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Sample records for mutants identify complex

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

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    Nowrousian, Minou; Teichert, Ines; Masloff, Sandra; Kück, Ulrich

    2012-02-01

    The study of mutants to elucidate gene functions has a long and successful history; however, to discover causative mutations in mutants that were generated by random mutagenesis often takes years of laboratory work and requires previously generated genetic and/or physical markers, or resources like DNA libraries for complementation. Here, we present an alternative method to identify defective genes in developmental mutants of the filamentous fungus Sordaria macrospora through Illumina/Solexa whole-genome sequencing. We sequenced pooled DNA from progeny of crosses of three mutants and the wild type and were able to pinpoint the causative mutations in the mutant strains through bioinformatics analysis. One mutant is a spore color mutant, and the mutated gene encodes a melanin biosynthesis enzyme. The causative mutation is a G to A change in the first base of an intron, leading to a splice defect. The second mutant carries an allelic mutation in the pro41 gene encoding a protein essential for sexual development. In the mutant, we detected a complex pattern of deletion/rearrangements at the pro41 locus. In the third mutant, a point mutation in the stop codon of a transcription factor-encoding gene leads to the production of immature fruiting bodies. For all mutants, transformation with a wild type-copy of the affected gene restored the wild-type phenotype. Our data demonstrate that whole-genome sequencing of mutant strains is a rapid method to identify developmental genes in an organism that can be genetically crossed and where a reference genome sequence is available, even without prior mapping information.

  2. The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

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    Sven Vilain

    2012-01-01

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

  3. High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.

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    Lochlainn, Seosamh Ó; Amoah, Stephen; Graham, Neil S; Alamer, Khalid; Rios, Juan J; Kurup, Smita; Stoute, Andrew; Hammond, John P; Østergaard, Lars; King, Graham J; White, Phillip J; Broadley, Martin R

    2011-12-08

    Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

  4. High Resolution Melt (HRM analysis is an efficient tool to genotype EMS mutants in complex crop genomes

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    Lochlainn Seosamh Ó

    2011-12-01

    Full Text Available Abstract Background Targeted Induced Loci Lesions IN Genomes (TILLING is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs and insertion/deletions (IN/DELs enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

  5. Genetic screens to identify new Notch pathway mutants in Drosophila.

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    Giagtzoglou, Nikolaos

    2014-01-01

    Notch signaling controls a wide range of developmental processes, including proliferation, apoptosis, and cell fate specification during both development and adult tissue homeostasis. The functional versatility of the Notch signaling pathway is tightly linked with the complexity of its regulation in different cellular contexts. To unravel the complexity of Notch signaling, it is important to identify the different components of the Notch signaling pathway. A powerful strategy to accomplish this task is based on genetic screens. Given that the developmental context of signaling is important, these screens should be customized to specific cell populations or tissues. Here, I describe how to perform F1 clonal forward genetic screens in Drosophila to identify novel components of the Notch signaling pathway. These screens combine a classical EMS (ethyl methanesulfonate) chemical mutagenesis protocol along with clonal analysis via FRT-mediated mitotic recombination. These F1 clonal screens allow rapid phenotypic screening within clones of mutant cells induced at specific developmental stages and in tissues of interest, bypassing the pleiotropic effects of isolated mutations. More importantly, since EMS mutations have been notoriously difficult to map to specific genes in the past, I briefly discuss mapping methods that allow rapid identification of the causative mutations.

  6. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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    Carlos A Santiviago

    2009-07-01

    Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

  7. Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

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    Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

    2003-07-30

    Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.

  8. Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

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    Derry Voisin

    2009-10-01

    Full Text Available Mutations in LACERATA (LCR, FIDDLEHEAD (FDH, and BODYGUARD (BDG cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis, of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE, which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

  9. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

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    Wright Anthony PH

    2010-01-01

    Full Text Available Abstract Background Histone acetyltransferase enzymes (HATs are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. Results We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression. Conclusions Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene.

  10. NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

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    Birdsall, B.; Andrews, J.; Ostler, G.; Tendler, S.J.B.; Feeney, J.; Roberts, G.C.K.; Davies, R.W.; Cheung, H.T.A.

    1989-01-01

    Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21 → Leu and Asp 26 → Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1 H, 13 C, and 31 P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1 H NMR studies of the Trp 21 → Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 angstrom away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21 → Leu mutant indicate that the delicately poised equilibria can be perturbed. Some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim

  11. A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response.

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    Dey, Arup; Wall, Daniel

    2014-12-01

    Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. AFM images of complexes between amylose and Aspergillus niger glucoamylase mutants, native and mutant starch binding domains: a model for the action of glucoamylase

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    Morris, V. M.; Gunning, A. P.; Faults, C. B.

    2005-01-01

    Atomic force microscopy has been used to investigate the complexes formed between high molecular weight amylose chains and Aspergillus niger glucoamylase mutants (E400Q and W52F), wild-type A. niger starch binding domains (SBDS), and mutant SBDs (W563K and W590K) lacking either of the two starch...

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

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    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

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    Farshad Farshidfar

    2017-03-01

    Full Text Available Cholangiocarcinoma (CCA is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

  15. Transcriptome profiling identifies genes and pathways deregulated upon floxuridine treatment in colorectal cancer cells harboring GOF mutant p53

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    Arindam Datta

    2016-06-01

    Full Text Available Mutation in TP53 is a common genetic alteration in human cancers. Certain tumor associated p53 missense mutants acquire gain-of-function (GOF properties and confer oncogenic phenotypes including enhanced chemoresistance. The colorectal cancers (CRC harboring mutant p53 are generally aggressive in nature and difficult to treat. To identify a potential gene expression signature of GOF mutant p53-driven acquired chemoresistance in CRC, we performed transcriptome profiling of floxuridine (FUdR treated SW480 cells expressing mutant p53R273H (GEO#: GSE77533. We obtained several genes differentially regulated between FUdR treated and untreated cells. Further, functional characterization and pathway analysis revealed significant enrichment of crucial biological processes and pathways upon FUdR treatment in SW480 cells. Our data suggest that in response to chemotherapeutics treatment, cancer cells with GOF mutant p53 can modulate key cellular pathways to withstand the cytotoxic effect of the drugs. The genes and pathways identified in the present study can be further validated and targeted for better chemotherapy response in colorectal cancer patients harboring mutant p53.

  16. Whole genome-wide transcript profiling to identify differentially expressed genes associated with seed field emergence in two soybean low phytate mutants.

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    Yuan, Fengjie; Yu, Xiaomin; Dong, Dekun; Yang, Qinghua; Fu, Xujun; Zhu, Shenlong; Zhu, Danhua

    2017-01-18

    Seed germination is important to soybean (Glycine max) growth and development, ultimately affecting soybean yield. A lower seed field emergence has been the main hindrance for breeding soybeans low in phytate. Although this reduction could be overcome by additional breeding and selection, the mechanisms of seed germination in different low phytate mutants remain unknown. In this study, we performed a comparative transcript analysis of two low phytate soybean mutants (TW-1 and TW-1-M), which have the same mutation, a 2 bp deletion in GmMIPS1, but show a significant difference in seed field emergence, TW-1-M was higher than that of TW-1 . Numerous genes analyzed by RNA-Seq showed markedly different expression levels between TW-1-M and TW-1 mutants. Approximately 30,000-35,000 read-mapped genes and ~21000-25000 expressed genes were identified for each library. There were ~3900-9200 differentially expressed genes (DEGs) in each contrast library, the number of up-regulated genes was similar with down-regulated genes in the mutant TW-1and TW-1-M. Gene ontology functional categories of DEGs indicated that the ethylene-mediated signaling pathway, the abscisic acid-mediated signaling pathway, response to hormone, ethylene biosynthetic process, ethylene metabolic process, regulation of hormone levels, and oxidation-reduction process, regulation of flavonoid biosynthetic process and regulation of abscisic acid-activated signaling pathway had high correlations with seed germination. In total, 2457 DEGs involved in the above functional categories were identified. Twenty-two genes with 20 biological functions were the most highly up/down- regulated (absolute value Log2FC >5) in the high field emergence mutant TW-1-M and were related to metabolic or signaling pathways. Fifty-seven genes with 36 biological functions had the greatest expression abundance (FRPM >100) in germination-related pathways. Seed germination in the soybean low phytate mutants is a very complex process

  17. Use of induced chlorophyll deficient mutants to identify 'heterotic blocks' in pearl millet chromosomes

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    Burton, G.W.

    1989-01-01

    Full text: Chlorophyll deficient mutant stocks induced in 'Tift 23' of pearl millet (Pennisetum americanum L. Leeke) were crossed with 'Tift 23' and 5 other normal inbreds to study the effect of these deleterious recessive genes on yield. The difference between near-isogenic S 1 (F 2 ) populations homozygous or heterozygous for the chlorophyll deficiency was not significant. However among 69 S 1 progenies from crosses with other inbreds the heterozygotes were higher yielding than the homozygotes in 53 cases, 15 of which were significant. A mutant like 'M5' identified a high yield 'heterotic block' in 'Inbred 104' and a very low yield 'heterotic block' in 'Inbred 186'. (author)

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

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

    2017-09-01

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

  19. Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

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    Jiang, Yongying; Ortiz de Montellano, Paul R

    2008-05-05

    Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

  20. Monomeric RC-LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides.

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    Adams, Peter G; Mothersole, David J; Ng, Irene W; Olsen, John D; Hunter, C Neil

    2011-09-01

    In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre-light-harvesting 1-PufX (RC-LH1-PufX) 'core' complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX(-)). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX(-) mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC-LH1-PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX(-) membranes, resulting in locally ordered clusters of monomeric RC-LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation. 2011 Elsevier B.V. All rights reserved.

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

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    Salinas, Thalia; Larosa, Véronique; Cardol, Pierre; Maréchal-Drouard, Laurence; Remacle, Claire

    2014-05-01

    Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Lipidomic Profiling of Lung Pleural Effusion Identifies Unique Metabotype for EGFR Mutants in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Ho, Ying Swan; Yip, Lian Yee; Basri, Nurhidayah; Chong, Vivian Su Hui; Teo, Chin Chye; Tan, Eddy; Lim, Kah Ling; Tan, Gek San; Yang, Xulei; Yeo, Si Yong; Koh, Mariko Si Yue; Devanand, Anantham; Takano, Angela; Tan, Eng Huat; Tan, Daniel Shao Weng; Lim, Tony Kiat Hon

    2016-10-14

    Cytology and histology forms the cornerstone for the diagnosis of non-small cell lung cancer (NSCLC) but obtaining sufficient tumour cells or tissue biopsies for these tests remains a challenge. We investigate the lipidome of lung pleural effusion (PE) for unique metabolic signatures to discriminate benign versus malignant PE and EGFR versus non-EGFR malignant subgroups to identify novel diagnostic markers that is independent of tumour cell availability. Using liquid chromatography mass spectrometry, we profiled the lipidomes of the PE of 30 benign and 41 malignant cases with or without EGFR mutation. Unsupervised principal component analysis revealed distinctive differences between the lipidomes of benign and malignant PE as well as between EGFR mutants and non-EGFR mutants. Docosapentaenoic acid and Docosahexaenoic acid gave superior sensitivity and specificity for detecting NSCLC when used singly. Additionally, several 20- and 22- carbon polyunsaturated fatty acids and phospholipid species were significantly elevated in the EGFR mutants compared to non-EGFR mutants. A 7-lipid panel showed great promise in the stratification of EGFR from non-EGFR malignant PE. Our data revealed novel lipid candidate markers in the non-cellular fraction of PE that holds potential to aid the diagnosis of benign, EGFR mutation positive and negative NSCLC.

