given pair of symbiotic partners (Petunia hybrida and R. irregularis), the entire range from mutually symbiotic to parasitic can be observed depending on the nutritional conditions. Taken together, these results reveal complex nutritional feedback mechanisms in the control of root colonization by arbuscular mycorrhizal fungi.

  13. Structural Motif-Based Homology Modeling of CYP27A1 and Site-Directed Mutational Analyses Affecting Vitamin D Hydroxylation

    PubMed Central

    Prosser, David E.; Guo, YuDing; Jia, Zongchao; Jones, Glenville

    2006-01-01

    Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D3 and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D3 structures into the active site of this model identified potential substrate contact residues in the F-helix, the β-3 sheet, and the β-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1α-hydroxyvitamin D3 to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the β-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the β-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404. PMID:16500955

  14. Mutation of the Rice Narrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport1[OA

    PubMed Central

    Qi, Jing; Qian, Qian; Bu, Qingyun; Li, Shuyu; Chen, Qian; Sun, Jiaqiang; Liang, Wenxing; Zhou, Yihua; Chu, Chengcai; Li, Xugang; Ren, Fugang; Palme, Klaus; Zhao, Bingran; Chen, Jinfeng; Chen, Mingsheng; Li, Chuanyou

    2008-01-01

    The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth. PMID:18562767

  15. Arsenic affects expression and processing of amyloid precursor protein (APP) in primary neuronal cells overexpressing the Swedish mutation of human APP.

    PubMed

    Zarazúa, Sergio; Bürger, Susanne; Delgado, Juan M; Jiménez-Capdeville, Maria E; Schliebs, Reinhard

    2011-06-01

    Arsenic poisoning due to contaminated water and soil, mining waste, glass manufacture, select agrochemicals, as well as sea food, affects millions of people world wide. Recently, an involvement of arsenic in Alzheimer's disease (AD) has been hypothesized (Gong and O'Bryant, 2010). The present study stresses the hypothesis whether sodium arsenite, and its main metabolite, dimethylarsinic acid (DMA), may affect expression and processing of the amyloid precursor protein (APP), using the cholinergic cell line SN56.B5.G4 and primary neuronal cells overexpressing the Swedish mutation of APP, as experimental approaches. Exposure of cholinergic SN56.B5.G4 cells with either sodium arsenite or DMA decreased cell viability in a concentration- and exposure-time dependent manner, and affected the activities of the cholinergic enzymes acetylcholinesterase and choline acetyltransferase. Both sodium arsenite and DMA exposure of SN56.B5.G4 cells resulted in enhanced level of APP, and sAPP in the membrane and cytosolic fractions, respectively. To reveal any effect of arsenic on APP processing, the amounts of APP cleavage products, sAPPβ, and β-amyloid (Aβ) peptides, released into the culture medium of primary neuronal cells derived from transgenic Tg2576 mice, were assessed by ELISA. Following exposure of neuronal cells by sodium arsenite for 12h, the membrane-bound APP level was enhanced, the amount of sAPPβ released into the culture medium was slightly higher, while the levels of Aβ peptides in the culture medium were considerably lower as compared to that assayed in the absence of any drug. The sodium arsenite-induced reduction of Aβ formation suggests an inhibition of the APP γ-cleavage step by arsenite. In contrast, DMA exposure of neuronal cells considerably increased formation of Aβ and sAPPβ, accompanied by enhanced membrane APP level. The DMA-induced changes in APP processing may be the result of the enhanced APP expression. Alternatively, increased Aβ production

  16. A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis.

    PubMed

    Javot, Hélène; Penmetsa, R Varma; Terzaghi, Nadia; Cook, Douglas R; Harrison, Maria J

    2007-01-30

    The arbuscular mycorrhizal (AM) symbiosis is a mutualistic endosymbiosis formed by plant roots and AM fungi. Most vascular flowering plants have the ability to form these associations, which have a significant impact on plant health and consequently on ecosystem function. Nutrient exchange is a central feature of the AM symbiosis, and AM fungi obtain carbon from their plant host while assisting the plant with the acquisition of phosphorus (as phosphate) from the soil. In the AM symbiosis, the fungus delivers P(i) to the root through specialized hyphae called arbuscules. The molecular mechanisms of P(i) and carbon transfer in the symbiosis are largely unknown, as are the mechanisms by which the plant regulates the symbiosis in response to its nutrient status. Plants possess many classes of P(i) transport proteins, including a unique clade (Pht1, subfamily I), members of which are expressed only in the AM symbiosis. Here, we show that MtPT4, a Medicago truncatula member of subfamily I, is essential for the acquisition of P(i) delivered by the AM fungus. However, more significantly, MtPT4 function is critical for AM symbiosis. Loss of MtPT4 function leads to premature death of the arbuscules; the fungus is unable to proliferate within the root, and symbiosis is terminated. Thus, P(i) transport is not only a benefit for the plant but is also a requirement for the AM symbiosis.

  17. Nitric oxide and coral bleaching: is peroxynitrite generation required for symbiosis collapse?

    PubMed

    Hawkins, Thomas D; Davy, Simon K

    2013-09-01

    The temperature-induced collapse ('bleaching') of the coral-dinoflagellate symbiosis is hypothesised to result from symbiont oxidative stress and a subsequent host innate immune-like response. This includes the production of nitric oxide (NO), which is involved in numerous microbial symbioses. Much of NO's cytotoxicity has been attributed to its conversion, in the presence of superoxide (O2(-)), to highly reactive peroxynitrite (ONOO(-)). However, ONOO(-) generation has yet to be observed in either a lower invertebrate or an intracellular mutualism. Using confocal laser scanning microscopy with the fluorescent ONOO(-) indicator aminophenyl fluorescein (APF), we observed strong evidence that ONOO(-) is generated in symbiotic Aiptasia pulchella under conditions known to induce thermal bleaching. However, a role for ONOO(-) in bleaching remains unclear as treatment with a peroxynitrite scavenger had no significant effect on thermal bleaching. Therefore, while ONOO(-) may have a potential for cytotoxicity, in vivo levels of the compound may be insufficient to affect bleaching.

  18. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    PubMed

    Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B; Kato, Takamitsu A; Shih, Hung-Ying; Xie, Xian-Jin; Wilson, Paul F; Brogan, John R; Kurimasa, Akihiro; Chen, David J; Bedford, Joel S; Chen, Benjamin P C

    2014-01-01

    We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  19. Arbuscular mycorrhizal symbiosis ameliorates the optimum quantum yield of photosystem II and reduces non-photochemical quenching in rice plants subjected to salt stress.

    PubMed

    Porcel, Rosa; Redondo-Gómez, Susana; Mateos-Naranjo, Enrique; Aroca, Ricardo; Garcia, Rosalva; Ruiz-Lozano, Juan Manuel

    2015-08-01

    Rice is the most important food crop in the world and is a primary source of food for more than half of the world population. However, salinity is considered the most common abiotic stress reducing its productivity. Soil salinity inhibits photosynthetic processes, which can induce an over-reduction of the reaction centres in photosystem II (PSII), damaging the photosynthetic machinery. The arbuscular mycorrhizal (AM) symbiosis may improve host plant tolerance to salinity, but it is not clear how the AM symbiosis affects the plant photosynthetic capacity, particularly the efficiency of PSII. This study aimed at determining the influence of the AM symbiosis on the performance of PSII in rice plants subjected to salinity. Photosynthetic activity, plant gas-exchange parameters, accumulation of photosynthetic pigments and rubisco activity and gene expression were also measured in order to analyse comprehensively the response of the photosynthetic processes to AM symbiosis and salinity. Results showed that the AM symbiosis enhanced the actual quantum yield of PSII photochemistry and reduced the quantum yield of non-photochemical quenching in rice plants subjected to salinity. AM rice plants maintained higher net photosynthetic rate, stomatal conductance and transpiration rate than nonAM plants. Thus, we propose that AM rice plants had a higher photochemical efficiency for CO2 fixation and solar energy utilization and this increases plant salt tolerance by preventing the injury to the photosystems reaction centres and by allowing a better utilization of light energy in photochemical processes. All these processes translated into higher photosynthetic and rubisco activities in AM rice plants and improved plant biomass production under salinity.

  20. The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells.

    PubMed

    Amornwichet, Napapat; Oike, Takahiro; Shibata, Atsushi; Nirodi, Chaitanya S; Ogiwara, Hideaki; Makino, Haruhiko; Kimura, Yuka; Hirota, Yuka; Isono, Mayu; Yoshida, Yukari; Ohno, Tatsuya; Kohno, Takashi; Nakano, Takashi

    2015-06-11

    Carbon-ion radiotherapy (CIRT) holds promise to treat inoperable locally-advanced non-small cell lung carcinoma (NSCLC), a disease poorly controlled by standard chemoradiotherapy using X-rays. Since CIRT is an extremely limited medical resource, selection of NSCLC patients likely to benefit from it is important; however, biological predictors of response to CIRT are ill-defined. The present study investigated the association between the mutational status of EGFR and KRAS, driver genes frequently mutated in NSCLC, and the relative biological effectiveness (RBE) of carbon-ion beams over X-rays. The assessment of 15 NSCLC lines of different EGFR/KRAS mutational status and that of isogenic NSCLC lines expressing wild-type or mutant EGFR revealed that EGFR-mutant NSCLC cells, but not KRAS-mutant cells, show low RBE. This was attributable to (i) the high X-ray sensitivity of EGFR-mutant cells, since EGFR mutation is associated with a defect in non-homologous end joining, a major pathway for DNA double-strand break (DSB) repair, and (ii) the strong cell-killing effect of carbon-ion beams due to poor repair of carbon-ion beam-induced DSBs regardless of EGFR mutation status. These data highlight the potential of EGFR mutation status as a predictor of response to CIRT, i.e., CIRT may show a high therapeutic index in EGFR mutation-negative NSCLC.

  1. Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system.

    PubMed

    Baldanti, Fausto; Michel, Detlef; Simoncini, Lavinia; Heuschmid, Maria; Zimmermann, Albert; Minisini, Rosalba; Schaarschmidt, Peter; Schmid, Thomas; Gerna, Giuseppe; Mertens, Thomas

    2002-04-01

    Mutations in the human cytomegalovirus (HCMV) UL97 phosphotransferase have been associated with ganciclovir (GCV) resistance due to an impairment of GCV monophosphorylation. Vaccinia virus recombinants (rVV) were generated that encoded different HCMV UL97 proteins (pUL97) with mutations previously detected in resistant HCMV clinical isolates at codons 460, 520, 592, 594, 595, 598 and 607. These rVVs allowed quantification of GCV phosphorylation catalyzed by the different mutated pUL97s. When compared to rVV-UL97 wild type, mean levels of residual intracellular GCV phosphorylation differed by a factor of 10 for the mutated UL97 proteins ranging from 5.2 to 51.8%. Mutations M460V (located in a UL97 region homologous to domain VIb of protein kinases) and H520Q (located in a cytomegalovirus-specific, functionally critical domain) were responsible for the lowest levels of residual GCV phosphorylation (9.3 and 5.2%). Mutations in a region homologous to the domain IX had a lower impact on GCV phosphorylation (15.8-51.8%). The relevance of pUL97 mutation G598S in inducing GCV resistance was demonstrated for the first time.

  2. The naturally occurring mutation Y197C does not affect the expression or signaling of the human histamine H3 receptor.

    PubMed

    Flores-Clemente, Cecilia; Escamilla-Sánchez, Juan; Arias, Juan-Manuel; Arias-Montaño, José-Antonio

    2017-02-22

    There is evidence for genetic polymorphism within the human histamine H3 receptor (hH3R), and a Tyr to Cys exchange at position 197 (Y197C), located in the amino terminus of the fifth transmembrane domain, has been reported. In this work we compared the expression and the pharmacological and signaling properties of wild-type (hH3RWT) and mutant (hH3RY197C) receptors transiently expressed in CHO-K1 cells. The hH3RY197C cDNA was created by overlap extension PCR amplification. Receptor expression and affinity were assessed by N-α-[methyl-(3)H]-histamine binding to cell membranes and intact cells. Receptor function was evaluated by stimulation of [(35)S]-GTPγS binding to cell membranes and by inhibition of forskolin-induced cAMP accumulation in intact cells. The hH3RWT and hH3RY197C were expressed at similar levels (761±68 and 663±66fmol/mg protein for membranes, and 13,434±1533 and 15,894±1884 receptors per cell, respectively). There were no significant differences in the affinities for H3R agonists or antagonists/inverse agonists between the hH3RWT and hH3RY197C, and the H3R agonist RAMH was similarly efficacious and potent to stimulate [(35)S]-GTPγS binding and to inhibit forskolin-induced cAMP accumulation. These results indicate that the Y197C mutation does not affect the expression, ligand affinity or signaling of the human H3 receptor.

  3. Impediment to symbiosis establishment between giant clams and Symbiodinium algae due to sterilization of seawater.

    PubMed

    Kurihara, Takeo; Yamada, Hideaki; Inoue, Ken; Iwai, Kenji; Hatta, Masayuki

    2013-01-01

    To survive the juvenile stage, giant clam juveniles need to establish a symbiotic relationship with the microalgae Symbiodinium occurring in the environment. The percentage of giant clam juveniles succeeding in symbiosis establishment ("symbiosis rate") is often low, which is problematic for seed producers. We investigated how and why symbiosis rates vary, depending on whether giant clam seeds are continuously reared in UV treated or non treated seawater. Results repeatedly demonstrated that symbiosis rates were lower for UV treated seawater than for non treated seawater. Symbiosis rates were also lower for autoclaved seawater and 0.2-µm filtered seawater than for non treated seawater. The decreased symbiosis rates in various sterilized seawater suggest the possibility that some factors helping symbiosis establishment in natural seawater are weakened owing to sterilization. The possible factors include vitality of giant clam seeds, since additional experiments revealed that survival rates of seeds reared alone without Symbiodinium were lower in sterilized seawater than in non treated seawater. In conclusion, UV treatment of seawater was found to lead to decreased symbiosis rates, which is due possibly to some adverse effects common to the various sterilization techniques and relates to the vitality of the giant clam seeds.

  4. Impediment to Symbiosis Establishment between Giant Clams and Symbiodinium Algae Due to Sterilization of Seawater

    PubMed Central

    Kurihara, Takeo; Yamada, Hideaki; Inoue, Ken; Iwai, Kenji; Hatta, Masayuki

    2013-01-01

    To survive the juvenile stage, giant clam juveniles need to establish a symbiotic relationship with the microalgae Symbiodinium occurring in the environment. The percentage of giant clam juveniles succeeding in symbiosis establishment (“symbiosis rate”) is often low, which is problematic for seed producers. We investigated how and why symbiosis rates vary, depending on whether giant clam seeds are continuously reared in UV treated or non treated seawater. Results repeatedly demonstrated that symbiosis rates were lower for UV treated seawater than for non treated seawater. Symbiosis rates were also lower for autoclaved seawater and 0.2-µm filtered seawater than for non treated seawater. The decreased symbiosis rates in various sterilized seawater suggest the possibility that some factors helping symbiosis establishment in natural seawater are weakened owing to sterilization. The possible factors include vitality of giant clam seeds, since additional experiments revealed that survival rates of seeds reared alone without Symbiodinium were lower in sterilized seawater than in non treated seawater. In conclusion, UV treatment of seawater was found to lead to decreased symbiosis rates, which is due possibly to some adverse effects common to the various sterilization techniques and relates to the vitality of the giant clam seeds. PMID:23613802

  5. Three mutations in Zea mays affecting zein accumulation: a comparison of zein polypeptides, in vitro synthesis and processing, mRNA levels, and genomic organization

    SciTech Connect

    Burr, F.A.; Burr, B.

    1982-07-01

    Researchers studied three mutations, opaque-2 (o2), opaque-7 (o7), and floury-2(fl2), each of which causes a depression in zein synthesis. Researchers examined the processing efficiencies of the rough endoplasmic reticulum membranes in vitro, the levels of RNA transcription using cloned zein probes, and the genomic organization of the zein sequences as possible sites for the genetic defects. The results obtained indicate that the steps in prezein translation and processing occurring on the protein body membranes are not accountable for the lowered zein content in any ofl the mutations. The o2 mutation that typically shows a paucity of 22.5-kdalton zein polypeptides was found to have a concomitant reduction in a particular subgroup of mRNAs coding for this size class. Southern analyses suggest that the 02 mutation is not the result of a large deletion of tandem-linked zein genes.

  6. Three mutations in Zea mays affecting zein accumulation: a comparison of zein polypeptides, in vitro synthesis and processing, mRNA levels, and genomic organization

    PubMed Central

    1982-01-01

    We studied three mutations, opaque-2 (o2), opaque-7 (o7), and floury- 2(fI2), each of which causes a depression in zein synthesis. We examined the processing efficiencies of the rough endoplasmic reticulum membranes in vitro, the levels of RNA transcription using cloned zein probes, and the genomic organization of the zein sequences as possible sites for the genetic defects. The results obtained indicate that the steps in prezein translation and processing occurring on the protein body membranes are not accountable for the lowered zein content in any of the mutations. The o2 mutation that typically shows a paucity of 22.5-kdalton zein polypeptides was found to have a concomitant reduction in a particular subgroup of mRNAs coding for this size class. Southern analyses suggest that the o2 mutation is not the result of a large deletion of tandem-linked zein genes. PMID:7119014

  7. Persistent virus and addiction modules: an engine of symbiosis.

    PubMed

    Villarreal, Luis P

    2016-06-01

    The giant DNA viruses are highly prevalent and have a particular affinity for the lytic infection of unicellular eukaryotic host. The giant viruses can also be infected by inhibitory virophage which can provide lysis protection to their host. The combined protective and destructive action of such viruses can define a general model (PD) of virus-mediated host survival. Here, I present a general model for role such viruses play in the evolution of host symbiosis. By considering how virus mixtures can participate in addiction modules, I provide a functional explanation for persistence of virus derived genetic 'junk' in their host genomic habitats.

  8. Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis.

    PubMed

    Bonfante, Paola; Genre, Andrea

    2010-07-27

    Mycorrhizal fungi are a heterogeneous group of diverse fungal taxa, associated with the roots of over 90% of all plant species. Recently, state-of-the-art molecular and genetic tools, coupled to high-throughput sequencing and advanced microscopy, have led to the genome and transcriptome analysis of several symbionts. Signalling pathways between plants and fungi have now been described and the identification of several novel nutrient transporters has revealed some of the cellular processes that underlie symbiosis. Thus, the contributions of each partner in a mycorrhizal association are starting to be unravelled. This new knowledge is now available for use in agricultural practices.

  9. IL36RN Mutations Affect Protein Expression and Function: A Basis for Genotype-Phenotype Correlation in Pustular Diseases.

    PubMed

    Tauber, Marie; Bal, Elodie; Pei, Xue-Yuan; Madrange, Marine; Khelil, Amel; Sahel, Houria; Zenati, Akila; Makrelouf, Mohamed; Boubridaa, Khaled; Chiali, Amel; Smahi, Naima; Otsmane, Farida; Bouajar, Bakar; Marrakchi, Slaheddine; Turki, Hamida; Bourrat, Emmanuelle; Viguier, Manuelle; Hamel, Yamina; Bachelez, Hervé; Smahi, Asma

    2016-09-01

    Homozygous or compound heterozygous IL36RN gene mutations underlie the pathogenesis of psoriasis-related pustular eruptions including generalized pustular psoriasis, palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau, and acute generalized exanthematous pustular eruption. We identified two unreported IL36RN homozygous mutations (c.41C>A/p.Ser14X and c.420_426del/p.Gly141MetfsX29) in patients with familial generalized pustular psoriasis. We analyzed the impact of a spectrum of IL36RN mutations on IL-36 receptor antagonist protein by using site-directed mutagenesis and expression in HEK293T cells. This enabled us to differentiate null mutations with complete absence of IL-36 receptor antagonist (the two previously unreported mutations, c.80T>C/p.Leu27Pro, c.28C>T/p.Arg10X, c.280G>T/p.Glu94X, c.368C>G/p.Thr123Arg, c.368C>T/p.Thr123Met, and c.227C>T/p.Pro76Leu) from mutations with decreased (c.95A>G/p.His32Arg, c.142C>T/p.Arg48Trp, and c.308C>T/p.Ser113Leu) or unchanged (c.304C>T/p.Arg102Trp and c.104A>G/p.Lys35Arg) protein expression. Functional assays measuring the impact of mutations on the capacity to repress IL-36-dependent activation of the NF-κB pathway showed complete functional impairment for null mutations, whereas partial or no impairment was observed for other mutations considered as hypomorphic. Finally, null mutations were associated with severe clinical phenotypes (generalized pustular psoriasis, acute generalized exanthematous pustular eruption), whereas hypomorphic mutations were identified in both localized (palmoplantar pustular psoriasis, acrodermatitis continua of Hallopeau) and generalized variants. These results provide a preliminary basis for genotype-phenotype correlation in patients with deficiency of the IL-36Ra (DITRA), and suggest the involvement of other factors in the modulation of clinical expression.

  10. [Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis]. Progress report

    SciTech Connect

    Guerinot, M.L.

    1992-06-01

    We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

  11. (Iron regulation of gene expression in the Bradyrhizobium japonicum/soybean symbiosis)

    SciTech Connect

    Guerinot, M.L.

    1992-01-01

    We wish to address the question of whether iron plays a regulatory role in the Bradyrhizobium japonicum/soybeam symbiosis. Iron may be an important regulatory signal in planta as the bacteria must acquire iron from their plant hosts and iron-containing proteins figure prominently in all nitrogen-fixing symbioses. For example, the bacterial partner is believed to synthesize the heme moiety of leghemoglobin, which may represent as much as 25--30% of the total soluble protein in an infected plant cell. For this reason, we have focused our attention on the regulation by iron of the first step in the bacterial heme biosynthetic pathway. The enzyme which catalyzes this step, 5-aminolevulinic acid synthase, is encoded by the hemA gene which we had previously cloned and sequenced. Specific objectives include: to define the cis-acting sequences which confer iron regulation on the B. japonicum hemA gene; to identify trans-acting factors which regulate the expression of hemA by iron; to identify new loci which are transcriptionally responsive to changes in iron availability; and to examine the effects of mutations in various known regulatory genes for their effect on the expression of hemA.

  12. Stars and Symbiosis: MicroRNA- and MicroRNA*-Mediated Transcript Cleavage Involved in Arbuscular Mycorrhizal Symbiosis1[W][OA

    PubMed Central

    Devers, Emanuel A.; Branscheid, Anja; May, Patrick; Krajinski, Franziska

    2011-01-01

    The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out high-throughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development. PMID:21571671

  13. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

    PubMed

    Zigler, J Samuel; Hodgkinson, Colin A; Wright, Megan; Klise, Andrew; Sundin, Olof; Broman, Karl W; Hejtmancik, Fielding; Huang, Hao; Patek, Bonnie; Sergeev, Yuri; Hose, Stacey; Brayton, Cory; Xaiodong, Jiao; Vasquez, David; Maragakis, Nicholas; Mori, Susumu; Goldman, David; Hoke, Ahmet; Sinha, Debasish

    2016-01-01

    A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs.

  14. A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems

    PubMed Central

    Zigler, J. Samuel; Hodgkinson, Colin A.; Wright, Megan; Klise, Andrew; Broman, Karl W.; Huang, Hao; Patek, Bonnie; Sergeev, Yuri; Hose, Stacey; Xaiodong, Jiao; Vasquez, David; Maragakis, Nicholas; Mori, Susumu; Goldman, David; Sinha, Debasish

    2016-01-01

    A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70–80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs

  15. Effects of multiple climate change factors on the tall fescue-fungal endophyte symbiosis: infection frequency and tissue chemistry.

    SciTech Connect

    Brosi, Glade; McCulley, Rebecca L; Bush, L P; Nelson, Jim A; Classen, Aimee T; Norby, Richard J

    2011-01-01

    Climate change (altered CO{sub 2}, warming, and precipitation) may affect plant-microbial interactions, such as the Lolium arundinaceum-Neotyphodium coenophialum symbiosis, to alter future ecosystem structure and function. To assess this possibility, tall fescue tillers were collected from an existing climate manipulation experiment in a constructed old-field community in Tennessee (USA). Endophyte infection frequency (EIF) was determined, and infected (E+) and uninfected (E-) tillers were analysed for tissue chemistry. The EIF of tall fescue was higher under elevated CO{sub 2} (91% infected) than with ambient CO{sub 2} (81%) but was not affected by warming or precipitation treatments. Within E+ tillers, elevated CO{sub 2} decreased alkaloid concentrations of both ergovaline and loline, by c. 30%; whereas warming increased loline concentrations 28% but had no effect on ergovaline. Independent of endophyte infection, elevated CO{sub 2} reduced concentrations of nitrogen, cellulose, hemicellulose, and lignin. These results suggest that elevated CO{sub 2}, more than changes in temperature or precipitation, may promote this grass-fungal symbiosis, leading to higher EIF in tall fescue in old-field communities. However, as all three climate factors are likely to change in the future, predicting the symbiotic response and resulting ecological consequences may be difficult and dependent on the specific atmospheric and climatic conditions encountered.