  3. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

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    Sukumar, Narayanasami [NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, Argonne, IL 60439 (United States); Dewanti, Asteriani [Department of Chemistry and Physics, Western Carolina University, Cullowhee, NC 28723 (United States); Merli, Angelo; Rossi, Gian Luigi [Department of Biochemistry and Molecular Biology, University of Parma, Parma (Italy); Mitra, Bharati [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Mathews, F. Scott, E-mail: mathews@biochem.wustl.edu [Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110 (United States); NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2009-06-01

    The crystal structure of the G81A mutant form of the chimera of (S)-mandelate dehydrogenase and of its complexes with two of its substrates reveal productive and non-productive modes of binding for the catalytic reaction. The structure also indicates the role of G81A in lowering the redox potential of the flavin co-factor leading to an ∼200-fold slower catalytic rate of substrate oxidation. (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed ∼100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 Å resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by

  4. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane

    International Nuclear Information System (INIS)

    Lahoda, Maryna; Chaloupkova, Radka; Stsiapanava, Alena; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-01-01

    A mutant of the haloalkane dehalogenase DhaA (DhaA31) from R. rhodochrous NCIMB 13064 and its complex with 1,2,3-trichloropropane were crystallized and the crystals diffracted to high resolution. Haloalkane dehalogenases hydrolyze carbon–halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure–function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP

  5. An extra early mutant of pigeonpea

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  6. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Science.gov (United States)

    Henderson, Morgan L; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  7. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase.

    Directory of Open Access Journals (Sweden)

    Morgan L Henderson

    Full Text Available Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs, located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs. Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation: RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA. In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

  8. Molecular analysis of mutants of the Neurospora adenylosuccinate ...

    Indian Academy of Sciences (India)

    2012-08-07

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

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

    Science.gov (United States)

    Tani, Tatsunori; Taguchi, Hisataka; Fujimori, Kazuhiro E; Sahara, Takehiko; Ohgiya, Satoru; Kamagata, Yoichi; Akamatsu, Takashi

    2016-10-01

    To clarify the mechanisms of xylitol utilization, three xylitol-assimilating mutants were isolated from recombinant Saccharomyces cerevisiae strains showing highly efficient xylose-utilization. The nucleotide sequences of the mutant genomes were analyzed and compared with those of the wild-type strains and the mutation sites were identified. gal80 mutations were common to all the mutants, and recessive to the wild-type allele. Hence we constructed a gal80Δ mutant and confirmed that the gal80Δ mutant showed a xylitol-assimilation phenotype. When the constructed gal80Δ mutant was crossed with the three isolated mutants, all diploid hybrids showed xylitol assimilation, indicating that the mutations were all located in the GAL80. We analyzed the role of the galactose permease Gal2, controlled by the regulatory protein Gal80, in assimilating xylitol. A gal2Δ gal80Δ double mutant did not show xylitol assimilation, whereas expression of GAL2 under the control of the TDH3 promoter in the GAL80 strain did result in assimilation. These data indicate that Gal2 was needed for xylitol assimilation in the wild-type strain. When the gal80 mutant with an initial cell concentration of A660 = 20 was used for batch fermentation in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C under oxygen limitation, the gal80 mutant consumed 100% of the xylose within 12 h, but xylitol within 100 h, indicating that xylose reductase is required for xylitol consumption in oxygen-limited conditions. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

    Directory of Open Access Journals (Sweden)

    Amsterdam Adam

    2006-06-01

    Full Text Available Abstract Background Craniofacial birth defects result from defects in cranial neural crest (NC patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1 signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. Results Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. Conclusion This work represents a systematic

  11. Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

    Science.gov (United States)

    Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

    2008-09-01

    We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  13. Reanalysis of parabiosis of obesity mutants in the age of leptin.

    Science.gov (United States)

    Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M

    2015-07-21

    In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

  14. Role of xanthophylls in light harvesting in green plants: a spectroscopic investigation of mutant LHCII and Lhcb pigment-protein complexes.

    Science.gov (United States)

    Fuciman, Marcel; Enriquez, Miriam M; Polívka, Tomáš; Dall'Osto, Luca; Bassi, Roberto; Frank, Harry A

    2012-03-29

    The spectroscopic properties and energy transfer dynamics of the protein-bound chlorophylls and xanthophylls in monomeric, major LHCII complexes, and minor Lhcb complexes from genetically altered Arabidopsis thaliana plants have been investigated using both steady-state and time-resolved absorption and fluorescence spectroscopic methods. The pigment-protein complexes that were studied contain Chl a, Chl b, and variable amounts of the xanthophylls, zeaxanthin (Z), violaxanthin (V), neoxanthin (N), and lutein (L). The complexes were derived from mutants of plants denoted npq1 (NVL), npq2lut2 (Z), aba4npq1lut2 (V), aba4npq1 (VL), npq1lut2 (NV), and npq2 (LZ). The data reveal specific singlet energy transfer routes and excited state spectra and dynamics that depend on the xanthophyll present in the complex.

  15. Dynamics of translocation and substrate binding in individual complexes formed with active site mutants of {phi}29 DNA polymerase.

    Science.gov (United States)

    Dahl, Joseph M; Wang, Hongyun; Lázaro, José M; Salas, Margarita; Lieberman, Kate R

    2014-03-07

    The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn(2+) rather than Mg(2+). The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.

  16. Mutational analysis of the yeast TRAPP subunit Trs20p identifies roles in endocytic recycling and sporulation.

    Directory of Open Access Journals (Sweden)

    Hichem Mahfouz

    Full Text Available Trs20p is a subunit of the evolutionarily conserved TRAPP (TRAnsport Protein Particle complex that mediates various aspects of membrane trafficking. Three TRAPP complexes have been identified in yeast with roles in ER-to-Golgi trafficking, post-Golgi and endosomal-to-Golgi transport and in autophagy. The role of Trs20p, which is essential for viability and a component of all three complexes, and how it might function within each TRAPP complex, has not been clarified to date. To begin to address the role of Trs20p we generated different mutants by random mutagenesis but, surprisingly, no defects were observed in diverse anterograde transport pathways or general secretion in Trs20 temperature-sensitive mutants. Instead, mutation of Trs20 led to defects in endocytic recycling and a block in sporulation/meiosis. The phenotypes of different mutants appear to be separable suggesting that the mutations affect the function of Trs20 in different TRAPP complexes.

  17. Enterocin A mutants identified by saturation mutagenesis enhance potency towards vancomycin-resistant Enterococci.

    Science.gov (United States)

    McClintock, Maria K; Kaznessis, Yiannis N; Hackel, Benjamin J

    2016-02-01

    Vancomycin-resistant Enterococci infections are a significant clinical problem. One proposed solution is to use probiotics, such as lactic acid bacteria, to produce antimicrobial peptides at the site of infection. Enterocin A, a class 2a bacteriocin, exhibits inhibitory activity against E. faecium and E. faecalis, which account for 86% of vancomycin-resistant Enterococci infections. In this study, we aimed to engineer enterocin A mutants with enhanced potency within a lactic acid bacterial production system. Peptide mutants resulting from saturation mutagenesis at sites A24 and T27 were efficiently screened in a 96-well plate assay for inhibition of pathogen growth. Several mutants exhibit increased potency relative to wild-type enterocin A in both liquid- and solid-medium growth assays. In particular, A24P and T27G exhibit enhanced inhibition of multiple strains of E. faecium and E. faecalis, including clinically isolated vancomycin-resistant strains. A24P and T27G enhance killing of E. faecium 8 by 13 ± 3- and 18 ± 4-fold, respectively. The engineered enterocin A/lactic acid bacteria systems offer significant potential to combat antibiotic-resistant infections. © 2015 Wiley Periodicals, Inc.

  18. Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora.

    Science.gov (United States)

    Nowrousian, Minou; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

    2005-04-01

    The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

  19. Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex.

    Science.gov (United States)

    Noll, Meenakshi; Battaile, Kevin P; Bateman, Raynard; Lax, Timothy P; Rathbun, Keany; Reifsteck, Carol; Bagby, Grover; Finegold, Milton; Olson, Susan; Grompe, Markus

    2002-07-01

    Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.

  20. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane.

    Science.gov (United States)

    Lahoda, Maryna; Chaloupkova, Radka; Stsiapanava, Alena; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-03-01

    Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.

  1. Chemotaxis-defective mutants of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Dusenbery, D B; Sheridan, R E; Russell, R L

    1975-06-01

    The technique of countercurrent separation has been used to isolate 17 independent chemotaxis-defective mutants of the nematode Caenorhabditis elegans. The mutants, selected to be relatively insensitive to the normally attractive salt NaCl, show varying degrees of residual sensitivity; some are actually weakly repelled by NaCl. The mutants are due to single gene defects, are autosomal and recessive, and identify at least five complementation groups.

  2. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    OpenAIRE

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

    2010-01-01

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

  3. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    Science.gov (United States)

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-05

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.

  4. Biochemical characterization and cellular effects of CADASIL mutants of NOTCH3.

    Directory of Open Access Journals (Sweden)

    He Meng

    Full Text Available Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL is the best understood cause of dominantly inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein accumulation and selective arterial smooth muscle degeneration. Previous studies show that NOTCH3 protein forms multimers. Here, we investigate protein interactions between NOTCH3 and other vascular Notch isoforms and characterize the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y mutant NOTCH3 form complexes which are more resistant to detergents than wild type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we found significant inhibition of mutant NOTCH3 clearance. In coculture assays of NOTCH function, overexpressed wild type and mutant NOTCH3 significantly repressed NOTCH-regulated smooth muscle transcripts and potently impaired the activity of three independent smooth muscle promoters. Wildtype and R90C recombinant NOTCH3 proteins applied to cell cultures also blocked canonical Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle cells.

  5. Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network.