  16. Nitric oxide detoxification in the rhizobia-legume symbiosis.

    PubMed

    Sánchez, Cristina; Cabrera, Juan J; Gates, Andrew J; Bedmar, Eulogio J; Richardson, David J; Delgado, María J

    2011-01-01

    NO (nitric oxide) is a signal molecule involved in diverse physiological processes in cells which can become very toxic under certain conditions determined by its rate of production and diffusion. Several studies have clearly shown the production of NO in early stages of rhizobia-legume symbiosis and in mature nodules. In functioning nodules, it has been demonstrated that NO, which has been reported as a potent inhibitor of nitrogenase activity, can bind Lb (leghaemoglobin) to form LbNOs (nitrosyl-leghaemoglobin complexes). These observations have led to the question of how nodules overcome the toxicity of NO. On the bacterial side, one candidate for NO detoxification in nodules is the respiratory Nor (NO reductase) that catalyses the reduction of NO to nitrous oxide. In addition, rhizobial fHbs (flavohaemoglobins) and single-domain Hbs which dioxygenate NO to form nitrate are candidates to detoxify NO under free-living and symbiotic conditions. On the plant side, sHbs (symbiotic Hbs) (Lb) and nsHbs (non-symbiotic Hbs) have been proposed to play important roles as modulators of NO levels in the rhizobia-legume symbiosis. In the present review, current knowledge of NO detoxification by legume-associated endosymbiotic bacteria is summarized.

  17. An ancient tripartite symbiosis of plants, ants and scale insects.

    PubMed

    Ueda, Shouhei; Quek, Swee-Peck; Itioka, Takao; Inamori, Keita; Sato, Yumiko; Murase, Kaori; Itino, Takao

    2008-10-22

    In the Asian tropics, a conspicuous radiation of Macaranga plants is inhabited by obligately associated Crematogaster ants tending Coccus (Coccidae) scale insects, forming a tripartite symbiosis. Recent phylogenetic studies have shown that the plants and the ants have been codiversifying over the past 16-20 million years (Myr). The prevalence of coccoids in ant-plant mutualisms suggest that they play an important role in the evolution of ant-plant symbioses. To determine whether the scale insects were involved in the evolutionary origin of the mutualism between Macaranga and Crematogaster, we constructed a cytochrome oxidase I (COI) gene phylogeny of the scale insects collected from myrmecophytic Macaranga and estimated their time of origin based on a COI molecular clock. The minimum age of the associated Coccus was estimated to be half that of the ants, at 7-9Myr, suggesting that they were latecomers in the evolutionary history of the symbiosis. Crematogaster mitochondrial DNA (mtDNA) lineages did not exhibit specificity towards Coccus mtDNA lineages, and the latter was not found to be specific towards Macaranga taxa, suggesting that patterns of associations in the scale insects are dictated by opportunity rather than by specialized adaptations to host plant traits.

  18. Diminished exoproteome of Frankia spp. in culture and symbiosis.

    PubMed

    Mastronunzio, J E; Huang, Y; Benson, D R

    2009-11-01

    Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N(2) fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

  19. Understanding resilience in industrial symbiosis networks: insights from network analysis.

    PubMed

    Chopra, Shauhrat S; Khanna, Vikas

    2014-08-01

    Industrial symbiotic networks are based on the principles of ecological systems where waste equals food, to develop synergistic networks. For example, industrial symbiosis (IS) at Kalundborg, Denmark, creates an exchange network of waste, water, and energy among companies based on contractual dependency. Since most of the industrial symbiotic networks are based on ad-hoc opportunities rather than strategic planning, gaining insight into disruptive scenarios is pivotal for understanding the balance of resilience and sustainability and developing heuristics for designing resilient IS networks. The present work focuses on understanding resilience as an emergent property of an IS network via a network-based approach with application to the Kalundborg Industrial Symbiosis (KIS). Results from network metrics and simulated disruptive scenarios reveal Asnaes power plant as the most critical node in the system. We also observe a decrease in the vulnerability of nodes and reduction in single points of failure in the system, suggesting an increase in the overall resilience of the KIS system from 1960 to 2010. Based on our findings, we recommend design strategies, such as increasing diversity, redundancy, and multi-functionality to ensure flexibility and plasticity, to develop resilient and sustainable industrial symbiotic networks.

  20. Metabolic and physiological interdependencies in the Bathymodiolus azoricus symbiosis

    PubMed Central

    Ponnudurai, Ruby; Kleiner, Manuel; Sayavedra, Lizbeth; Petersen, Jillian M; Moche, Martin; Otto, Andreas; Becher, Dörte; Takeuchi, Takeshi; Satoh, Noriyuki; Dubilier, Nicole; Schweder, Thomas; Markert, Stephanie

    2017-01-01

    The hydrothermal vent mussel Bathymodiolus azoricus lives in an intimate symbiosis with two types of chemosynthetic Gammaproteobacteria in its gills: a sulfur oxidizer and a methane oxidizer. Despite numerous investigations over the last decades, the degree of interdependence between the three symbiotic partners, their individual metabolic contributions, as well as the mechanism of carbon transfer from the symbionts to the host are poorly understood. We used a combination of proteomics and genomics to investigate the physiology and metabolism of the individual symbiotic partners. Our study revealed that key metabolic functions are most likely accomplished jointly by B. azoricus and its symbionts: (1) CO2 is pre-concentrated by the host for carbon fixation by the sulfur-oxidizing symbiont, and (2) the host replenishes essential biosynthetic TCA cycle intermediates for the sulfur-oxidizing symbiont. In return (3), the sulfur oxidizer may compensate for the host's putative deficiency in amino acid and cofactor biosynthesis. We also identified numerous ‘symbiosis-specific' host proteins by comparing symbiont-containing and symbiont-free host tissues and symbiont fractions. These proteins included a large complement of host digestive enzymes in the gill that are likely involved in symbiont digestion and carbon transfer from the symbionts to the host. PMID:27801908

  1. Discaria trinervis - Frankia symbiosis promotion by saprophytic actinomycetes.

    PubMed

    Solans, Mariana

    2007-06-01

    The influence of saprophytic actinomycetes strains on the Discaria trinervis - Frankia actinorhizal symbiosis was investigated. Three strains out of 122 isolated from the rhizosphere and rhizoplane of D. trinervis with multiple enzymatic activities, were selected for plant growth experiments: Streptomyces (BCRU-MM40), Actinoplanes (BCRU-ME3) and Micromonospora (BCRU-MM18). Inoculated seedlings of Discaria trinervis were grown in glass tubes with vermiculite-sand for 12 weeks. They were inoculated either with a single saprophytic strain or a combination of one or two of them together with the symbiotic N(2) fixing strain Frankia BCU110501. The saprophytic strains were applied in two experimental series, i.e. mycelium + supernatant simultaneously or mycelium and supernatant (growth medium free of cells) separately. Micromonospora strain MM18 showed a direct promotion effect on shoot growth, when plants were inoculated with mycelium and supernatant together. Streptomyces strain MM40 and Actinoplanes strain ME3 promoted the actinorhizal symbiosis with Frankia and consequently the development of plant shoots, when supernatant was involved as inoculum. It is supposed, that the strains MM18, MM40 and ME3 produce bioactive metabolites, which are released into the culture medium. The saprophytic strains studied could be considered as "promoting or helper rhizoactinomycetes" of the actinorhizal plant D. trinervis.

  2. Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

    PubMed Central

    Zgadzaj, Rafal; Garrido-Oter, Ruben; Jensen, Dorthe Bodker; Koprivova, Anna; Schulze-Lefert, Paul; Radutoiu, Simona

    2016-01-01

    Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance. PMID:27864511

  3. Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis with Sinorhizobium meliloti by preventing nitrogen fixation inhibition.

    PubMed

    Palma, F; López-Gómez, M; Tejera, N A; Lluch, C

    2013-07-01

    In this work we have investigated the contribution of pretreatment with 0.1 and 0.5mM salicylic acid (SA) to the protection against salt stress in root nodules of Medicago sativa in symbiosis with Sinorhizobium meliloti. SA alleviated the inhibition induced by salinity in the plant growth and photosynthetic capacity of M. sativa-S. meliloti symbiosis. In addition, SA prevented the inhibition of the nitrogen fixation capacity under salt stress since nodule biomass was not affected by salinity in SA pretreated plants. Antioxidant enzymes peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), dehidroascorbate reductase (DHAR) and glutathione reductase (GR), key in the main pathway that scavenges H2O2 in plants, were induced by SA pretreatments which suggest that SA may participate in the redox balance in root nodules under salt stress. Catalase activity (CAT) was inhibited around 40% by SA which could be behind the increase of H2O2 detected in nodules of plants pretreated with SA. The accumulation of polyamines (PAs) synthesized in response to salinity was prevented by SA which together with the induction of 1-aminocyclopropane-l-carboxylic acid (ACC) content suggest the prevalence of the ethylene signaling pathway induced by SA in detriment of the synthesis of PAs. In conclusion, SA alleviated the negative effect of salt stress in the M. sativa-S. meliloti symbiosis through the increased level of nodule biomass and the induction of the nodular antioxidant metabolism under salt stress. The H2O2 accumulation and the PAs inhibition induced by SA in nodules of M. sativa suggest that SA activates a hypersensitive response dependent on ethylene.

  4. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige; Sakurai, Takeshi

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  5. Mutational And Structural Studies Of The PixD BLUF Output Signal That Affects Light-Regulated Interactions With PixE

    PubMed Central

    Yuan, Hua; Dragnea, Vladimira; Wu, Qiong; Gardner, Kevin H.; Bauer, Carl E.

    2011-01-01

    PixD (Slr1694) is a BLUF (blue-light using FAD) photoreceptor used by the cyanobacterium Synechocystis sp. PCC6803 to control phototaxis toward blue light. In this study we probe the involvement of a conserved Tyr8-Gln50-Met93 triad in promoting an output signal upon blue light excitation of the bound flavin. Analysis of acrylamide quenching of Trp91 fluorescence shows that the side chain of this residue remains partially solvent exposed in both the lit and dark states. Mutational analysis demonstrates that substitution mutations at Tyr8 and Gln50 result in the loss of the photocycle while a mutation of Met93 does not appreciably disturb the formation of the light excited state and only minimally accelerates its decay from 5.7 s to 4.5 s. However, mutations in Tyr8, Gln50 and Met93 disrupt the ability of PixD dimers to interact with PixE to form a higher ordered PixD10-PixE5 complex, which is indicative of a lit conformational state. Solution NMR spectroscopy and X-ray crystallographic analyses confirm that a Tyr8 to Phe mutation is locked in a pseudo light excited state revealing flexible areas in PixD that likely constitute part of an output signal upon light excitation of wild type PixD. PMID:21688827

  6. A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective Sinorhizobium meliloti symbiosis with alfalfa.

    PubMed

    Yurgel, Svetlana N; Kahn, Michael L

    2008-12-02

    The nitrogen-fixing symbiosis between rhizobia and legume plants is a model of coevolved nutritional complementation. The plants reduce atmospheric CO(2) by photosynthesis and provide carbon compounds to symbiotically associated bacteria; the rhizobia use these compounds to reduce (fix) atmospheric N(2) to ammonia, a form of nitrogen the plants can use. A key feature of symbiotic N(2) fixation is that N(2) fixation is uncoupled from bacterial nitrogen stress metabolism so that the rhizobia generate "excess" ammonia and release this ammonia to the plant. In the symbiosis between Sinorhizobium meliloti and alfalfa, mutations in GlnD, the major bacterial nitrogen stress response sensor protein, led to a symbiosis in which nitrogen was fixed (Fix(+)) but was not effective (Eff(-)) in substantially increasing plant growth. Fixed (15)N(2) was transported to the shoots, but most fixed (15)N was not present in the plant after 24 h. Analysis of free-living S. meliloti strains with mutations in genes related to nitrogen stress response regulation (glnD, glnB, ntrC, and ntrA) showed that catabolism of various nitrogen-containing compounds depended on the NtrC and GlnD components of the nitrogen stress response cascade. However, only mutants of GlnD with an amino terminal deletion had the unusual Fix(+)Eff(-) symbiotic phenotype, and the data suggest that these glnD mutants export fixed nitrogen in a form that the plants cannot use. These results indicate that bacterial nitrogen stress regulation is important to symbiotic productivity and suggest that GlnD may act in a novel way to influence symbiotic behavior.