    Science.gov (United States)

    Wei, Shu; Gruber, Margaret Y; Yu, Bianyun; Gao, Ming-Jun; Khachatourians, George G; Hegedus, Dwayne D; Parkin, Isobel A P; Hannoufa, Abdelali

    2012-09-18

    The Arabidopsis microRNA156 (miR156) regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known. In this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT) ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated) SPL15 (SPL15m) largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n) and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro interaction between DNA-binding SBP domain of SPL15

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

    Directory of Open Access Journals (Sweden)

    Fei Yuan

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

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

    Science.gov (United States)

    Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

    2018-06-01

    There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

  8. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2011-03-01

    Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

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

    Science.gov (United States)

    Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

    2016-10-07

    CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

  10. A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

    NARCIS (Netherlands)

    Damveld, R.A.; Franken, A.; Arentshorst, M.; Punt, P.J.; Klis, F.M.; van den Hondel, C.A.M.J.J.; Ram, A.F.J.

    2008-01-01

    To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  12. Mutant DnaAs of Escherichia coli that are refractory to negative control.

    Science.gov (United States)

    Chodavarapu, Sundari; Felczak, Magdalena M; Simmons, Lyle A; Murillo, Alec; Kaguni, Jon M

    2013-12-01

    DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

  13. Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

    Science.gov (United States)

    Komatsu, Setsuko; Nanjo, Yohei; Nishimura, Minoru

    2013-02-21

    Flooding stress of soybean is a serious problem because it reduces growth; however, flooding-tolerant cultivars have not been identified. To analyze the flooding tolerance mechanism of soybean, the flooding-tolerant mutant was isolated and analyzed using a proteomic technique. Flooding-tolerance tests were repeated five times using gamma-ray irradiated soybeans, whose root growth (M6 stage) was not suppressed even under flooding stress. Two-day-old wild-type and mutant plants were subjected to flooding stress for 2days, and proteins were identified using a gel-based proteomic technique. In wild-type under flooding stress, levels of proteins related to development, protein synthesis/degradation, secondary metabolism, and the cell wall changed; however, these proteins did not markedly differ in the mutant. In contrast, an increased number of fermentation-related proteins were identified in the mutant under flooding stress. The root tips of mutant plants were not affected by flooding stress, even though the wild-type plants had damaged root. Alcohol dehydrogenase activity in the mutant increased at an early stage of flooding stress compared with that of the wild-type. Taken together, these results suggest that activation of the fermentation system in the early stages of flooding may be an important factor for the acquisition of flooding tolerance in soybean. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.

    Directory of Open Access Journals (Sweden)

    David Ramonet

    2011-04-01

    Full Text Available Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene cause late-onset, autosomal dominant familial Parkinson's disease (PD and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s through which familial mutations precipitate neuronal degeneration and PD.

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

    Directory of Open Access Journals (Sweden)

    David Chan-Rodriguez

    2018-02-01

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

  16. A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

    NARCIS (Netherlands)

    Damveld, R.A.; Franken, A.; Arentshorst, M.; Punt, P.J.; Klis, F.M.; Hondel, C.A.M.J.J. van den; Ram, A.F.J.

    2008-01-01

    To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative a-glucan

  17. DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

    International Nuclear Information System (INIS)

    Cuthill, S.; Poellinger, L.

    1988-01-01

    The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-01-01

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

  19. Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant.

    Science.gov (United States)

    Carlisle, L; Steel, K; Forge, A

    1990-11-01

    Deafness in the viable dominant spotting mouse mutant is due to a primary defect of the stria vascularis which results in absence of the positive endocochlear potential in scala media. Endocochlear potentials were measured and the structure of stria vascularis of mutants with potentials close to zero was compared with that in normal littermate controls by use of morphometric methods. The stria vascularis was significantly thinner in mutants. Marginal cells were not significantly different from controls in terms of volume density or intramembrane particle density but the network density of tight junctions was significantly reduced in the mutants. A virtual absence of gap junctions between basal cells and marginal or intermediate cells was observed, but intramembrane particle density and junctional complexes between adjacent basal cells were not different from controls. The volume density of basal cells was significantly greater in mutants. Intermediate cells accounted for a significantly smaller volume density of the stria vascularis in mutants and had a lower density of intramembrane particles than controls. Melanocytes were not identified in the stria vascularis of mutants. These results suggest that communication between marginal, intermediate and basal cells might be important to the normal function of the stria vascularis.

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

    International Nuclear Information System (INIS)

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

    1999-01-01

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

  1. Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156-controlled gene network

    Directory of Open Access Journals (Sweden)

    Wei Shu

    2012-09-01

    Full Text Available Abstract Background The Arabidopsis microRNA156 (miR156 regulates 11 members of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL family by base pairing to complementary target mRNAs. Each SPL gene further regulates a set of other genes; thus, miR156 controls numerous genes through a complex gene regulation network. Increased axillary branching occurs in transgenic Arabidopsis overexpressing miR156b, similar to that observed in loss-of-function max3 and max4 mutants with lesions in carotenoid cleavage dioxygenases. Arabidopsis miR156b was found to enhance carotenoid levels and reproductive shoot branching when expressed in Brassica napus, suggesting a link between miR156b expression and carotenoid metabolism. However, details of the miR156 regulatory network of SPL genes related to carotenoid metabolism are not known. Results In this study, an Arabidopsis T-DNA enhancer mutant, sk156, was identified due to its altered branching and trichome morphology and increased seed carotenoid levels compared to wild type (WT ecovar Columbia. Enhanced miR156b expression due to the 35S enhancers present on the T-DNA insert was responsible for these phenotypes. Constitutive and leaf primodium-specific expression of a miR156-insensitive (mutated SPL15 (SPL15m largely restored WT seed carotenoid levels and plant morphology when expressed in sk156. The Arabidopsis native miR156-sensitive SPL15 (SPL15n and SPL15m driven by a native SPL15 promoter did not restore the WT phenotype in sk156. Our findings suggest that SPL15 function is somewhat redundant with other SPL family members, which collectively affect plant phenotypes. Moreover, substantially decreased miR156b transcript levels in sk156 expressing SPL15m, together with the presence of multiple repeats of SPL-binding GTAC core sequence close to the miR156b transcription start site, suggested feedback regulation of miR156b expression by SPL15. This was supported by the demonstration of specific in vitro

  2. Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

    Science.gov (United States)

    Soto-Suárez, Mauricio; Serrato, Antonio J; Rojas-González, José A; Bautista, Rocío; Sahrawy, Mariam

    2016-12-01

    During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

  3. The Neurospora rca-1 gene complements an Aspergillus flbD sporulation mutant but has no identifiable role in Neurospora sporulation.

    OpenAIRE

    Shen, W C; Wieser, J; Adams, T H; Ebbole, D J

    1998-01-01

    The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused coni...

  4. Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

    NARCIS (Netherlands)

    Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

    2003-01-01

    Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

  5. Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

    Directory of Open Access Journals (Sweden)

    Seok-Hyung Kim

    2013-07-01

    Tuberous sclerosis complex (TSC is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1 kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors.

  6. Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

    International Nuclear Information System (INIS)

    Chandra, P. Manish; Brannigan, James A.; Prabhune, Asmita; Pundle, Archana; Turkenburg, Johan P.; Dodson, G. Guy; Suresh, C. G.

    2004-01-01

    The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants. The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme

  7. Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma.

    Science.gov (United States)

    Di Pietro, Chiara; Marazziti, Daniela; La Sala, Gina; Abbaszadeh, Zeinab; Golini, Elisabetta; Matteoni, Rafaele; Tocchini-Valentini, Glauco P

    2017-01-01

    Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.

  8. Comparative production of cellulases by mutants of Trichoderma parceramosume PTCC5140

    Directory of Open Access Journals (Sweden)

    Hoda Nouri

    2017-06-01

    Discussion and conclusion: Evaluation of cellulase production in mutant strains of Trichoderma parceramosume PTCC 5140 showed that use of chemical mutagenesis with 2 to 11 fold increasing in enzyme activity is a potent method to improve cellulase complex activity. In the current study, obtained mutant strains could be introduced as a potent cellulase producer for further studies in bioconversion processes.

  9. Identifying modular relations in complex brain networks

    DEFF Research Database (Denmark)

    Andersen, Kasper Winther; Mørup, Morten; Siebner, Hartwig

    2012-01-01

    We evaluate the infinite relational model (IRM) against two simpler alternative nonparametric Bayesian models for identifying structures in multi subject brain networks. The models are evaluated for their ability to predict new data and infer reproducible structures. Prediction and reproducibility...... and obtains comparable reproducibility and predictability. For resting state functional magnetic resonance imaging data from 30 healthy controls the IRM model is also superior to the two simpler alternatives, suggesting that brain networks indeed exhibit universal complex relational structure...

  10. Commercialization Of Orchid Mutants For Floriculture Industry

    International Nuclear Information System (INIS)

    Sakinah Ariffin; Zaiton Ahmad

    2014-01-01

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

  11. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

    Science.gov (United States)

    Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

  12. Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

    International Nuclear Information System (INIS)

    Kaefer, E.; Mayor, O.

    1986-01-01

    To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or γ-rays. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. (Auth.)

  13. Mutant fatty acid desaturase and methods for directed mutagenesis

    Science.gov (United States)

    Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  14. Characterization of a Weak Allele of Zebrafish cloche Mutant

    Science.gov (United States)

    Ma, Ning; Huang, Zhibin; Chen, Xiaohui; He, Fei; Wang, Kun; Liu, Wei; Zhao, Linfeng; Xu, Xiangmin; Liao, Wangjun; Ruan, Hua; Luo, Shenqiu; Zhang, Wenqing

    2011-01-01

    Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene. PMID:22132109

  15. Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  16. Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Steven D [ORNL; Guss, Adam M [ORNL; Karpinets, Tatiana V [ORNL; Parks, Jerry M [ORNL; Smolin, Nikolai [ORNL; Yang, Shihui [ORNL; Land, Miriam L [ORNL; Klingeman, Dawn Marie [ORNL; Bhandiwad, Ashwini [Thayer School of Engineering at Dartmouth; Rodriguez, Jr., Miguel [ORNL; Raman, Babu [Dow Chemical Company, The; Shao, Xiongjun [Thayer School of Engineering at Dartmouth; Mielenz, Jonathan R [ORNL; Smith, Jeremy C [ORNL; Keller, Martin [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2011-01-01

    Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

  17. Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9.

    Science.gov (United States)

    Willson, T A; Nagley, P

    1987-09-01

    This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9

  18. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    Directory of Open Access Journals (Sweden)

    Charalampos Rallis

    2014-01-01

    Target of rapamycin complex 1 (TORC1, which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

  19. Photosynthetic characterization of a rolled leaf mutant of rice ( Oryza ...

    African Journals Online (AJOL)

    A new rolling leaf rice mutant was identified which showed an apparently straighter longitudinal shape normal transverse rolling characters at all developing stages. The chlorophyll contents per fresh weight of this mutant leaves were lower than those of wild-type. The electron transfer rate (ETR) and photochemical ...

  20. In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB

    DEFF Research Database (Denmark)

    Hediger, Martin Robert; De Vico, Luca; Rannes, Julie Bille

    2013-01-01

    Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., 2012) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barriers......, the qualitative activity of 15 out of 22 mutants is correctly predicted. The method identifies four of the six most active mutants with ≥3-fold wild type activity and seven out of the eight least active mutants with ≤0.5-fold wild type activity. The method is further used to screen all sterically possible (386......) double-, triple- and quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified....

  1. A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis.

    Science.gov (United States)

    Liu, Ying; Wu, Ting; Song, Jian; Chen, Xuelian; Zhang, Yu; Wan, Yu

    2013-03-11

    Distinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange™ mutagenesis. Although Dpn I digestion can eliminate methylated parental (WT) DNA, the efficiency is not satisfying due to the existence of hemi-methylated DNA in the PCR products, which is resistant to Dpn I. The present study designed a novel critical annealing temperature (T(c))-PCR to replace Dpn I digestion for more perfect mutant distinguishing, in which part-overlapping primers containing mutation(s) were used to reduce initial concentration of template DNA in mutagenic PCR. A T(c)-PCR with the same mutagenic primers was performed without Dpn I digestion. The T(c) for each pair of the primers was identified by gradient PCR. The relationship between PCR-identified T(c) and T(m) of the primers was analyzed and modeled with correlation and regression. Gradient PCR identified a T(c) for each of 14 tested mutagenic primers, which could discriminate mismatched parental molecules and undesired mutants from desired mutants. The PCR-identified T(c) was correlated to the primer's T(m) (r = 0.804, P<0.0001). Thus, in practical applications, the T(c) can be easily calculated with a regression equation, T(c)= 48.81 + 0.253*T(m). The new protocol introduced a novel T(c)-PCR method for mutant screening which can more efficiently and accurately select against parental molecules and undesired mutations in mutagenic sequence segments.

  2. Analysis of AtCry1 and Mutants

    Science.gov (United States)

    Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

    Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

  3. Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

    Science.gov (United States)

    Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

    2017-01-31

    The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

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

    International Nuclear Information System (INIS)

    Wu, T.; Cao, J.

    2015-01-01

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

  5. Absence-like and tonic seizures in aspartoacylase/attractin double-mutant mice.

    Science.gov (United States)

    Gohma, Hiroshi; Kuramoto, Takashi; Matalon, Reuben; Surendran, Sankar; Tyring, Stephen; Kitada, Kazuhiro; Sasa, Masashi; Serikawa, Tadao

    2007-04-01

    The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn(mg-3J) mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.

  6. Identification of some Rice Mutants using Morphological and Molecular Methods

    International Nuclear Information System (INIS)

    Sobieh, S.E.S.

    2006-01-01

    This investigation was conducted at the experimental farm of plant research department, nuclear research center, atomic energy authority, abu zaabal in order to verify four rice genotypes i.e sakha 102, giza 178, high yielding mutant (Ms 6) and high yielding mutant (MG 16). the (UPOV) rice descriptor was used to identify the germplasm morphologically .Molecular RAPD-PCR was used to identify genetic variability on the molecular level for the tested genotypes. 1- the results indicated that according to (UPOV) rice descriptor eight morphological traits were completely different between mutant Ms 6 in comparison with the parent sakha 102 and mut. MG 16 in comparison parent giza 178, the traits were ; stem thickness, stem length, panicle length, 1000-grain weight, grain length, grain width decorticated grain length and decorticated grain width. 2- using 10 arbitrary primers, through four rice genotypes on the molecular level using RAPD markers. the size of the amplified fragments were ranged from 0.201 to 2.036 k bp. two primers OPB-13 and OPB-16 showed no polymorphism among genotypes tested. 3- the total number of amplicons produced by the 8 polymorphic RAPD profiels was 66. the total number of monomorphic amplicons was 32. however, the total number of polymorphic amplicons was 34. 4- the percentage of polymorphism ranged from (22.22%) for primer OPA-18 to (90%) for primer OPB-11. 5-the highest genetic similarity (90.3%) was between sakha 102 and high yielding mut. (Ms 6). the genetic similarity (75.5%) was between giza 178 and high yielding mut.(MG 16). 6- one positive unique marker amplified by OPA-18 Primer identified the high yielding mutant Ms 6 but three positive unique markers amplified by OPB-17 primer and OPA-18 primer identified the high yielding mutant MG 16

  7. Collection of rice mutants and application studies of their agronomic characters

    International Nuclear Information System (INIS)

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

    1993-01-01

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

  8. ARID1B is a specific vulnerability in ARID1A-mutant cancers.

    Science.gov (United States)

    Helming, Katherine C; Wang, Xiaofeng; Wilson, Boris G; Vazquez, Francisca; Haswell, Jeffrey R; Manchester, Haley E; Kim, Youngha; Kryukov, Gregory V; Ghandi, Mahmoud; Aguirre, Andrew J; Jagani, Zainab; Wang, Zhong; Garraway, Levi A; Hahn, William C; Roberts, Charles W M

    2014-03-01

    Recent studies have revealed that ARID1A, encoding AT-rich interactive domain 1A (SWI-like), is frequently mutated across a variety of human cancers and also has bona fide tumor suppressor properties. Consequently, identification of vulnerabilities conferred by ARID1A mutation would have major relevance for human cancer. Here, using a broad screening approach, we identify ARID1B, an ARID1A homolog whose gene product is mutually exclusive with ARID1A in SWI/SNF complexes, as the number 1 gene preferentially required for the survival of ARID1A-mutant cancer cell lines. We show that loss of ARID1B in ARID1A-deficient backgrounds destabilizes SWI/SNF and impairs proliferation in both cancer cells and primary cells. We also find that ARID1A and ARID1B are frequently co-mutated in cancer but that ARID1A-deficient cancers retain at least one functional ARID1B allele. These results suggest that loss of ARID1A and ARID1B alleles cooperatively promotes cancer formation but also results in a unique functional dependence. The results further identify ARID1B as a potential therapeutic target for ARID1A-mutant cancers.

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

    International Nuclear Information System (INIS)

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

    1980-01-01

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

  10. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.

    1989-01-01

    : DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6......-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent...

  11. Mutants of Streptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites

    International Nuclear Information System (INIS)

    Blumauerova, M.; Stajner, K.; Pokorny, V.; Hostalek, Z.; Vanek, Z.

    1978-01-01

    Mutants of Streptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, γ-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described in Streptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, zeta-rhodomycinone and glycosides of epsilon-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases. (author)

  12. Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

    Directory of Open Access Journals (Sweden)

    Anja Wartenberg

    2014-12-01

    Full Text Available Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

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

    Science.gov (United States)

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

    2017-10-01

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

  14. Data on quantification of signaling pathways activated by KIT and PDGFRA mutants

    Directory of Open Access Journals (Sweden)

    Christelle Bahlawane

    2016-12-01

    Full Text Available The present data are related to the article entitled “Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling” (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016 [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells. Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf. We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

  15. Identifying partial topology of complex dynamical networks via a pinning mechanism

    Science.gov (United States)

    Zhu, Shuaibing; Zhou, Jin; Lu, Jun-an

    2018-04-01

    In this paper, we study the problem of identifying the partial topology of complex dynamical networks via a pinning mechanism. By using the network synchronization theory and the adaptive feedback controlling method, we propose a method which can greatly reduce the number of nodes and observers in the response network. Particularly, this method can also identify the whole topology of complex networks. A theorem is established rigorously, from which some corollaries are also derived in order to make our method more cost-effective. Several numerical examples are provided to verify the effectiveness of the proposed method. In the simulation, an approach is also given to avoid possible identification failure caused by inner synchronization of the drive network.

  16. Molecular dynamics simulations of Hsp40 J-domain mutants identifies disruption of the critical HPD-motif as the key factor for impaired curing in vivo of the yeast prion [URE3].

    Science.gov (United States)

    Xue, You-Lin; Wang, Hao; Riedy, Michael; Roberts, Brittany-Lee; Sun, Yuna; Song, Yong-Bo; Jones, Gary W; Masison, Daniel C; Song, Youtao

    2018-05-01

    Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.

  17. Repair of gamma radiation damage in wild type and a radiation sensitive mutant of Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Mizuma, Nagayo

    1989-01-01

    In an effort to examine production and repair of radiation-induced single and double strand breaks in the DNA, a repair-deficient wild type and a repair-deficient mutant, UV17, of Deinococcus radiodurans were subjected to Co-60 gamma irradiation at a dose rate of 6.3 kGy/hr for wild type and 3.9 kGy/hr for UV17 mutant. The shoulder of the curve of UV17 mutant was narrow but existed with the intercept of 0.7 kGy and the corresponding value of the wild type was 4.2 kGy. Mutant cells exhibited about 6 fold increases in sensitivity for the shoulder relative to the wild type. The D 37 doses in the wild type and the mutant were 0.57 kGy and 0.25 kGy, respectively. From the survival curves, difference in the sensitivity between two strains was mainly due to difference of repair capacity than the number of radiation sensitive target. Sedimentation rate of the main component in the irradiated cells of UV17 mutant increased almost to the level of unirradiated control by the postincubation at 30deg C for 3 hrs. The results indicated that this sensitive mutant also exhibited an ability to restore single strand breaks after exposure to a sublethal dose of 0.6 kGy. When restitution of double strand breaks was analyzed by sedimentation in a neutral sucrose gradient, the wild type showed restitution to DNA-membrane complex from large part of the breaks. For UV17 mutant, the apparent increase in DNA-membrane complex formation was seen after 3 hours incubation. Large part of the decrease in the activities of peak 2 was recovered in the peak 1 for the wild type. For the mutant, there was little restitution to peak 1. Almost free DNA component in UV17 mutant, therefore, was merely degraded into shorter pieces. Restoration of DNA-membrane complex from free DNA derived from gamma-ray induced double strand scission involved closely in the repair of gamma-induced damage and survival. (N.K.)

  18. Isolation and Characterization of Two Cellulose Morphology Mutants of Gluconacetobacter hansenii ATCC23769 Producing Cellulose with Lower Crystallinity

    Science.gov (United States)

    Deng, Ying; Nagachar, Nivedita; Fang, Lin; Luan, Xin; Catchmark, Jeffrey M.; Tien, Ming; Kao, Teh-hui

    2015-01-01

    Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the

  19. Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

    Directory of Open Access Journals (Sweden)

    Ying Deng

    Full Text Available Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of β-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC. These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of

  20. EPR analysis of cyanide complexes of wild-type human neuroglobin and mutants in comparison to horse heart myoglobin.

    Science.gov (United States)

    Van Doorslaer, Sabine; Trandafir, Florin; Harmer, Jeffrey R; Moens, Luc; Dewilde, Sylvia

    2014-06-01

    Electron paramagnetic resonance (EPR) data reveal large differences between the ferric ((13)C-)cyanide complexes of wild-type human neuroglobin (NGB) and its H64Q and F28L point mutants and the cyanide complexes of mammalian myo- and haemoglobin. The point mutations, which involve residues comprising the distal haem pocket in NGB, induce smaller, but still significant changes, related to changes in the stabilization of the cyanide ligand. Furthermore, for the first time, the full (13)C hyperfine tensor of the cyanide carbon of cyanide-ligated horse heart myoglobin (hhMb) was determined using Davies ENDOR (electron nuclear double resonance). Disagreement of these experimental data with earlier predictions based on (13)C NMR data and a theoretical model reveal significant flaws in the model assumptions. The same ENDOR procedure allowed also partial determination of the corresponding (13)C hyperfine tensor of cyanide-ligated NGB and H64QNGB. These (13)C parameters differ significantly from those of cyanide-ligated hhMb and challenge our current theoretical understanding of how the haem environment influences the magnetic parameters obtained by EPR and NMR in cyanide-ligated haem proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Induced mutants for the improvement of sesame and hybrid seed production

    International Nuclear Information System (INIS)

    Murty, G.S.S.