  7. Identification of novel mutations in HFE, HFE2, TfR2, and SLC40A1 genes in Chinese patients affected by hereditary hemochromatosis.

    PubMed

    Wang, Yongwei; Du, Yali; Liu, Gang; Guo, Shanshan; Hou, Bo; Jiang, Xianyong; Han, Bing; Chang, Yanzhong; Nie, Guangjun

    2017-04-01

    Hereditary hemochromatosis (HH) is a group of inherited iron-overload disorders associated with pathogenic defects in the genes encoding hemochromatosis (HFE), hemojuvelin (HJV/HFE2), hepcidin (HAMP), transferrin receptor 2 (TfR2), and ferroportin (FPN1/SLC40A1) proteins, and the clinical features are well described. However, there have been only a few detailed reports of HH in Chinese populations. Thus, there is insufficient patient information for population-based analyses in Chinese populations or comparative studies among different ethical groups. In the current work, we describe eight Chinese cases of hereditary hemochromatosis. Gene sequencing results revealed eight mutations (five novel mutations) in HFE, HFE2, TfR2, and SLC40A1 genes in these Chinese HH patients. In addition, we used Polymorphism Phenotyping v2 (Polyphen), Sorting Intolerant From Tolerant (SIFT), and a sequence alignment program to predict the molecular consequences of missense mutations.

  8. [Homozygous E387K (1159G>A) mutation of the CYP1B1 gene in a Roma boy affected with primary congenital glaucoma. Case report].

    PubMed

    Vogt, Gábor; Kádasi, Ľudevit Lajos; Czeizel, Endre

    2014-08-17

    Primary congenital glaucoma was diagnosed in a son (born in 2009) of a healthy, non-consanguineous Roma couple. This couple terminated their next two pregnancies because of the 25% recurrence risk of this autosomal recessive ophthalmological abnormality. Molecular genetic analysis showed the homozygote E387K mutation of the CYP1B1 gene in the proband and the presence of this gene mutation in heterozygous form in both parents. This gene mutation is characteristic for Slovakian Roma population. There are two objectives of this case report. On one hand this finding indicates the genetic relationship of Slovakian and Hungarian Romas. On the other hand, the couple plans to have further pregnancies, and prenatal genetic test may help to assess the possible recurrence risk of this hereditary disease.

  9. Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk.

    PubMed

    Mestawet, T A; Girma, A; Adnøy, T; Devold, T G; Vegarud, G E

    2013-08-01

    Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the αs1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5' untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (αs1-, αs2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln77→Pro77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val15 and AGA→AGG in Arg100 of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5' UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of αs1-CN (15.32g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln77→Pro77 was significantly higher than in milks of heterozygous (CA; 9.05g/kg) and reference (AA; 7.61g/kg) genotype animals. The αs2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the

  10. A novel mutation affecting the arginine-137 residue of AVPR2 in dizygous twins leads to nephrogenic diabetes insipidus and attenuated urine exosome aquaporin-2.

    PubMed

    Hinrichs, Gitte R; Hansen, Louise H; Nielsen, Maria R; Fagerberg, Christina; Dieperink, Hans; Rittig, Søren; Jensen, Boye L

    2016-04-01

    Mutations in the vasopressin V2 receptor gene AVPR2 may cause X-linked nephrogenic diabetes insipidus by defective apical insertion of aquaporin-2 in the renal collecting duct principal cell. Substitution mutations with exchange of arginine at codon 137 can cause nephrogenic syndrome of inappropriate antidiuresis or congenital X-linked nephrogenic diabetes insipidus. We present a novel mutation in codon 137 within AVPR2 with substitution of glycine for arginine in male dizygotic twins. Nephrogenic diabetes insipidus was demonstrated by water deprivation test and resistance to vasopressin administration. While a similar urine exosome release rate was shown between probands and controls by western blotting for the marker ALIX, there was a selective decrease in exosome aquaporin-2 versus aquaporin-1 protein in probands compared to controls.

  11. Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene.

    PubMed

    Aznarez, Isabel; Chan, Elayne M; Zielenski, Julian; Blencowe, Benjamin J; Tsui, Lap-Chee

    2003-08-15

    Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

  12. Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

    PubMed

    Zhu, Jian; Cui, Gang; Chen, Ming; Xu, Qinian; Wang, Xiuyun; Zhou, Dai; Lv, Shengxiang; Fu, Linshan; Wang, Zhong; Zuo, Jianling

    2013-05-01

    Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

  13. ida4-1, ida4-2, and ida4-3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms.

    PubMed Central

    LeDizet, M; Piperno, G

    1995-01-01

    We recently determined the nucleotide sequence of the gene encoding p28, a light chain of inner dynein arms of Chlamydomonas axonemes. Here, we show that p28 is the protein encoded by the IDA4 locus. p28, and the dynein heavy chains normally associated with it, are completely absent from the flagella and cell bodies of three allelic strains of ida4, named ida4-1, ida4-2, and ida4-3. We determined the nucleotide sequence of the three alleles of the p28 gene and found in each case a single nucleotide change, affecting the splice sites of the first, second, and fourth introns, respectively. Reverse transcriptase-polymerase chain reaction amplification of RNAs prepared from ida4 cells confirmed that these mutations prevent the correct splicing of the affected introns, thereby blocking the synthesis of full-length p28. These are the first intron splicing mutations described in Chlamydomonas and the first inner dynein arm mutations characterized at the molecular level. The absence in ida4 axonemes of the dynein heavy chains normally found in association with p28 suggests that p28 is necessary for stable assembly of a subset of inner dynein arms or for the binding of these arms to the microtubule doublets. Images PMID:7579690

  14. Getting at the "what" and the "how" in symbiosis.

    PubMed

    Newton, Irene L G

    2017-02-01

    Symbioses are ubiquitous and have had a tremendous impact on the evolution of life on the planet. Indeed, endosymbiosis lead to the generation of the first eukaryotic cell and from that point onwards, eukaryotes have interacted with the other domains of life, sometimes forming persistent and necessary relationships that span generations. However, because the majority of hosts and symbionts are not easily manipulated, the intricate details of these symbioses, an understanding of the molecular underpinnings of these interactions, have not been elucidated. It is difficult to ask questions about the details of a host-microbe symbiosis if either member cannot be cultured, genetically manipulated, or even housed in a laboratory. Several technological advances in recent years may address these difficulties, making it easier for researchers to ask mechanistic questions in symbiotic systems.

  15. Long-distance transport of signals during symbiosis

    PubMed Central

    Xie, Zhi-Ping; Illana, Antonio

    2011-01-01

    Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions. PMID:21455020

  16. Nuclear energy and waste management pyroprocess for system symbiosis

    NASA Astrophysics Data System (ADS)

    Ogawa, Toru; Minato, Kazuo; Okamoto, Yoshihiro; Nishihara, Kenji

    2007-01-01

    The actinide management has become a key issue in nuclear energy. Recovering and fissioning transuranium elements reduce the long-term proliferation risks and the environmental burden. The better way of waste management will be made by system symbiosis: a combination of light-water reactor and fast reactor and/or accelerator-driven transmutation system should be sought. The new recycling technology should be able to achieve good economy with smaller plants, which can process fuels from different types of reactors on a common technical basis. Ease in handling the higher heat load of transuranium nuclides is also important. Pyroprocesses with the use of molten salts are regarded as the strong candidate for such recycling technology. In JAEA, the first laboratory for the high-temperature chemistry of Am and Cm has been established. The fundamental data will be combined with the computer code for predicting the molten-salts electrolytic processes.

  17. Bacterial Molecular Signals in the Sinorhizobium fredii-Soybean Symbiosis

    PubMed Central

    López-Baena, Francisco J.; Ruiz-Sainz, José E.; Rodríguez-Carvajal, Miguel A.; Vinardell, José M.

    2016-01-01

    Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system. PMID:27213334

  18. 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.

  19. Species specificity of symbiosis and secondary metabolism in ascidians.

    PubMed

    Tianero, Ma Diarey B; Kwan, Jason C; Wyche, Thomas P; Presson, Angela P; Koch, Michael; Barrows, Louis R; Bugni, Tim S; Schmidt, Eric W

    2015-03-01

    Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these 'chemical symbioses'. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=-0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and

  20. Lichen symbiosis: nature's high yielding machines for induced hydrogen production.

    PubMed

    Papazi, Aikaterini; Kastanaki, Elizabeth; Pirintsos, Stergios; Kotzabasis, Kiriakos

    2015-01-01

    Hydrogen is a promising future energy source. Although the ability of green algae to produce hydrogen has long been recognized (since 1939) and several biotechnological applications have been attempted, the greatest obstacle, being the O2-sensitivity of the hydrogenase enzyme, has not yet been overcome. In the present contribution, 75 years after the first report on algal hydrogen production, taking advantage of a natural mechanism of oxygen balance, we demonstrate high hydrogen yields by lichens. Lichens have been selected as the ideal organisms in nature for hydrogen production, since they consist of a mycobiont and a photobiont in symbiosis. It has been hypothesized that the mycobiont's and photobiont's consumption of oxygen (increase of COX and AOX proteins of mitochondrial respiratory pathways and PTOX protein of chrolorespiration) establishes the required anoxic conditions for the activation of the phycobiont's hydrogenase in a closed system. Our results clearly supported the above hypothesis, showing that lichens have the ability to activate appropriate bioenergetic pathways depending on the specific incubation conditions. Under light conditions, they successfully use the PSII-dependent and the PSII-independent pathways (decrease of D1 protein and parallel increase of PSaA protein) to transfer electrons to hydrogenase, while under dark conditions, lichens use the PFOR enzyme and the dark fermentative pathway to supply electrons to hydrogenase. These advantages of lichen symbiosis in combination with their ability to survive in extreme environments (while in a dry state) constitute them as unique and valuable hydrogen producing natural factories and pave the way for future biotechnological applications.

  1. Arbuscular mycorrhizal fungi in terms of symbiosis-parasitism continuum.

    PubMed

    Schmidt, B; Gaşpar, S; Camen, D; Ciobanu, I; Sumălan, R

    2011-01-01

    adverse climatic conditions, like temperature shock at the beginning of growing period modified the nature of symbiosis. In this case, the physiological parameters were reduced at colonized plants, while usual, constant growing conditions permitted the normal, efficient and beneficial development of symbiosis.

  2. Origin of Chemical Diversity in Prochloron-Tunicate Symbiosis

    PubMed Central

    Lin, Zhenjian; Torres, Joshua P.; Tianero, M. Diarey; Kwan, Jason C.

    2016-01-01

    ABSTRACT Diversity-generating metabolism leads to the evolution of many different chemicals in living organisms. Here, by examining a marine symbiosis, we provide a precise evolutionary model of how nature generates a family of novel chemicals, the cyanobactins. We show that tunicates and their symbiotic Prochloron cyanobacteria share congruent phylogenies, indicating that Prochloron phylogeny is related to host phylogeny and not to external habitat or geography. We observe that Prochloron exchanges discrete functional genetic modules for cyanobactin secondary metabolite biosynthesis in an otherwise conserved genetic background. The module exchange leads to gain or loss of discrete chemical functional groups. Because the underlying enzymes exhibit broad substrate tolerance, discrete exchange of substrates and enzymes between Prochloron strains leads to the rapid generation of chemical novelty. These results have implications in choosing biochemical pathways and enzymes for engineered or combinatorial biosynthesis. IMPORTANCE While most biosynthetic pathways lead to one or a few products, a subset of pathways are diversity generating and are capable of producing thousands to millions of derivatives. This property is highly useful in biotechnology since it enables biochemical or synthetic biological methods to create desired chemicals. A fundamental question has been how nature itself creates this chemical diversity. Here, by examining the symbiosis between coral reef animals and bacteria, we describe the genetic basis of chemical variation with unprecedented precision. New compounds from the cyanobactin family are created by either varying the substrate or importing needed enzymatic functions from other organisms or via both mechanisms. This natural process matches successful laboratory strategies to engineer the biosynthesis of new chemicals and teaches a new strategy to direct biosynthesis. PMID:27037119

  3. Species specificity of symbiosis and secondary metabolism in ascidians

    PubMed Central

    Tianero, Ma Diarey B; Kwan, Jason C; Wyche, Thomas P; Presson, Angela P; Koch, Michael; Barrows, Louis R; Bugni, Tim S; Schmidt, Eric W

    2015-01-01

    Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these ‘chemical symbioses'. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and

  4. Trans-generational specificity within a cnidarian-algal symbiosis

    NASA Astrophysics Data System (ADS)

    Poland, D. M.; Coffroth, M. A.