    2001-01-01

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

  2. Molecular Genetic Identification Of Some Flax Mutants

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  3. Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

    Science.gov (United States)

    Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

    2013-01-01

    The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

  4. Modified Starch of Sorghum Mutant Line Zh-30 for High Fiber Muffin Products

    Directory of Open Access Journals (Sweden)

    D.D.S. Santosa

    2009-01-01

    Full Text Available Sorghum mutant line Zh-30 is a breeding line developed at the Center for the Application of Isotope and Radiation Technology, BATAN by using mutation techniques. Gamma irradiation with the dose of 300 Gy was used to induce sorghum genetic variation. Through selection processes in several generations, the mutant line Zh-30 was identified to have better agronomic characteristics, better grain quality and higher grain yield than the original variety. Research on modified starch quality of this mutant line was done to identify its potential use in food industry. Functionality of pregelatinized, hydroxypropyl and crosslinked starch of this mutant line (Mutant TexInstant 30 has been studied for its use in high fiber muffin products. Characteristics of high fiber muffins containing 1.50; 3.50 and 5.50% of Mutant Tex-Instant 30 replacement levels to wheat flour were evaluated using both sensory panel and physical test methods. With regard to the sensory parameters, the high fiber muffins containing 1.50 - 5.50 % Mutant Tex-Instant 30 in general were not significantly different compared to the standard reference muffin. Results of physical evaluations showed that all Mutant Tex-Instant 30 containing products retained more moisture during baking than the standard reference. Tenderness of all products decreased at similar rate following 24 and 48 hr of room temperature storage and seven days at freezer temperature. These results suggested that sorghum mutant line Zh-30 starch could be modified and potentially used in food industry as a subtitute of wheat flour.

  5. Modified Starch of Sorghum Mutant Line Zh-30 For High Fiber Muffin Products

    International Nuclear Information System (INIS)

    Santosa, D. D. S; Human, S

    2009-01-01

    Sorghum mutant line Zh-30 is a breeding line developed at the Center for the Application of Isotope and Radiation Technology, BATAN by using mutation techniques. Gamma irradiation with the dose of 300 Gy was used to induce sorghum genetic variation. Through selection processes in several generations, the mutant line Zh-30 was identified to have better agronomic characteristics, better grain quality and higher grain yield than the original variety. Research on modified starch quality of this mutant line was done to identify its potential use in food industry. Functionality of pregelatinized, hydroxypropyl and crosslinked starch of this mutant line (Mutant TexInstant 30) has been studied for its use in high fiber muffin products. Characteristics of high fiber muffins containing 1.50; 3.50 and 5.50% of Mutant Tex-Instant 30 replacement levels to wheat flour were evaluated using both sensory panel and physical test methods. With regard to the sensory parameters, the high fiber muffins containing 1.50 - 5.50 % Mutant Tex-Instant 30 in general were not significantly different compared to the standard reference muffin. Results of physical evaluations showed that all Mutant Tex-Instant 30 containing products retained more moisture during baking than the standard reference. Tenderness of all products decreased at similar rate following 24 and 48 hr of room temperature storage and seven days at freezer temperature. These results suggested that sorghum mutant line Zh-30 starch could be modified and potentially used in food industry as a subtitute of wheat flour (author)

  6. Isolating human DNA repair genes using rodent-cell mutants

    International Nuclear Information System (INIS)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-01-01

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

  7. Resonance Raman spectroscopy of xanthophylls in pigment mutant thylakoid membranes of pea.

    Science.gov (United States)

    Andreeva, Atanaska; Stoitchkova, Katerina; Busheva, Mira; Apostolova, Emilia; Várkonyi, Zsuzsanna; Garab, Gyözö

    Low-temperature resonance Raman spectroscopy was used to study the changes in the molecular structure and configuration of the major xanthophylls in thylakoid membranes isolated from mutants of pea with modified pigment content and altered structural organization of their pigment-protein complexes. The Raman spectra contained four known groups of bands, nu(1)-nu(4), which could be assigned to originate mainly from the long wavelength absorbing lutein and neoxanthin upon 514.5 nm and at 488 nm excitations, respectively. The overall configuration of these bound xanthophyll molecules in the mutants appeared to be similar to the wild type, and the configuration in the wild type was almost identical with that in the isolated main chlorophyll a/b light harvesting protein complex of photosystem II (LHCII). Significant differences were found mainly in the region of nu(4) (around 960 cm(-1)), which suggest that the macroorganization of PS II-LHCII supercomplexes and/or of the LHCII-only domains are modified in the mutants compared to the wild type. Copyright 2004 Wiley Periodicals, Inc. Biopolymers, 2004

  8. Biodiversity in the cyclic competition system of three species according to the emergence of mutant species

    Science.gov (United States)

    Park, Junpyo

    2018-05-01

    Understanding mechanisms which promote or hinder existing ecosystems are important issues in ecological sciences. In addition to fundamental interactions such as competition and migration among native species, existing ecosystems can be easily disturbed by external factors, and the emergence of new species may be an example in such cases. The new species which does not exist in a current ecosystem can be regarded as either alien species entered from outside or mutant species born by mutation in existing normal species. Recently, as existing ecosystems are getting influenced by various physical/chemical external factors, mutation due to anthropogenic and environmental factors can occur more frequently and is thus attracting much attention for the maintenance of ecosystems. In this paper, we consider emergences of mutant species among self-competing three species in the cyclic dominance. By defining mutation as the birth of mutant species, we investigate how mutant species can affect biodiversity in the existing ecosystem. Through microscopic and macroscopic approaches, we have found that the society of existing normal species can be disturbed by mutant species either the society is maintained accompanying with the coexistence of all species or jeopardized by occupying of mutant species. Due to the birth of mutant species, the existing society may be more complex by constituting two different groups of normal and mutant species, and our results can be contributed to analyze complex ecosystems of many species. We hope our findings may propose a new insight on mutation in cyclic competition systems of many species.

  9. Defective glycinergic synaptic transmission in zebrafish motility mutants

    Directory of Open Access Journals (Sweden)

    Hiromi Hirata

    2010-01-01

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

  10. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    Science.gov (United States)

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

    2009-01-01

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

  11. Competitive Interactions Between Incompatible Mutants of the Social Bacterium Myxococcus xanthus DK1622

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    Ya Gong

    2018-06-01

    Full Text Available Due to the high similarity in their requirements for space and food, close bacterial relatives may be each other's strongest competitors. Close bacterial relatives often form visible boundaries to separate their swarming colonies, a phenomenon termed colony-merger incompatibility. While bacterial species are known to have many incompatible strains, it is largely unclear which traits lead to multiple incompatibilities and the interactions between multiple incompatible siblings. To investigate the competitive interactions of closely related incompatible strains, we mutated Myxococcus xanthus DK1622, a predatory bacterium with complex social behavior. From 3392 random transposon mutations, we obtained 11 self-identification (SI deficient mutants that formed unmerged colony boundaries with the ancestral strain. The mutations were at nine loci with unknown functions and formed nine independent SI mutants. Compared with their ancestral strain, most of the SI mutants showed reduced growth, swarming and development abilities, but some remained unchanged from their monocultures. When pairwise mixed with their ancestral strain for co-cultivation, these mutants exhibited improved, reduced or unchanged competitive abilities compared with the ancestral strain. The sporulation efficiencies were affected by the DK1622 partner, ranging from almost complete inhibition to 360% stimulation. The differences in competitive growth between the SI mutants and DK1622 were highly correlated with the differences in their sporulation efficiencies. However, the competitive efficiencies of the mutants in mixture were inconsistent with their growth or sporulation abilities in monocultures. We propose that the colony-merger incompatibility in M. xanthus is associated with multiple independent genetic loci, and the incompatible strains hold competitive interaction abilities, which probably determine the complex relationships between multiple incompatible M. xanthus strains and

  12. Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

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    Xueyong Zhu

    2015-11-01

    Full Text Available Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

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    Koichi Sato

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

  15. Connexin mutants and cataracts

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    Eric C Beyer

    2013-04-01

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

  16. Temperature-sensitive mutants of fowl plague virus: isolation and genetic characterization

    International Nuclear Information System (INIS)

    Almond, J.W.; McGeoch, D.; Barry, R.D.

    1979-01-01

    Forty-nine temperature-sensitive mutants of fowl plague virus (FPV) strain Rostock and four ts mutants of FPV-strain Dobson were isolated by utilizing two methods of plaque screening, after either spontaneous or chemically induced mutagenesis. Twenty-nine of the FPV-Rostock mutants were further characterized by genetic recombination studies and were found to fall into six high frequency recombination groups. The genome segment carrying the ts mutation in each group was identified by analyzing the gene composition of ts + recombinants generated from crosses between representatives of each group and ts mutants of FPV-Dobson. It was concluded that the six groups correspond to mutations in six different genome segments, namely, those coding for the P 1 , P 2 , P 3 , HA, NP, and NS proteins

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

    Directory of Open Access Journals (Sweden)

    Man Hei Cheng

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

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

    Science.gov (United States)

    Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

    2014-09-01

    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

  19. Characterization of novel Sorghum brown midrib mutants from an EMS-mutagenized population.

    Science.gov (United States)

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

    2014-09-02

    Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles. Copyright © 2014 Sattler et al.

  20. Defective nucleolar localization and dominant interfering properties of a parafibromin L95P missense mutant causing the hyperparathyroidism-jaw tumor syndrome

    Science.gov (United States)

    Panicker, Leelamma M.; Zhang, Jian-Hua; Dagur, Pradeep K.; Gastinger, Matthew J.; Simonds, William F.