    2017-03-01

    Ocean warming and other anthropogenic stresses threaten the symbiosis between tropical reef cnidarians and their dinoflagellate endosymbionts ( Symbiodinium). Offspring of many cnidarians acquire their algal symbionts from the environment, and such flexibility could allow corals to respond to environmental changes between generations. To investigate the effect of both habitat and host genotype on symbiont acquisition, we transplanted aposymbiotic offspring of the common Caribbean octocoral Briareum asbestinum to (1) an environmentally different habitat that lacked B. asbestinum and (2) an environmentally similar habitat where local adults harbored Symbiodinium phylotypes that differed from parental colonies. Symbiont acquisition and establishment of symbioses over time was followed using a within-clade DNA marker (23S chloroplast rDNA) and a within-phylotype marker (unique alleles at a single microsatellite locus). Early in the symbiosis, B. asbestinum juveniles harbored multiple symbiont phylotypes, regardless of source (parent or site). However, with time ( 4 yr), offspring established symbioses with the symbiont phylotype dominant in the parental colonies, regardless of transplant location. Within-phylotype analyses of the symbionts revealed a similar pattern, with offspring acquiring the allelic variant common in symbionts in the parental population regardless of the environment in which the offspring was reared. These data suggest that in this host species, host-symbiont specificity is a genetically determined trait. If this level of specificity is widespread among other symbiotic cnidarians, many cnidarian-algal symbioses may not be able to respond to rapid, climate change-associated environmental changes by means of between-generation switching of symbionts.

  5. Unfolding the secrets of coral–algal symbiosis

    PubMed Central

    Rosic, Nedeljka; Ling, Edmund Yew Siang; Chan, Chon-Kit Kenneth; Lee, Hong Ching; Kaniewska, Paulina; Edwards, David; Dove, Sophie; Hoegh-Guldberg, Ove

    2015-01-01

    Dinoflagellates from the genus Symbiodinium form a mutualistic symbiotic relationship with reef-building corals. Here we applied massively parallel Illumina sequencing to assess genetic similarity and diversity among four phylogenetically diverse dinoflagellate clades (A, B, C and D) that are commonly associated with corals. We obtained more than 30 000 predicted genes for each Symbiodinium clade, with a majority of the aligned transcripts corresponding to sequence data sets of symbiotic dinoflagellates and <2% of sequences having bacterial or other foreign origin. We report 1053 genes, orthologous among four Symbiodinium clades, that share a high level of sequence identity to known proteins from the SwissProt (SP) database. Approximately 80% of the transcripts aligning to the 1053 SP genes were unique to Symbiodinium species and did not align to other dinoflagellates and unrelated eukaryotic transcriptomes/genomes. Six pathways were common to all four Symbiodinium clades including the phosphatidylinositol signaling system and inositol phosphate metabolism pathways. The list of Symbiodinium transcripts common to all four clades included conserved genes such as heat shock proteins (Hsp70 and Hsp90), calmodulin, actin and tubulin, several ribosomal, photosynthetic and cytochrome genes and chloroplast-based heme-containing cytochrome P450, involved in the biosynthesis of xanthophylls. Antioxidant genes, which are important in stress responses, were also preserved, as were a number of calcium-dependent and calcium/calmodulin-dependent protein kinases that may play a role in the establishment of symbiosis. Our findings disclose new knowledge about the genetic uniqueness of symbiotic dinoflagellates and provide a list of homologous genes important for the foundation of coral–algal symbiosis. PMID:25343511

  6. FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

    PubMed

    Peterlongo, Paolo; Catucci, Irene; Colombo, Mara; Caleca, Laura; Mucaki, Eliseos; Bogliolo, Massimo; Marin, Maria; Damiola, Francesca; Bernard, Loris; Pensotti, Valeria; Volorio, Sara; Dall'Olio, Valentina; Meindl, Alfons; Bartram, Claus; Sutter, Christian; Surowy, Harald; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M; Mitchell, Gillian; James, Paul A; Thompson, Ella; Marchetti, Marina; Verzeroli, Cristina; Tartari, Carmen; Capone, Gabriele Lorenzo; Putignano, Anna Laura; Genuardi, Maurizio; Medici, Veronica; Marchi, Isabella; Federico, Massimo; Tognazzo, Silvia; Matricardi, Laura; Agata, Simona; Dolcetti, Riccardo; Della Puppa, Lara; Cini, Giulia; Gismondi, Viviana; Viassolo, Valeria; Perfumo, Chiara; Mencarelli, Maria Antonietta; Baldassarri, Margherita; Peissel, Bernard; Roversi, Gaia; Silvestri, Valentina; Rizzolo, Piera; Spina, Francesca; Vivanet, Caterina; Tibiletti, Maria Grazia; Caligo, Maria Adelaide; Gambino, Gaetana; Tommasi, Stefania; Pilato, Brunella; Tondini, Carlo; Corna, Chiara; Bonanni, Bernardo; Barile, Monica; Osorio, Ana; Benitez, Javier; Balestrino, Luisa; Ottini, Laura; Manoukian, Siranoush; Pierotti, Marco A; Renieri, Alessandra; Varesco, Liliana; Couch, Fergus J; Wang, Xianshu; Devilee, Peter; Hilbers, Florentine S; van Asperen, Christi J; Viel, Alessandra; Montagna, Marco; Cortesi, Laura; Diez, Orland; Balmaña, Judith; Hauke, Jan; Schmutzler, Rita K; Papi, Laura; Pujana, Miguel Angel; Lázaro, Conxi; Falanga, Anna; Offit, Kenneth; Vijai, Joseph; Campbell, Ian; Burwinkel, Barbara; Kvist, Anders; Ehrencrona, Hans; Mazoyer, Sylvie; Pizzamiglio, Sara; Verderio, Paolo; Surralles, Jordi; Rogan, Peter K; Radice, Paolo

    2015-09-15

    Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

  7. FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

    PubMed Central

    Peterlongo, Paolo; Catucci, Irene; Colombo, Mara; Caleca, Laura; Mucaki, Eliseos; Bogliolo, Massimo; Marin, Maria; Damiola, Francesca; Bernard, Loris; Pensotti, Valeria; Volorio, Sara; Dall'Olio, Valentina; Meindl, Alfons; Bartram, Claus; Sutter, Christian; Surowy, Harald; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M.; Mitchell, Gillian; James, Paul A.; Thompson, Ella; Marchetti, Marina; Verzeroli, Cristina; Tartari, Carmen; Capone, Gabriele Lorenzo; Putignano, Anna Laura; Genuardi, Maurizio; Medici, Veronica; Marchi, Isabella; Federico, Massimo; Tognazzo, Silvia; Matricardi, Laura; Agata, Simona; Dolcetti, Riccardo; Puppa, Lara Della; Cini, Giulia; Gismondi, Viviana; Viassolo, Valeria; Perfumo, Chiara; Mencarelli, Maria Antonietta; Baldassarri, Margherita; Peissel, Bernard; Roversi, Gaia; Silvestri, Valentina; Rizzolo, Piera; Spina, Francesca; Vivanet, Caterina; Tibiletti, Maria Grazia; Caligo, Maria Adelaide; Gambino, Gaetana; Tommasi, Stefania; Pilato, Brunella; Tondini, Carlo; Corna, Chiara; Bonanni, Bernardo; Barile, Monica; Osorio, Ana; Benitez, Javier; Balestrino, Luisa; Ottini, Laura; Manoukian, Siranoush; Pierotti, Marco A.; Renieri, Alessandra; Varesco, Liliana; Couch, Fergus J.; Wang, Xianshu; Devilee, Peter; Hilbers, Florentine S.; van Asperen, Christi J.; Viel, Alessandra; Montagna, Marco; Cortesi, Laura; Diez, Orland; Balmaña, Judith; Hauke, Jan; Schmutzler, Rita K.; Papi, Laura; Pujana, Miguel Angel; Lázaro, Conxi; Falanga, Anna; Offit, Kenneth; Vijai, Joseph; Campbell, Ian; Burwinkel, Barbara; Kvist, Anders; Ehrencrona, Hans; Mazoyer, Sylvie; Pizzamiglio, Sara; Verderio, Paolo; Surralles, Jordi; Rogan, Peter K.; Radice, Paolo

    2015-01-01

    Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28–12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04–12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09–13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer. PMID:26130695

  8. Deletions within a defective suppressor-mutator element in maize affect the frequency and developmental timing of its excision from the bronze locus.

    PubMed Central

    Schiefelbein, J W; Raboy, V; Fedoroff, N V; Nelson, O E

    1985-01-01

    Six independent derivatives of the bz-m13 allele, which contains a 2.2-kilobase-pair defective Suppressor-mutator (dSpm) insertion at the bronze (bz) locus, have been isolated and analyzed. The derivatives were selected for alterations in the frequency and timing of somatic reversion; such derivatives have previously been analyzed genetically and designated "changes in state" by McClintock [McClintock, B. (1955) Carnegie Inst. Washington, Yearb. 54, 245-255]. All of the derivatives analyzed in the present study revert substantially later in development than the original insertion mutation and some show a very low frequency of reversion as well. All of the derivatives contain insertions at the same site as the parent bz-m13 allele. Deletions of 400-1300 base pairs were found in the dSpm elements in four of the six derivatives; the remaining derivatives could not be distinguished structurally from the original mutant allele. The results suggest that changes in the frequency and developmental timing of excision are attributable to alterations in the dSpm element. Furthermore, these data suggest that DNA sequences near the ends of the element are important for responding to the two transacting functions supplied by the transposition-competent Suppressor-mutator (Spm) element. Images PMID:2991894

  9. Cloning and insertional inactivation of the dye (sfrA) gene, mutation of which affects sex factor F expression and dye sensitivity of Escherichia coli K-12.

    PubMed Central

    Buxton, R S; Drury, L S

    1983-01-01

    Deletions of the Escherichia coli K-12 chromosome between trpR and thr render the bacterium sensitive to the dye toluidine blue (Dye-), and if male (Hfr or F'), the strain is sterile (Fex-), failing to donate F' or chromosomal markers and resistant to male-specific phages as a consequence of its inability to elaborate F pili. A 6-kilobase SalI fragment of E. coli chromosomal DNA cloned into the plasmid pBR322 has been shown to complement both the Dye- and Fex- phenotypes. Insertion of the transposon gamma delta (Tn1000) into a specific part of this plasmid invariably results in both the Dye- and Fex- phenotypes, indicating that these phenotypes derive from mutation in a single gene. Complementation tests between such insertions and sfrA4, a previously isolated mutation resulting in a Fex- phenotype and reported to code for a transcriptional control factor for F (L. Beutin, P. A. Manning, M. Achtman, and N. Willetts, J. Bacteriol. 145:840-844, 1981), indicated that dye and sfrA4 were mutations in a single cistron. It is proposed that the dye (sfrA) gene product is necessary not only for efficient transcription of the F factor genes, but also for some component(s) of the bacterial envelope, loss of which results in sensitivity to toluidine blue. PMID:6304010

  10. Pathogenic mutations associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy differently affect Jagged1 binding and Notch3 activity via the RBP/JK signaling Pathway.

    PubMed

    Joutel, Anne; Monet, Marie; Domenga, Valérie; Riant, Florence; Tournier-Lasserve, Elisabeth

    2004-02-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterized by the degeneration of smooth-muscle cells in small cerebral arteries. CADASIL is caused by mutations in NOTCH3, one of the four mammalian homologs to the Drosophila melanogaster NOTCH gene. Disease-associated mutations are distributed throughout the 34 epidermal growth factor-like repeats (EGFRs) that compose the extracellular domain of the Notch3 receptor and result in a loss or a gain of a cysteine residue in one of these EGFRs. In human adults, Notch3 expression is highly restricted to vascular smooth-muscle cells. In patients with CADASIL, there is an abnormal accumulation of Notch3 in the vessel. Molecular pathways linking NOTCH3 mutations to degeneration of vascular smooth-muscle cells are as yet poorly understood. In this study, we investigated the effect of CADASIL mutations on Notch3 activity. We studied five naturally occurring mutations: R90C and C212S, located in the previously identified mutational hotspot EGFR2-5; C428S, shown in this study to be located in the ligand-binding domain EGFR10-11; and C542Y and R1006C, located in EGFR13 and EGFR26, respectively. All five mutant proteins were correctly processed. The C428S and C542Y mutant receptors exhibited a significant reduction in Jagged1-induced transcriptional activity of a RBP/JK responsive luciferase reporter, relative to wild-type Notch3. Impaired signaling activity of these two mutants arose through different mechanisms; the C428S mutant lost its Jagged1-binding ability, whereas C542Y retained it but exhibited an impaired presentation to the cell surface. In contrast, the R90C, C212S, and R1006C mutants retained the ability to bind Jagged1 and were associated with apparently normal levels of signaling activity. We conclude that mutations in Notch3 differently affect Jagged1 binding and Notch3 signaling via the RBP/JK pathway.

  11. Positive Gene Regulation by a Natural Protective miRNA Enables Arbuscular Mycorrhizal Symbiosis.

    PubMed

    Couzigou, Jean-Malo; Lauressergues, Dominique; André, Olivier; Gutjahr, Caroline; Guillotin, Bruno; Bécard, Guillaume; Combier, Jean-Philippe

    2017-01-11

    Arbuscular mycorrhizal (AM) symbiosis associates most plants with fungi of the phylum Glomeromycota. The fungus penetrates into roots and forms within cortical cell branched structures called arbuscules for nutrient exchange. We discovered that miR171b has a mismatched cleavage site and is unable to downregulate the miR171 family target gene, LOM1 (LOST MERISTEMS 1). This mismatched cleavage site is conserved among plants that establish AM symbiosis, but not in non-mycotrophic plants. Unlike other members of the miR171 family, miR171b stimulates AM symbiosis and is expressed specifically in root cells that contain arbuscules. MiR171b protects LOM1 from negative regulation by other miR171 family members. These findings uncover a unique mechanism of positive post-transcriptional regulation of gene expression by miRNAs and demonstrate its relevance for the establishment of AM symbiosis.

  12. Landau and Lifshitz' formulation of Le Chatelier's principle: an insight into symbiosis?

    PubMed

    Halabi, T

    2013-12-01

    A correspondence allows application of Landau and Lifshitz' formulation of Le Chatelier's principle from statistical physics to a simple 2-D model of biological symbiosis. The insight: symbionts stabilize the occupation of narrow peaks on fitness landscape.

  13. [One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

    PubMed

    Liu, Yin-Xing; Xiong, Dong-Sheng; Fan, Dong-Mei; Shao, Xiao-Feng; Xu, Yuan-Fu; Zhu, Zhen-Ping; Yang, Chun-Zheng

    2003-05-01

    Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.

  14. The engine of the reef: photobiology of the coral–algal symbiosis

    PubMed Central

    Roth, Melissa S.

    2014-01-01

    Coral reef ecosystems thrive in tropical oligotrophic oceans because of the relationship between corals and endosymbiotic dinoflagellate algae called Symbiodinium. Symbiodinium convert sunlight and carbon dioxide into organic carbon and oxygen to fuel coral growth and calcification, creating habitat for these diverse and productive ecosystems. Light is thus a key regulating factor shaping the productivity, physiology, and ecology of the coral holobiont. Similar to all oxygenic photoautotrophs, Symbiodinium must safely harvest sunlight for photosynthesis and dissipate excess energy to prevent oxidative stress. Oxidative stress is caused by environmental stressors such as those associated with global climate change, and ultimately leads to breakdown of the coral–algal symbiosis known as coral bleaching. Recently, large-scale coral bleaching events have become pervasive and frequent threatening and endangering coral reefs. Because the coral–algal symbiosis is the biological engine producing the reef, the future of coral reef ecosystems depends on the ecophysiology of the symbiosis. This review examines the photobiology of the coral–algal symbiosis with particular focus on the photophysiological responses and timescales of corals and Symbiodinium. Additionally, this review summarizes the light environment and its dynamics, the vulnerability of the symbiosis to oxidative stress, the abiotic and biotic factors influencing photosynthesis, the diversity of the coral–algal symbiosis, and recent advances in the field. Studies integrating physiology with the developing “omics” fields will provide new insights into the coral–algal symbiosis. Greater physiological and ecological understanding of the coral–algal symbiosis is needed for protection and conservation of coral reefs. PMID:25202301

  15. Origin and Evolution of Nitrogen Fixation Genes on Symbiosis Islands and Plasmid in Bradyrhizobium

    PubMed Central

    Okubo, Takashi; Piromyou, Pongdet; Tittabutr, Panlada; Teaumroong, Neung; Minamisawa, Kiwamu

    2016-01-01

    The nitrogen fixation (nif) genes of nodule-forming Bradyrhizobium strains are generally located on symbiosis islands or symbiosis plasmids, suggesting that these genes have been transferred laterally. The nif genes of rhizobial and non-rhizobial Bradyrhizobium strains were compared in order to infer the evolutionary histories of nif genes. Based on all codon positions, the phylogenetic tree of concatenated nifD and nifK sequences showed that nifDK on symbiosis islands formed a different clade from nifDK on non-symbiotic loci (located outside of symbiosis islands and plasmids) with elongated branches; however, these genes were located in close proximity, when only the 1st and 2nd codon positions were analyzed. The guanine (G) and cytosine (C) content of the 3rd codon position of nifDK on symbiosis islands was lower than that on non-symbiotic loci. These results suggest that nif genes on symbiosis islands were derived from the non-symbiotic loci of Bradyrhizobium or closely related strains and have evolved toward a lower GC content with a higher substitution rate than the ancestral state. Meanwhile, nifDK on symbiosis plasmids clustered with nifDK on non-symbiotic loci in the tree representing all codon positions, and the GC content of symbiotic and non-symbiotic loci were similar. These results suggest that nif genes on symbiosis plasmids were derived from the non-symbiotic loci of Bradyrhizobium and have evolved with a similar evolutionary pattern and rate as the ancestral state. PMID:27431195

  16. Origin and Evolution of Nitrogen Fixation Genes on Symbiosis Islands and Plasmid in Bradyrhizobium.

    PubMed

    Okubo, Takashi; Piromyou, Pongdet; Tittabutr, Panlada; Teaumroong, Neung; Minamisawa, Kiwamu

    2016-09-29

    The nitrogen fixation (nif) genes of nodule-forming Bradyrhizobium strains are generally located on symbiosis islands or symbiosis plasmids, suggesting that these genes have been transferred laterally. The nif genes of rhizobial and non-rhizobial Bradyrhizobium strains were compared in order to infer the evolutionary histories of nif genes. Based on all codon positions, the phylogenetic tree of concatenated nifD and nifK sequences showed that nifDK on symbiosis islands formed a different clade from nifDK on non-symbiotic loci (located outside of symbiosis islands and plasmids) with elongated branches; however, these genes were located in close proximity, when only the 1st and 2nd codon positions were analyzed. The guanine (G) and cytosine (C) content of the 3rd codon position of nifDK on symbiosis islands was lower than that on non-symbiotic loci. These results suggest that nif genes on symbiosis islands were derived from the non-symbiotic loci of Bradyrhizobium or closely related strains and have evolved toward a lower GC content with a higher substitution rate than the ancestral state. Meanwhile, nifDK on symbiosis plasmids clustered with nifDK on non-symbiotic loci in the tree representing all codon positions, and the GC content of symbiotic and non-symbiotic loci were similar. These results suggest that nif genes on symbiosis plasmids were derived from the non-symbiotic loci of Bradyrhizobium and have evolved with a similar evolutionary pattern and rate as the ancestral state.

  17. Carbon availability triggers fungal nitrogen uptake and transport in arbuscular mycorrhizal symbiosis.

    PubMed

    Fellbaum, Carl R; Gachomo, Emma W; Beesetty, Yugandhar; Choudhari, Sulbha; Strahan, Gary D; Pfeffer, Philip E; Kiers, E Toby; Bücking, Heike

    2012-02-14

    The arbuscular mycorrhizal (AM) symbiosis, formed between the majority of land plants and ubiquitous soil fungi of the phylum Glomeromycota, is responsible for massive nutrient transfer and global carbon sequestration. AM fungi take up nutrients from the soil and exchange them against photosynthetically fixed carbon (C) from the host. Recent studies have demonstrated that reciprocal reward strategies by plant and fungal partners guarantee a "fair trade" of phosphorus against C between partners [Kiers ET, et al. (2011) Science 333:880-882], but whether a similar reward mechanism also controls nitrogen (N) flux in the AM symbiosis is not known. Using mycorrhizal root organ cultures, we manipulated the C supply to the host and fungus and followed the uptake and transport of N sources in the AM symbiosis, the enzymatic activities of arginase and urease, and fungal gene expression in the extraradical and intraradical mycelium. We found that the C supply of the host plant triggers the uptake and transport of N in the symbiosis, and that the increase in N transport is orchestrated by changes in fungal gene expression. N transport in the symbiosis is stimulated only when the C is delivered by the host across the mycorrhizal interface, not when C is supplied directly to the fungal extraradical mycelium in the form of acetate. These findings support the importance of C flux from the root to the fungus as a key trigger for N uptake and transport and provide insight into the N transport regulation in the AM symbiosis.

  18. Book review of Insect Symbiosis. Volume 2. Bourtzis, K.A. and Miller, T.A. editros. 2006 CRC Press, Taylor and Francis Group, Boca Raton, FL, 276 pp. ISBN 0-8493-1286-8

    SciTech Connect

    Hoy, M.A.

    2007-03-15

    There are several definitions of symbiosis, but in this book it involves an association where one organism (the symbiont) lives within or on the body of another organism (the host), regardless of the actual effect on the host. Some symbioses are mutualistic, some parasitic, and some involve commensalism, in which one partner derives some benefit without either harming or benefiting the other. This is the second volume in this exciting and rapidly advancing topic by these editors. The first volume was published in 2003 and during the intervening three years additional data have been produced that make this book a useful addition to your library. The first book provided chapters that provided an overview of insect symbiosis, discussions of the primary aphid symbiont Buchnera and other aphid symbionts, symbiosis in tsetse, symbionts in the weevil Sitophilus , the possible use of paratransgenic symbionts of Rhodnius prolixis to prevent disease transmission, bark beetle and fungal symbiosis, symbionts of tephritid fruit flies, symbionts affecting termite behavior, an overview of microsporidia as symbionts (parasites?) of insects, an overview of a newly discovered bacterium that causes sex-ratio distortion in insects and mites (from the Bacteroides group), symbionts that selectively kill male insects, and several chapters on the ubiquitous endosymbiont Wolbachia.

  19. Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii.

    PubMed

    Boretsky, Yuriy R; Protchenko, Olga V; Prokopiv, Tetiana M; Mukalov, Igor O; Fedorovych, Daria V; Sibirny, Andriy A

    2007-10-01

    Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.