    2011-01-01

    The hyperparathyroidism-jaw tumor syndrome (HPT-JT) is a familial cancer syndrome that can result from germline inactivation of HRPT2/CDC73, a putative tumor suppressor gene that encodes parafibromin, a component of the transcriptional regulatory PAF1 complex with homology to the yeast protein Cdc73p. The vast majority of HRPT2/CDC73 germline mutations identified have been truncation or frameshift mutations, and loss-of-function due to missense mutation is rare. We report here a kindred with HPT-JT due to a germline L95P missense mutation in parafibromin. The mutant parafibromin was studied in vitro to understand the basis of its presumed loss-of-function. When transfected in cultured cells the L95P mutant was expressed to a lower level than wild-type parafibromin, a difference that was not overcome by inhibition of the proteasome degradation pathway. The L95P mutant parafibromin retained the ability to assemble with endogenous PAF1 complex components as evidenced by co-immunoprecipitation. Analysis of subcellular localization showed that the L95P mutant was markedly deficient in nucleolar localization compared to the wild-type, an impairment likely resulting from disruption of a putative nucleolar localization signal immediately upstream of the L95P mutation. Transfection of the L95P parafibromin mutant, but not the wild type, enhanced cell-cycle progression and increased cell survival in NIH-3T3 and HEK 293 cells, resulting apparently from dominant interference with endogenous parafibromin action. The simultaneous loss of nucleolar localization and acquisition of a growth stimulatory phenotype with the L95P mutation raise the possibility that parafibromin must interact with targets in the nucleolus to fully execute its tumor suppressor functions. PMID:20304979

  1. Sodium azide mutagenesis in wheat: Mutants with golden glumes

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  2. A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation.

    Science.gov (United States)

    Kupfer, G; Naf, D; Garcia-Higuera, I; Wasik, J; Cheng, A; Yamashita, T; Tipping, A; Morgan, N; Mathew, C G; D'Andrea, A D

    1999-04-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but the function of the encoded FA proteins remains unknown. We recently demonstrated that the FANCA and FANCC proteins bind and form a nuclear complex. In the current study, we identified a homozygous mutation in the FANCA gene (3329A>C) in an Egyptian FA patient from a consanguineous family. This mutant FANCA allele is predicted to encode a mutant FANCA protein, FANCA(H1110P), in which histidine 1110 is changed to proline. Initially, we characterized the FANCA(H1110P) protein, expressed in an Epstein Barr virus (EBV)-immortalized lymphoblast line derived from the patient. Unlike wild-type FANCA protein expressed in normal lymphoblasts, FANCA(H1110P) was not phosphorylated and failed to bind to FANCC. To test directly the effect of this mutation on FANCA function, we used retroviral-mediated transduction to express either wild-type FANCA or FANCA(H1110P) protein in the FA-A fibroblast line, GM6914. Unlike wild-type FANCA, the mutant protein failed to complement the mitomycin C sensitivity of these cells. In addition, the FANCA(H1110P) protein was defective in nuclear accumulation in the transduced cells. The characteristics of this mutant protein underscore the importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation in the function of the FA pathway.

  3. Regulatory Mechanisms of a Highly Pectinolytic Mutant of Penicillium occitanis and Functional Analysis of a Candidate Gene in the Plant Pathogen Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Gustavo Bravo-Ruiz

    2017-09-01

    Full Text Available Penicillium occitanis is a model system for enzymatic regulation. A mutant strain exhibiting constitutive overproduction of different pectinolytic enzymes both under inducing (pectin or repressing conditions (glucose was previously isolated after chemical mutagenesis. In order to identify the molecular basis of this regulatory mechanism, the genomes of the wild type and the derived mutant strain were sequenced and compared, providing the first reference genome for this species. We used a phylogenomic approach to compare P. occitanis with other pectinolytic fungi and to trace expansions of gene families involved in carbohydrate degradation. Genome comparison between wild type and mutant identified seven mutations associated with predicted proteins. The most likely candidate was a mutation in a highly conserved serine residue of a conserved fungal protein containing a GAL4-like Zn2Cys6 binuclear cluster DNA-binding domain and a fungus-specific transcription factor regulatory middle homology region. To functionally characterize the role of this candidate gene, the mutation was recapitulated in the predicted orthologue Fusarium oxysporum, a vascular wilt pathogen which secretes a wide array of plant cell wall degrading enzymes, including polygalacturonases, pectate lyases, xylanases and proteases, all of which contribute to infection. However, neither the null mutant nor a mutant carrying the analogous point mutation exhibited a deregulation of pectinolytic enzymes. The availability, annotation and phylogenomic analysis of the P. occitanis genome sequence represents an important resource for understanding the evolution and biology of this species, and sets the basis for the discovery of new genes of biotechnological interest for the degradation of complex polysaccharides.

  4. Analysis of human HPRT- deletion mutants by the microarray-CGH (comparative genomic hybridization)

    International Nuclear Information System (INIS)

    Kodaira, M.; Sasaki, K.; Tagawa, H.; Omine, H.; Kushiro, J.; Takahashi, N.; Katayama, H.

    2003-01-01

    We are trying to evaluate genetic effects of radiation on human using mutation frequency as an indicator. For the efficient detection of mutations, it is important to understand the mechanism and the characteristics of radiation-induced mutations. We have started the analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants induced by X-ray in order to clarify the deletion size and the mutation-distribution. We analyzed 39 human X-ray induced HPRT-deletion mutants by using the microarray-CGH. The array for this analysis contains 57 BAC clones covering as much as possible of the 4Mb of the 5' side and 10Mb of the 3' side of the HPRT gene based on the NCBI genome database. DNA from parent strain and each HPRT-mutant strain are labeled with Cy5 and Cy3 respectively, and were mixed and hybridized on the array. Fluorescent intensity ratio of the obtained spots was analyzed using software we developed to identify clones corresponding to the deletion region. The deletion in these strains ranged up to 3.5 Mb on the 5' side and 6 Mb on the 3' side of the HPRT gene. Deletions in 13 strains ended around BAC clones located at about 3 Mb on the 5' side. On the 3' side, deletions extended up to the specific clones located at 1.5 Mb in 11 strains. The mutations seem to be complex on the 3' end of deletion; some accompanied duplications with deletions and others could not be explained by one mutation event. We need to confirm these results, taking into account the experimental reproducibility and the accuracy of the published genetic map. The results of the research using the microarray-CGH help us to search the regions where deletions are easily induced and to identify the factors affecting the range of deletions

  5. A sorghum (Sorghum bicolor mutant with altered carbon isotope ratio.

    Directory of Open Access Journals (Sweden)

    Govinda Rizal

    Full Text Available Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor mutant with a low δ13C characteristic. A mutant (named Mut33 with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT. The back-cross (BC1F1 progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 μmol CO2.mol-1air and the maximum rate of photosynthesis was less than 5μmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used

  6. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B

    2014-07-01

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

  7. Induced high yielding mutant in green gram (Vigna radiata (L.) Wilczek)

    International Nuclear Information System (INIS)

    Pulivarthi, H.R.; Mary, T.N.

    1987-01-01

    Green gram (mungbean) plays a significant role in meeting the protein requirements in India, with its predominantly vegetarian population. Therefore, an attempt was made to induce desirable mutants. Dry seed of cultivar 'Pusa 105' were irradiated with gamma rays ranging from 10 to 50 krad. A high yielding mutant (Hy I) identified in the M 4 generation from 40 krad dose, has shown significant increases in the number of pods/plants, number of branches/plant, and yield/plant. Further work is in progress. Comparison of the mutant HyI with the parent cultivar Pusa 105 is given

  8. Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. | Office of Cancer Genomics

    Science.gov (United States)

    A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers.

  9. Dissecting the sequential assembly and localization of intraflagellar transport particle complex B in Chlamydomonas.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Richey

    Full Text Available Intraflagellar transport (IFT, the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism.

  10. High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

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    Olga Mestre

    Full Text Available Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS. Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

  11. Evaluation of induced mutants of wheat for resistance to fungal diseases

    International Nuclear Information System (INIS)

    Barriga B, P.; Fuentes P, R.; Andrade S, N.; Seeman F, P.

    1990-01-01

    Evaluation of induced mutants of wheat for resistance to fungal diseases. Seeds of spring wheat cultivars Austral and Huenufen were exposed to gamma radiation in doses of 0.10 and 0.25 KGy with the objective of producing genotypes resistant to the main fungal diseases, with a high protein content and grain yield, for the southern region of Chile (39 sup(o)-44 sup(o) Latitude south). The selection process and evaluation up to the generation M sub(8) has made possible to identify mutants with a higher protein content and grain yield. Progress made in improving resistance to Puccinia striiformis and tolerance to Septoria spp., has also been important. Some selected mutants, conditioned to their future performance, could be directly used as commercial varieties and other mutants, on crosses with regionally adapted cultivars. (author)

  12. Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants.

    Science.gov (United States)

    Käfer, E; Mayor, O

    1986-07-01

    To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV

  13. Mutant heterosis in rice

    International Nuclear Information System (INIS)

    1987-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  15. Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

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    Xianjun Shen

    Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

  16. ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS

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    Kavita Rajesh Pandey

    2016-09-01

    Full Text Available Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII obtained in previous studies (Dubey and Vakil, 2010, were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell.

  17. Andrographolide induces degradation of mutant p53 via activation of Hsp70.

    Science.gov (United States)

    Sato, Hirofumi; Hiraki, Masatsugu; Namba, Takushi; Egawa, Noriyuki; Baba, Koichi; Tanaka, Tomokazu; Noshiro, Hirokazu

    2018-05-22

    The tumor suppressor gene p53 encodes a transcription factor that regulates various cellular functions, including DNA repair, apoptosis and cell cycle progression. Approximately half of all human cancers carry mutations in p53 that lead to loss of tumor suppressor function or gain of functions that promote the cancer phenotype. Thus, targeting mutant p53 as an anticancer therapy has attracted considerable attention. In the current study, a small-molecule screen identified andrographlide (ANDRO) as a mutant p53 suppressor. The effects of ANDRO, a small molecule isolated from the Chinese herb Andrographis paniculata, on tumor cells carrying wild-type or mutant p53 were examined. ANDRO suppressed expression of mutant p53, induced expression of the cyclin-dependent kinase inhibitor p21 and pro-apoptotic proteins genes, and inhibited the growth of cancer cells harboring mutant p53. ANDRO also induced expression of the heat-shock protein (Hsp70) and increased binding between Hsp70 and mutant p53 protein, thus promoting proteasomal degradation of p53. These results provide novel insights into the mechanisms regulating the function of mutant p53 and suggest that activation of Hsp70 may be a new strategy for the treatment of cancers harboring mutant p53.

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

    Directory of Open Access Journals (Sweden)

    Ming Liu

    2010-10-01

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

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

    Science.gov (United States)

    Schein, Stanley J.

    1976-01-01

    The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the `high-barium' solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (`extreme' pawns) to nearly wild-type reversal behavior (`partial' pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of Kung et al. (1975). All of the pwA and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the pwA mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A–B, A–C, B–C), identified in the exautogamous progeny of crosses between `partial' mutants, exhibited a more extreme non-reversing phenotype than either of their single-mutant (`partial' pawn) parents.———Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (Schein, Bennett and Katz 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electro-physiology of the pawns demonstrate that the pwA and pwB genes have different effects on calcium activation. PMID:1001878

  20. Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552

    DEFF Research Database (Denmark)

    Jensen, Lise Torp; Ladefoged, Søren; Birkelund, Svend

    1995-01-01

    monoclonal antibody (MAb) 552. The epitope for MAb 552 was localized at the repeated part of the protein. The gene encoding Lmp1 is part of a transcriptional complex that contains 9.5 direct repeats of 471 bp each. Pure cultures of mutant strains were obtained by subcloning, and three mutants were...

  1. Genetic background of Prop1(df) mutants provides remarkable protection against hypothyroidism-induced hearing impairment.

    Science.gov (United States)

    Fang, Qing; Giordimaina, Alicia M; Dolan, David F; Camper, Sally A; Mustapha, Mirna

    2012-04-01

    Hypothyroidism is a cause of genetic and environmentally induced deafness. The sensitivity of cochlear development and function to thyroid hormone (TH) mandates understanding TH action in this sensory organ. Prop1(df) and Pou1f1(dw) mutant mice carry mutations in different pituitary transcription factors, each resulting in pituitary thyrotropin deficiency. Despite the same lack of detectable serum TH, these mutants have very different hearing abilities: Prop1(df) mutants are mildly affected, while Pou1f1(dw) mutants are completely deaf. Genetic studies show that this difference is attributable to the genetic backgrounds. Using embryo transfer, we discovered that factors intrinsic to the fetus are the major contributor to this difference, not maternal effects. We analyzed Prop1(df) mutants to identify processes in cochlear development that are disrupted in other hypothyroid animal models but protected in Prop1(df) mutants by the genetic background. The development of outer hair cell (OHC) function is delayed, but Prestin and KCNQ4 immunostaining appear normal in mature Prop1(df) mutants. The endocochlear potential and KCNJ10 immunostaining in the stria vascularis are indistinguishable from wild type, and no differences in neurofilament or synaptophysin staining are evident in Prop1(df) mutants. The synaptic vesicle protein otoferlin normally shifts expression from OHC to IHC as temporary afferent fibers beneath the OHC regress postnatally. Prop1(df) mutants exhibit persistent, abnormal expression of otoferlin in apical OHC, suggesting delayed maturation of synaptic function. Thus, the genetic background of Prop1(df) mutants is remarkably protective for most functions affected in other hypothyroid mice. The Prop1(df) mutant is an attractive model for identifying the genes that protect against deafness.

  2. High-content screening of yeast mutant libraries by shotgun lipidomics

    DEFF Research Database (Denmark)

    Tarasov, Kirill; Stefanko, Adam; Casanovas, Albert

    2014-01-01

    To identify proteins with a functional role in lipid metabolism and homeostasis we designed a high-throughput platform for high-content lipidomic screening of yeast mutant libraries. To this end, we combined culturing and lipid extraction in 96-well format, automated direct infusion...... factor KAR4 precipitated distinct lipid metabolic phenotypes. These results demonstrate that the high-throughput shotgun lipidomics platform is a valid and complementary proxy for high-content screening of yeast mutant libraries....... nanoelectrospray ionization, high-resolution Orbitrap mass spectrometry, and a dedicated data processing framework to support lipid phenotyping across hundreds of Saccharomyces cerevisiae mutants. Our novel approach revealed that the absence of genes with unknown function YBR141C and YJR015W, and the transcription...

  3. Screening and identification of respiration deficiency mutants of yeasts (Saccharomyces Cerevisiae) induced by heavy ion irradiation

    International Nuclear Information System (INIS)

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

    2006-01-01

    A screen of respiration deficiency mutants of Saccharomyces Cerevisiae induced by 5.19 MeV/u 22 Ne 5- ion irradiation is studied. Some respiration deficiency mutants, which are white colony phenotype in the selective culture of TTC medium, are obtained. The mutants are effectively identified by means of a new and simplified restriction analysis method. (authors)

  4. Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Tibshirani, Michael; Zhao, Beibei; Gentil, Benoit J; Minotti, Sandra; Marques, Christine; Keith, Julia; Rogaeva, Ekaterina; Zinman, Lorne; Rouaux, Caroline; Robertson, Janice; Durham, Heather D

    2017-11-01

    Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with paralysis resulting from dysfunction and loss of motor neurons. A common neuropathological finding is attrition of motor neuron dendrites, which make central connections vital to motor control. The chromatin remodelling complex, neuronal Brahma-related gene 1 (Brg1)-associated factor complex (nBAF), is critical for neuronal differentiation, dendritic extension and synaptic function. We have identified loss of the crucial nBAF subunits Brg1, Brg1-associated factor 53b and calcium responsive transactivator in cultured motor neurons expressing FUS or TAR-DNA Binding Protein 43 (TDP-43) mutants linked to familial ALS. When plasmids encoding wild-type or mutant human FUS or TDP-43 were expressed in motor neurons of dissociated spinal cord cultures prepared from E13 mice, mutant proteins in particular accumulated in the cytoplasm. Immunolabelling of nBAF subunits was reduced in proportion to loss of nuclear FUS or TDP-43 and depletion of Brg1 was associated with nuclear retention of Brg1 mRNA. Dendritic attrition (loss of intermediate and terminal dendritic branches) occurred in motor neurons expressing mutant, but not wild-type, FUS or TDP-43. This attrition was delayed by ectopic over-expression of Brg1 and was reproduced by inhibiting Brg1 activity either through genetic manipulation or treatment with the chemical inhibitor, (E)-1-(2-Hydroxyphenyl)-3-((1R, 4R)-5-(pyridin-2-yl)-2, 5-diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one, demonstrating the importance of Brg1 to maintenance of dendritic architecture. Loss of nBAF subunits was also documented in spinal motor neurons in autopsy tissue from familial amyotrophic sclerosis (chromosome 9 open reading frame 72 with G4C2 nucleotide expansion) and from sporadic cases with no identified mutation, pointing to dysfunction of nBAF chromatin remodelling in multiple forms of ALS. © The Author 2017. Published by Oxford University Press. All rights reserved

  5. Studies on leaf mutants of Pea. (Part) I. Morphology, performance and somatic chromosomes

    International Nuclear Information System (INIS)

    Kaul, M.L.H.; Anjali, A.

    1988-01-01

    Three recessive non-allelic mutant genes alter foliar morphology of pea when present singly and in combination. Gene acacia replaces tendrils by a terminal leaflet, afila replaces leaflets by tendrils and cochleata replaces stipules by spoon shaped appendages. In combination, these genes drastically alter leaf morphology; plants can be identified only after flowering. The mutant genes influence shoot height, floral organ number, maturity period, grain yield and seed protein production; inter- and intra-genotypic variability in certain metric traits is significant. Influence of cochleata gene over floral form and function is considerable. In terms of seed yield and protein content, breeding value of all the mutants except of acacia is low because these mutant genes represent foreign untuned genes in pea genome. Segregation deficit is maximum in triple gene mutant with highly impaired fertility and low seed production. Somatic chromosome number in all the mutants and recombinants is 14; in morphology the chromosomes do not differ from the initial line, Bonneville. (author). 9 refs., 4 tabs

  6. Stress Marker Signatures in Lesion Mimic Single and Double Mutants Identify a Crucial Leaf Age-Dependent Salicylic Acid Related Defense Signal.

    Science.gov (United States)

    Kaurilind, Eve; Brosché, Mikael

    2017-01-01

    Plants are exposed to abiotic and biotic stress conditions throughout their lifespans that activates various defense programs. Programmed cell death (PCD) is an extreme defense strategy the plant uses to manage unfavorable environments as well as during developmentally induced senescence. Here we investigated the role of leaf age on the regulation of defense gene expression in Arabidopsis thaliana. Two lesion mimic mutants with misregulated cell death, catalase2 (cat2) and defense no death1 (dnd1) were used together with several double mutants to dissect signaling pathways regulating defense gene expression associated with cell death and leaf age. PCD marker genes showed leaf age dependent expression, with the highest expression in old leaves. The salicylic acid (SA) biosynthesis mutant salicylic acid induction deficient2 (sid2) had reduced expression of PCD marker genes in the cat2 sid2 double mutant demonstrating the importance of SA biosynthesis in regulation of defense gene expression. While the auxin- and jasmonic acid (JA)- insensitive auxin resistant1 (axr1) double mutant cat2 axr1 also led to decreased expression of PCD markers; the expression of several marker genes for SA signaling (ISOCHORISMATE SYNTHASE 1, PR1 and PR2) were additionally decreased in cat2 axr1 compared to cat2. The reduced expression of these SA markers genes in cat2 axr1 implicates AXR1 as a regulator of SA signaling in addition to its known role in auxin and JA signaling. Overall, the current study reinforces the important role of SA signaling in regulation of leaf age-related transcript signatures.

  7. No evidence for cardiac dysfunction in Kif6 mutant mice.

    Directory of Open Access Journals (Sweden)

    Abdul Hameed

    Full Text Available A KIF6 variant in man has been reported to be associated with adverse cardiovascular outcomes after myocardial infarction. No clear biological or physiological data exist for Kif6. We sought to investigate the impact of a deleterious KIF6 mutation on cardiac function in mice. Kif6 mutant mice were generated and verified. Cardiac function was assessed by serial echocardiography at baseline, after ageing and after exercise. Lipid levels were also measured. No discernable adverse lipid or cardiac phenotype was detected in Kif6 mutant mice. These data suggest that dysfunction of Kif6 is linked to other more complex biological/biochemical parameters or is unlikely to be of material consequence in cardiac function.

  8. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-01-01

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs

  9. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

    Directory of Open Access Journals (Sweden)

    Irini Topalidou

    2016-05-01

    Full Text Available The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

  10. The chloroplasts membrane phospholipids of Chlamydomonas reinhardii mutant not forming the Photosystem 2

    International Nuclear Information System (INIS)

    Trusova, V.M.; Ladygin, V.G.; Mezentsev, V.V.; Molchanov, M.I.

    1987-01-01

    Study on a component composition and physical state of photosynthetic membranes of Chlamydomonas chloroplasts of the wild type and mutant A-110 with disturbance of electron transfer chain in the photosystem 2 region permitted to conclude that 170 A diameter particles localized on the internal hydrophobic surface of membrane chips are deleted with respect to phosphatidylglycerin. The results obtained permit to suggest that the formation of protein-lipid complexes containing phosphatidylglycerins is suppressed in mutant A-110 which is not capable of the lamellar system differentation in

  11. Characterization Of Laccase T-DNA Mutants In Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Andersen, Jeppe Reitan; Asp, Torben; Mansfield, Shawn

    2009-01-01

    Laccases (P-diphenol:O2 oxidoreductase; EC 1.10.3.2), also termed laccase-like multicopper oxidases, are blue copper-containing oxidases which comprise multigene families in plants. In the Arabidopsis thaliana genome, 17 laccase genes (LAC1 to LAC17) have been annotated. To identify laccases...... for LAC15 T-DNA mutant seeds and an approximate 24 hour delay in germination was observed for these seeds. An approximate 20% reduction in glucose, galactose, and xylose was observed in primary stem cell walls of the LAC2 T-DNA mutants while similar relative increases in xylose were observed for LAC8...