  20. A Chemotaxis Receptor Modulates Nodulation during the Azorhizobium caulinodans-Sesbania rostrata Symbiosis

    PubMed Central

    Jiang, Nan; Liu, Wei; Li, Yan; Wu, Hailong; Zhang, Zhenhai; Elmerich, Claudine

    2016-01-01

    ABSTRACT Azorhizobium caulinodans ORS571 is a free-living nitrogen-fixing bacterium which can induce nitrogen-fixing nodules both on the root and the stem of its legume host Sesbania rostrata. This bacterium, which is an obligate aerobe that moves by means of a polar flagellum, possesses a single chemotaxis signal transduction pathway. The objective of this work was to examine the role that chemotaxis and aerotaxis play in the lifestyle of the bacterium in free-living and symbiotic conditions. In bacterial chemotaxis, chemoreceptors sense environmental changes and transmit this information to the chemotactic machinery to guide motile bacteria to preferred niches. Here, we characterized a chemoreceptor of A. caulinodans containing an N-terminal PAS domain, named IcpB. IcpB is a soluble heme-binding protein that localized at the cell poles. An icpB mutant strain was impaired in sensing oxygen gradients and in chemotaxis response to organic acids. Compared to the wild-type strain, the icpB mutant strain was also affected in the production of extracellular polysaccharides and impaired in flocculation. When inoculated alone, the icpB mutant induced nodules on S. rostrata, but the nodules formed were smaller and had reduced N2-fixing activity. The icpB mutant failed to nodulate its host when inoculated competitively with the wild-type strain. Together, the results identify chemotaxis and sensing of oxygen by IcpB as key regulators of the A. caulinodans-S. rostrata symbiosis. IMPORTANCE Bacterial chemotaxis has been implicated in the establishment of various plant-microbe associations, including that of rhizobial symbionts with their legume host. The exact signal(s) detected by the motile bacteria that guide them to their plant hosts remain poorly characterized. Azorhizobium caulinodans ORS571 is a diazotroph that is a motile and chemotactic rhizobial symbiont of Sesbania rostrata, where it forms nitrogen-fixing nodules on both the roots and the stems of the legume host

  1. The micro-RNA72c-APETALA2-1 node as a key regulator of the common bean-Rhizobium etli nitrogen fixation symbiosis.

    PubMed

    Nova-Franco, Bárbara; Íñiguez, Luis P; Valdés-López, Oswaldo; Alvarado-Affantranger, Xochitl; Leija, Alfonso; Fuentes, Sara I; Ramírez, Mario; Paul, Sujay; Reyes, José L; Girard, Lourdes; Hernández, Georgina

    2015-05-01

    Micro-RNAs are recognized as important posttranscriptional regulators in plants. The relevance of micro-RNAs as regulators of the legume-rhizobia nitrogen-fixing symbiosis is emerging. The objective of this work was to functionally characterize the role of micro-RNA172 (miR172) and its conserved target APETALA2 (AP2) transcription factor in the common bean (Phaseolus vulgaris)-Rhizobium etli symbiosis. Our expression analysis revealed that mature miR172c increased upon rhizobial infection and continued increasing during nodule development, reaching its maximum in mature nodules and decaying in senescent nodules. The expression of AP2-1 target showed a negative correlation with miR172c expression. A drastic decrease in miR172c and high AP2-1 mRNA levels were observed in ineffective nodules. Phenotypic analysis of composite bean plants with transgenic roots overexpressing miR172c or a mutated AP2-1 insensitive to miR172c cleavage demonstrated the pivotal regulatory role of the miR172 node in the common bean-rhizobia symbiosis. Increased miR172 resulted in improved root growth, increased rhizobial infection, increased expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen fixation. In addition, these plants showed decreased sensitivity to nitrate inhibition of nodulation. Through transcriptome analysis, we identified 114 common bean genes that coexpressed with AP2-1 and proposed these as being targets for transcriptional activation by AP2-1. Several of these genes are related to nodule senescence, and we propose that they have to be silenced, through miR172c-induced AP2-1 cleavage, in active mature nodules. Our work sets the basis for exploring the miR172-mediated improvement of symbiotic nitrogen fixation in common bean, the most important grain legume for human consumption.

  2. The Micro-RNA172c-APETALA2-1 Node as a Key Regulator of the Common Bean-Rhizobium etli Nitrogen Fixation Symbiosis1[OPEN

    PubMed Central

    Nova-Franco, Bárbara; Íñiguez, Luis P.; Valdés-López, Oswaldo; Leija, Alfonso; Fuentes, Sara I.; Ramírez, Mario; Paul, Sujay

    2015-01-01

    Micro-RNAs are recognized as important posttranscriptional regulators in plants. The relevance of micro-RNAs as regulators of the legume-rhizobia nitrogen-fixing symbiosis is emerging. The objective of this work was to functionally characterize the role of micro-RNA172 (miR172) and its conserved target APETALA2 (AP2) transcription factor in the common bean (Phaseolus vulgaris)-Rhizobium etli symbiosis. Our expression analysis revealed that mature miR172c increased upon rhizobial infection and continued increasing during nodule development, reaching its maximum in mature nodules and decaying in senescent nodules. The expression of AP2-1 target showed a negative correlation with miR172c expression. A drastic decrease in miR172c and high AP2-1 mRNA levels were observed in ineffective nodules. Phenotypic analysis of composite bean plants with transgenic roots overexpressing miR172c or a mutated AP2-1 insensitive to miR172c cleavage demonstrated the pivotal regulatory role of the miR172 node in the common bean-rhizobia symbiosis. Increased miR172 resulted in improved root growth, increased rhizobial infection, increased expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen fixation. In addition, these plants showed decreased sensitivity to nitrate inhibition of nodulation. Through transcriptome analysis, we identified 114 common bean genes that coexpressed with AP2-1 and proposed these as being targets for transcriptional activation by AP2-1. Several of these genes are related to nodule senescence, and we propose that they have to be silenced, through miR172c-induced AP2-1 cleavage, in active mature nodules. Our work sets the basis for exploring the miR172-mediated improvement of symbiotic nitrogen fixation in common bean, the most important grain legume for human consumption. PMID:25739700

  3. The R215W mutation in NBS1 impairs {gamma}-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients

    SciTech Connect

    Masi, Alessandra di Viganotti, Mara; Polticelli, Fabio; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio

    2008-05-09

    Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by chromosomal instability and hypersensitivity to ionising radiation. Compound heterozygous 657del5/R215W NBS patients display a clinical phenotype more severe than the majority of NBS patients homozygous for the 657del5 mutation. The NBS1 protein, mutated in NBS patients, contains a FHA/BRCT domain necessary for the DNA-double strand break (DSB) damage response. Recently, a second BRCT domain has been identified, however, its role is still unknown. Here, we demonstrate that the R215W mutation in NBS1 impairs histone {gamma}-H2AX binding after induction of DNA damage, leading to a delay in DNA-DSB rejoining. Molecular modelling reveals that the 215 residue of NBS1 is located between the two BRCT domains, affecting their relative orientation that appears critical for {gamma}-H2AX binding. Present data represent the first evidence for the role of NBS1 tandem BRCT domains in {gamma}-H2AX recognition, and could explain the severe phenotype observed in 657del5/R215W NBS patients.

  4. Auxin Perception Is Required for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis1[W

    PubMed Central

    Etemadi, Mohammad; Gutjahr, Caroline; Couzigou, Jean-Malo; Zouine, Mohamed; Lauressergues, Dominique; Timmers, Antonius; Audran, Corinne; Bouzayen, Mondher; Bécard, Guillaume; Combier, Jean-Philippe

    2014-01-01

    Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells. PMID:25096975

  5. A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.

    PubMed

    Nanjareddy, Kalpana; Blanco, Lourdes; Arthikala, Manoj-Kumar; Alvarado-Affantranger, Xóchitl; Quinto, Carmen; Sánchez, Federico; Lara, Miguel

    2016-11-01

    The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR's role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean.

  6. Altered Carbohydrates Allocation by Associated Bacteria-fungi Interactions in a Bark Beetle-microbe Symbiosis.

    PubMed

    Zhou, Fangyuan; Lou, Qiaozhe; Wang, Bo; Xu, Letian; Cheng, Chihang; Lu, Min; Sun, Jianghua

    2016-02-03

    Insect-microbe interaction is a key area of research in multiplayer symbiosis, yet little is known about the role of microbe-microbe interactions in insect-microbe symbioses. The red turpentine beetle (RTB) has destroyed millions of healthy pines in China and forms context-dependent relationships with associated fungi. The adult-associated fungus Leptographium procerum have played key roles in RTB colonization. However, common fungal associates (L. procerum and Ophiostoma minus) with RTB larvae compete for carbohydrates. Here, we report that dominant bacteria associated with RTB larvae buffer the competition by inhibiting the growth and D-glucose consumption of O. minus. However, they didn't inhibit the growth of L. procerum and forced this fungus to consume D-pinitol before consuming D-glucose, even though D-glucose was available and a better carbon source not only for L. procerum but also for RTB larvae and associated bacteria. This suggests the most frequently isolated bacteria associated with RTB larvae could affect fungal growth and the sequence of carbohydrate consumption. Thus, this regulates carbohydrate allocation in the RTB larva-microbe community, which may in turn benefit RTB larvae development. We also discuss the mechanism of carbohydrate allocation in the RTB larva-microbe community, and its potential contribution to the maintenance of a symbiotic community.

  7. Altered Carbohydrates Allocation by Associated Bacteria-fungi Interactions in a Bark Beetle-microbe Symbiosis

    PubMed Central

    Zhou, Fangyuan; Lou, Qiaozhe; Wang, Bo; Xu, Letian; Cheng, Chihang; Lu, Min; Sun, Jianghua

    2016-01-01

    Insect-microbe interaction is a key area of research in multiplayer symbiosis, yet little is known about the role of microbe-microbe interactions in insect-microbe symbioses. The red turpentine beetle (RTB) has destroyed millions of healthy pines in China and forms context-dependent relationships with associated fungi. The adult-associated fungus Leptographium procerum have played key roles in RTB colonization. However, common fungal associates (L. procerum and Ophiostoma minus) with RTB larvae compete for carbohydrates. Here, we report that dominant bacteria associated with RTB larvae buffer the competition by inhibiting the growth and D-glucose consumption of O. minus. However, they didn’t inhibit the growth of L. procerum and forced this fungus to consume D-pinitol before consuming D-glucose, even though D-glucose was available and a better carbon source not only for L. procerum but also for RTB larvae and associated bacteria. This suggests the most frequently isolated bacteria associated with RTB larvae could affect fungal growth and the sequence of carbohydrate consumption. Thus, this regulates carbohydrate allocation in the RTB larva-microbe community, which may in turn benefit RTB larvae development. We also discuss the mechanism of carbohydrate allocation in the RTB larva-microbe community, and its potential contribution to the maintenance of a symbiotic community. PMID:26839264

  8. Friend or foe? A behavioral and stable isotopic investigation of an ant-plant symbiosis.

    PubMed

    Tillberg, Chadwick V

    2004-08-01

    In ant-plant symbioses, the behavior of ant inhabitants affects the nature of the interaction, ranging from mutualism to parasitism. Mutualistic species confer a benefit to the plant, while parasites reap the benefits of the interaction without reciprocating, potentially resulting in a negative impact on the host plant. Using the ant-plant symbiosis between Cordia alliodora and its ant inhabitants as a model system, I examine the costs and benefits of habitation by the four most common ant inhabitants at La Selva Biological Station, Costa Rica. Costs are measured by counting coccoids associated with each ant species. Benefits include patrolling behavior, effectiveness at locating resources, and recruitment response. I also compare the diets of the four ant species using stable isotope analysis of nitrogen (N) and carbon (C). Ants varied in their rates of association with coccoids, performance of beneficial behaviors, and diet. These differences in cost, benefit, and diet among the ant species suggest differences in the nature of the symbiotic relationship between C. alliodora and its ants. Two of the ant species behave in a mutualistic manner, while the other two ant species appear to be parasites of the mutualism. I determined that the mutualistic ants feed at a higher trophic level than the parasitic ants. Behavioral and dietary evidence indicate the protective role of the mutualists, and suggest that the parasitic ants do not protect the plant by consuming herbivores.

  9. Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

    PubMed

    Gremillion, S K; Harris, S D; Jackson-Hayes, L; Kaminskyj, S G W; Loprete, D M; Gauthier, A C; Mercer, S; Ravita, A J; Hill, T W

    2014-12-01

    We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus.

  10. Common mycorrhizal networks and their effect on the bargaining power of the fungal partner in the arbuscular mycorrhizal symbiosis

    PubMed Central

    Bücking, Heike; Mensah, Jerry A.; Fellbaum, Carl R.

    2016-01-01

    ABSTRACT Arbuscular mycorrhizal (AM) fungi form mutualistic interactions with the majority of land plants, including some of the most important crop species. The fungus takes up nutrients from the soil, and transfers these nutrients to the mycorrhizal interface in the root, where these nutrients are exchanged against carbon from the host. AM fungi form extensive hyphal networks in the soil and connect with their network multiple host plants. These common mycorrhizal networks (CMNs) play a critical role in the long-distance transport of nutrients through soil ecosystems and allow the exchange of signals between the interconnected plants. CMNs affect the survival, fitness, and competitiveness of the fungal and plant species that interact via these networks, but how the resource transport within these CMNs is controlled is largely unknown. We discuss the significance of CMNs for plant communities and for the bargaining power of the fungal partner in the AM symbiosis. PMID:27066184

  11. A mutation in cnot8, component of the Ccr4-not complex regulating transcript stability, affects expression levels of developmental regulators and reveals a role of Fgf3 in development of caudal hypothalamic dopaminergic neurons.