  12. Newly identified CHO ERCC3/XPB mutations and phenotype characterization

    Science.gov (United States)

    Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

    2010-01-01

    Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596

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

    International Nuclear Information System (INIS)

    Balchiuniene, L.

    1995-01-01

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

  14. Dearth and Delayed Maturation of Testicular Germ Cells in Fanconi Anemia E Mutant Male Mice.

    Directory of Open Access Journals (Sweden)

    Chun Fu

    Full Text Available After using a self-inactivating lentivirus for non-targeted insertional mutagenesis in mice, we identified a transgenic family with a recessive mutation that resulted in reduced fertility in homozygous transgenic mice. The lentiviral integration site was amplified by inverse PCR. Sequencing revealed that integration had occurred in intron 8 of the mouse Fance gene, which encodes the Fanconi anemia E (Fance protein. Fanconi anemia (FA proteins play pivotal roles in cellular responses to DNA damage and Fance acts as a molecular bridge between the FA core complex and Fancd2. To investigate the reduced fertility in the mutant males, we analyzed postnatal development of testicular germ cells. At one week after birth, most tubules in the mutant testes contained few or no germ cells. Over the next 2-3 weeks, germ cells accumulated in a limited number of tubules, so that some tubules contained germ cells around the full periphery of the tubule. Once sufficient numbers of germ cells had accumulated, they began to undergo the later stages of spermatogenesis. Immunoassays revealed that the Fancd2 protein accumulated around the periphery of the nucleus in normal developing spermatocytes, but we did not detect a similar localization of Fancd2 in the Fance mutant testes. Our assays indicate that although Fance mutant males are germ cell deficient at birth, the extant germ cells can proliferate and, if they reach a threshold density, can differentiate into mature sperm. Analogous to previous studies of FA genes in mice, our results show that the Fance protein plays an important, but not absolutely essential, role in the initial developmental expansion of the male germ line.

  15. Promising rice mutants

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    Science.gov (United States)

    Godon, C; Krapp, A; Leydecker, M T; Daniel-Vedele, F; Caboche, M

    1996-02-25

    This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.

  17. Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing.

    Science.gov (United States)

    Huo, Heqiang; Henry, Isabelle M; Coppoolse, Eric R; Verhoef-Post, Miriam; Schut, Johan W; de Rooij, Han; Vogelaar, Aat; Joosen, Ronny V L; Woudenberg, Leo; Comai, Luca; Bradford, Kent J

    2016-11-01

    Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  18. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    Directory of Open Access Journals (Sweden)

    Jun Ren

    2014-01-01

    Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

  19. Gamma ray induced mutants in Coleus

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-07-01

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

  20. Gamma ray induced mutants in Coleus

    International Nuclear Information System (INIS)

    Vasudevan, K.; Jos, J.S.

    1988-01-01

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

  1. Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex.

    Science.gov (United States)

    Otsuki, T; Furukawa, Y; Ikeda, K; Endo, H; Yamashita, T; Shinohara, A; Iwamatsu, A; Ozawa, K; Liu, J M

    2001-11-01

    Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer. We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF complex. We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA. Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling. Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1. The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair.

  2. Oncogenic Signaling by Leukemia-Associated Mutant Cbl Proteins

    Science.gov (United States)

    Nadeau, Scott; An, Wei; Palermo, Nick; Feng, Dan; Ahmad, Gulzar; Dong, Lin; Borgstahl, Gloria E. O.; Natarajan, Amarnath; Naramura, Mayumi; Band, Vimla; Band, Hamid

    2013-01-01

    Members of the Cbl protein family (Cbl, Cbl-b, and Cbl-c) are E3 ubiquitin ligases that have emerged as critical negative regulators of protein tyrosine kinase (PTK) signaling. This function reflects their ability to directly interact with activated PTKs and to target them as well as their associated signaling components for ubiquitination. Given the critical roles of PTK signaling in driving oncogenesis, recent studies in animal models and genetic analyses in human cancer have firmly established that Cbl proteins function as tumor suppressors. Missense mutations or small in-frame deletions within the regions of Cbl protein that are essential for its E3 activity have been identified in nearly 5% of leukemia patients with myelodysplastic/myeloproliferative disorders. Based on evidence from cell culture studies, in vivo models and clinical data, we discuss the potential signaling mechanisms of mutant Cbl-driven oncogenesis. Mechanistic insights into oncogenic Cbl mutants and associated animal models are likely to enhance our understanding of normal hematopoietic stem cell homeostasis and provide avenues for targeted therapy of mutant Cbl-driven cancers. PMID:23997989

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

    Science.gov (United States)

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

    1985-01-01

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

  4. Induction and isolation of DNA transformation mutants in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Hegerich, P.A.; Bruschi, C.V.

    1987-01-01

    The objective of this research was to induce and isolate mutants of the yeast Saccharomyces cerevisiae which have become transformable by purified plasmid DNA. Non-transformable yeast cells were mutagenized by ultraviolet light using a 65% lethal dose (480 ergs/mm 2 ). After a period of overnight liquid holding recovery, the irradiated cells were subjected to DNA transformation using our CaCl 2 protocol with the multi-marker shuttle plasmid pBB carrying the LEU 2 leucine gene. Following transformation the colonies that grew on selective leucineless medium were identified and subjected to further genetic analysis. From a total of 1 x 10 9 cells the authors have isolated 7 colonies deriving from putative mutants that have acquired the capability to uptake plasmid DNA. The transformants were cured from the plasmid by its mitotic loss on non-selective medium, then re-transformed to verify their genetic competence to give rise to a number of transformants comparable to transformable strains. We have identified and isolated one mutant, coded trs-1, which is able to reproduce a frequency of transformation comparable with the tranformable control. They, therefore, conclude that this mutant is specific for plasmid DNA transformation and that the mutation is mitotically stable

  5. Responses of Soybean Mutant Lines to Aluminium under In Vitro and In Vivo Condition

    International Nuclear Information System (INIS)

    Yuliasti; Sudarsono

    2011-01-01

    The main limited factors of soybean plants expansion in acid soil are Aluminium (Al) toxicity and low pH. The best approach to solve this problem is by using Al tolerance variety. In vitro or in vivo selections using selective media containing AlCl 3 and induced callus embryonic of mutant lines are reliable methods to develop a new variety. The objectives of this research are to evaluate response of soybean genotypes against AlCl 3 under in vitro and in vivo condition. Addition of 15 part per million (ppm) AlCl 3 into in vitro and in vivo media severely affected plant growth. G3 soybean mutant line was identified as more tolerant than the control soybean cultivar Tanggamus. This mutant line was able to survive under more severe AlCl 3 concentrations (15 ppm) under in vitro conditions. Under in vivo conditions, G1 and G4 mutants were also identified as more tolerant than Tanggamus since they produced more pods and higher dry seed weigh per plant. Moreover, G4 mutant line also produced more dry seed weight per plant than Tanggamus when they were grown on soil containing high Al concentration 8.1 me/100 gr = 81 ppm Al +3 . (author)

  6. Isolation and partial characterization of a mutant of Bacillus thuringiensis producing melanin Isolamento e caracterização parcial de um mutante de Bacillus thuringiensis produtor de melanina

    Directory of Open Access Journals (Sweden)

    Gislayne T. Vilas-Bôas

    2005-09-01

    Full Text Available A mutant (407-P of Bacillus thuringiensis subsp. thuringiensis strain 407 producing a melanin was obtained after treatment with the mutagenic agent ethyl-methane-sulfonate. Several microbiological and biochemical properties of the two strains were analyzed and the results were similar. The mutant 407-P was also incorporated into non-sterilized soil samples, recovered, easily identified, and quantified, what enables its use in ecology of B. thuringiensis.Um mutante (407-P da linhagem Bacillus thuringiensis subsp. thuringiensis 407 produtor de melanina foi obtido após tratamento com o agente mutagênico etil-metano-sulfonato. Diversas propriedades microbiológicas e bioquímicas das duas linhagens foram analisadas e os resultados foram similares. O mutante 407-P foi incorporado em amostras de solo não esterilizado, recuperado, facilmente identificado e quantificado, possibilitando seu uso em estudos de ecologia de B. thuringiensis.

  7. Identification and quantification of flavonoids in yellow grain mutant of rice (Oryza sativa L.).

    Science.gov (United States)

    Kim, Backki; Woo, Sunmin; Kim, Mi-Jung; Kwon, Soon-Wook; Lee, Joohyun; Sung, Sang Hyun; Koh, Hee-Jong

    2018-02-15

    Flavonoids are naturally occurring phenolic compounds with potential health-promoting activities. Although anthocyanins and phenolic acids in coloured rice have been investigated, few studies have focused on flavonoids. Herein, we analysed flavonoids in a yellow grain rice mutant using UHPLC-DAD-ESI-Q-TOF-MS, and identified 19 flavonoids by comparing retention times and accurate mass measurements. Among them, six flavonoids, isoorientin, isoorientin 2″-O-glucoside, vitexin 2″-O-glucoside, isovitexin, isoscoparin 2″-O-glucoside and isoscoparin, were isolated and fully identified from the yellow grain rice mutant, and the levels were significantly higher than wild-type, with isoorientin particularly abundant in mutant embryo. Significant differences in total phenolic compounds and antioxidant activity were observed in mutant rice by DPPH, FRAP and TEAC assays. The results suggest that the representative six flavonoids may play an important role in colouration and antioxidant activity of embryo and endosperm tissue. The findings provide insight into flavonoid biosynthesis and the possibility of improving functionality in rice. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  9. The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene

    NARCIS (Netherlands)

    Soppe, W.J.J.; Jacobsen, S.E.; Alonso-Blanco, C.; Jackson, J.P.; Kakutani, T.; Koornneef, M.; Peeters, A.J.M.

    2000-01-01

    The transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants. FWA was identified by loss-of-function mutations in normally flowering revertants of the fwa mutant, and it encodes a homeodomain-containing transcription factor. The DNA sequence of wild-type and fwa mutant alleles

  10. Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

    Science.gov (United States)

    Suzuki, Keisuke; Mizushima, Hiroto; Abe, Hiroyuki; Iwamoto, Ryo; Nakamura, Haruki; Mekada, Eisuke

    2015-05-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  11. Selection of high hectolitre weight mutants of winter wheat

    International Nuclear Information System (INIS)

    Crowley, C.; Jones, P.

    1989-01-01

    Grain quality in wheat includes hectolitre weight (HLW) besides protein content and thousand-grain weight (TGW). The British winter wheat variety ''Guardian'' has a very high yield potential. Although the long grain of ''Guardian'' results in a desirable high TGW the HLW is too low. To select mutants exhibiting increased HLW the character was first analyzed to identify traits that could more easily be screened for using M 2 seeds. In comparison of 6 wheat cultivars, correlation analyses