    PubMed

    Koch, Peter; Löhr, Heiko B; Driever, Wolfgang

    2014-01-01

    While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of

  12. A Mutation in cnot8, Component of the Ccr4-Not Complex Regulating Transcript Stability, Affects Expression Levels of Developmental Regulators and Reveals a Role of Fgf3 in Development of Caudal Hypothalamic Dopaminergic Neurons

    PubMed Central

    Koch, Peter; Löhr, Heiko B.; Driever, Wolfgang

    2014-01-01

    While regulation of the activity of developmental control genes at the transcriptional level as well as by specific miRNA-based degradation are intensively studied, little is known whether general cellular mechanisms controlling mRNA decay may contribute to differential stability of mRNAs of developmental control genes. Here, we investigate whether a mutation in the deadenylation dependent mRNA decay pathway may reveal differential effects on developmental mechanisms, using dopaminergic differentiation in the zebrafish brain as model system. In a zebrafish genetic screen aimed at identifying genes controlling dopaminergic neuron development we isolated the m1061 mutation that selectively caused increased dopaminergic differentiation in the caudal hypothalamus, while other dopaminergic groups were not affected. Positional cloning revealed that m1061 causes a premature stop codon in the cnot8 open reading frame. Cnot8 is a component of the Ccr4-Not complex and displays deadenylase activity, which is required for removal of the poly (A) tail in bulk mRNA turnover. Analyses of expression of developmental regulators indicate that loss of Cnot8 activity results in increased mRNA in situ hybridization signal levels for a subset of developmental control genes. We show that in the area of caudal hypothalamic dopaminergic differentiation, mRNA levels for several components of the FGF signaling pathway, including Fgf3, FGF receptors, and FGF target genes, are increased. Pharmacological inhibition of FGF signaling or a mutation in the fgf3 gene can compensate the gain of caudal hypothalamic dopaminergic neurons in cnot8m1061 mutants, indicating a role for Fgf3 in control of development of this dopaminergic population. The cnot8m1061 mutant phenotype provides an in vivo system to study roles of the Cnot8 deadenylase component of the mRNA decay pathway in vertebrate development. Our data indicate that attenuation of Cnot8 activity differentially affects mRNA levels of

  13. mRNA decay during herpes simplex virus (HSV) infections: mutations that affect translation of an mRNA influence the sites at which it is cleaved by the HSV virion host shutoff (Vhs) protein.

    PubMed

    Shiflett, Lora A; Read, G Sullivan

    2013-01-01

    During lytic infections, the herpes simplex virus (HSV) virion host shutoff (Vhs) endoribonuclease degrades many host and viral mRNAs. Within infected cells it cuts mRNAs at preferred sites, including some in regions of translation initiation. Vhs binds the translation initiation factors eIF4H, eIF4AI, and eIF4AII, suggesting that its mRNA degradative function is somehow linked to translation. To explore how Vhs is targeted to preferred sites, we examined the in vitro degradation of a target mRNA in rabbit reticulocyte lysates containing in vitro-translated Vhs. Vhs caused rapid degradation of mRNAs beginning with cleavages at sites in the first 250 nucleotides, including a number near the start codon and in the 5' untranslated region. Ligation of the ends to form a circular mRNA inhibited Vhs cleavage at the same sites at which it cuts capped linear molecules. This was not due to an inability to cut any circular RNA, since Vhs cuts circular mRNAs containing an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) at the same sites as linear molecules with the IRES. Cutting linear mRNAs at preferred sites was augmented by the presence of a 5' cap. Moreover, mutations that altered the 5' proximal AUG abolished Vhs cleavage at nearby sites, while mutations that changed sequences surrounding the AUG to improve their match to the Kozak consensus sequence enhanced Vhs cutting near the start codon. The results indicate that mutations in an mRNA that affect its translation affect the sites at which it is cut by Vhs and suggest that Vhs is directed to its preferred cut sites during translation initiation.

  14. Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Seo, Susan M; Birukou, Ivan; Brennan, Richard G; Kaatz, Glenn W

    2015-03-01

    The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates.

  15. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    SciTech Connect

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng -Hua; Zhang, Jin -Song

    2016-01-08

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.

  16. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

    PubMed Central

    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  17. Global distribution and vertical patterns of a prymnesiophyte-cyanobacteria obligate symbiosis.

    PubMed

    Cabello, Ana M; Cornejo-Castillo, Francisco M; Raho, Nicolas; Blasco, Dolors; Vidal, Montserrat; Audic, Stéphane; de Vargas, Colomban; Latasa, Mikel; Acinas, Silvia G; Massana, Ramon

    2016-03-01

    A marine symbiosis has been recently discovered between prymnesiophyte species and the unicellular diazotrophic cyanobacterium UCYN-A. At least two different UCYN-A phylotypes exist, the clade UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and the clade UCYN-A2 in symbiosis with the larger Braarudosphaera bigelowii. We targeted the prymnesiophyte-UCYN-A1 symbiosis by double CARD-FISH (catalyzed reporter deposition-fluorescence in situ hybridization) and analyzed its abundance in surface samples from the MALASPINA circumnavigation expedition. Our use of a specific probe for the prymnesiophyte partner allowed us to verify that this algal species virtually always carried the UCYN-A symbiont, indicating that the association was also obligate for the host. The prymnesiophyte-UCYN-A1 symbiosis was detected in all ocean basins, displaying a patchy distribution with abundances (up to 500 cells ml(-1)) that could vary orders of magnitude. Additional vertical profiles taken at the NE Atlantic showed that this symbiosis occupied the upper water column and disappeared towards the Deep Chlorophyll Maximum, where the biomass of the prymnesiophyte assemblage peaked. Moreover, sequences of both prymnesiophyte partners were searched within a large 18S rDNA metabarcoding data set from the Tara-Oceans expedition around the world. This sequence-based analysis supported the patchy distribution of the UCYN-A1 host observed by CARD-FISH and highlighted an unexpected homogeneous distribution (at low relative abundance) of B. bigelowii in the open ocean. Our results demonstrate that partners are always in symbiosis in nature and show contrasted ecological patterns of the two related lineages.

  18. Global distribution and vertical patterns of a prymnesiophyte–cyanobacteria obligate symbiosis

    PubMed Central

    Cabello, Ana M; Cornejo-Castillo, Francisco M; Raho, Nicolas; Blasco, Dolors; Vidal, Montserrat; Audic, Stéphane; de Vargas, Colomban; Latasa, Mikel; Acinas, Silvia G; Massana, Ramon

    2016-01-01

    A marine symbiosis has been recently discovered between prymnesiophyte species and the unicellular diazotrophic cyanobacterium UCYN-A. At least two different UCYN-A phylotypes exist, the clade UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and the clade UCYN-A2 in symbiosis with the larger Braarudosphaera bigelowii. We targeted the prymnesiophyte–UCYN-A1 symbiosis by double CARD-FISH (catalyzed reporter deposition-fluorescence in situ hybridization) and analyzed its abundance in surface samples from the MALASPINA circumnavigation expedition. Our use of a specific probe for the prymnesiophyte partner allowed us to verify that this algal species virtually always carried the UCYN-A symbiont, indicating that the association was also obligate for the host. The prymnesiophyte–UCYN-A1 symbiosis was detected in all ocean basins, displaying a patchy distribution with abundances (up to 500 cells ml−1) that could vary orders of magnitude. Additional vertical profiles taken at the NE Atlantic showed that this symbiosis occupied the upper water column and disappeared towards the Deep Chlorophyll Maximum, where the biomass of the prymnesiophyte assemblage peaked. Moreover, sequences of both prymnesiophyte partners were searched within a large 18S rDNA metabarcoding data set from the Tara-Oceans expedition around the world. This sequence-based analysis supported the patchy distribution of the UCYN-A1 host observed by CARD-FISH and highlighted an unexpected homogeneous distribution (at low relative abundance) of B. bigelowii in the open ocean. Our results demonstrate that partners are always in symbiosis in nature and show contrasted ecological patterns of the two related lineages. PMID:26405830

  19. Emergy-based assessment on industrial symbiosis: a case of Shenyang Economic and Technological Development Zone.

    PubMed

    Geng, Yong; Liu, Zuoxi; Xue, Bing; Dong, Huijuan; Fujita, Tsuyoshi; Chiu, Anthony

    2014-12-01

    Industrial symbiosis is the sharing of services, utility, and by-product resources among industries. This is usually made in order to add value, reduce costs, and improve the environment, and therefore has been taken as an effective approach for developing an eco-industrial park, improving resource efficiency, and reducing pollutant emission. Most conventional evaluation approaches ignored the contribution of natural ecosystem to the development of industrial symbiosis and cannot reveal the interrelations between economic development and environmental protection, leading to a need of an innovative evaluation method. Under such a circumstance, we present an emergy analysis-based evaluation method by employing a case study at Shenyang Economic and Technological Development Zone (SETDZ). Specific emergy indicators on industrial symbiosis, including emergy savings and emdollar value of total emergy savings, were developed so that the holistic picture of industrial symbiosis can be presented. Research results show that nonrenewable inputs, imported resource inputs, and associated services could be saved by 89.3, 32.51, and 15.7 %, and the ratio of emergy savings to emergy of the total energy used would be about 25.58 %, and the ratio of the emdollar value of total emergy savings to the total gross regional product (GRP) of SETDZ would be 34.38 % through the implementation of industrial symbiosis. In general, research results indicate that industrial symbiosis could effectively reduce material and energy consumption and improve the overall eco-efficiency. Such a method can provide policy insights to industrial park managers so that they can raise appropriate strategies on developing eco-industrial parks. Useful strategies include identifying more potential industrial symbiosis opportunities, optimizing energy structure, increasing industrial efficiency, recovering local ecosystems, and improving public and industrial awareness of eco-industrial park policies.

  20. Systematic screening for mutations in the 5{prime}-regulatory region of the human dopamine D{sub 1} receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder

    SciTech Connect

    Cichon, S.; Noethen, M.M.; Stoeber, G.

    1996-07-26

    A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5{prime}-flanking region of the dopamine D{sub 1} receptor (DRD1) gene. This region has previously been shown to contain a functional promoter. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5{prime}-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder. 31 refs., 3 tabs.

  1. Symbiosis lost: imperfect vertical transmission of fungal endophytes in grasses.

    PubMed

    Afkhami, Michelle E; Rudgers, Jennifer A

    2008-09-01

    Vertically transmitted symbionts associate with some of the most ecologically dominant species on Earth, and their fixation has led to major evolutionary transitions (e.g., the development of mitochondria). Theory predicts that exclusive vertical transmission should favor mutualism and generate high frequencies of symbiosis in host populations. However, host populations often support lower-than-expected symbiont frequencies. Imperfect transmission (i.e., symbiont is not transmitted to all offspring) can reduce symbiont frequency, but for most beneficial symbionts it is unknown whether vertical transmission can be imperfect or during which life-history stage the symbiont is lost. Using quantitative natural history surveys of fungal endophytes in grasses, we show that transmission was imperfect in at least one stage for all seven host species examined. Endophytes were lost at all possible stages: within adult plants, from adult tillers to seeds, and from seeds to seedlings. Despite this loss, uninfected seeds failed to germinate in some species, resulting in perfect transmission to seedlings. The type and degree of loss differed among host populations and species and between endophyte genera. Populations with lower endophyte frequencies had higher rates of loss. Our results indicate new directions for understanding cooperation and conflict in symbioses and suggest mechanisms for host sanctions against costly symbionts.

  2. Algal ancestor of land plants was preadapted for symbiosis.

    PubMed

    Delaux, Pierre-Marc; Radhakrishnan, Guru V; Jayaraman, Dhileepkumar; Cheema, Jitender; Malbreil, Mathilde; Volkening, Jeremy D; Sekimoto, Hiroyuki; Nishiyama, Tomoaki; Melkonian, Michael; Pokorny, Lisa; Rothfels, Carl J; Sederoff, Heike Winter; Stevenson, Dennis W; Surek, Barbara; Zhang, Yong; Sussman, Michael R; Dunand, Christophe; Morris, Richard J; Roux, Christophe; Wong, Gane Ka-Shu; Oldroyd, Giles E D; Ané, Jean-Michel

    2015-10-27

    Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

  3. A novel type of thioredoxin dedicated to symbiosis in legumes.

    PubMed

    Alkhalfioui, Fatima; Renard, Michelle; Frendo, Pierre; Keichinger, Corinne; Meyer, Yves; Gelhaye, Eric; Hirasawa, Masakazu; Knaff, David B; Ritzenthaler, Christophe; Montrichard, Françoise

    2008-09-01

    Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula o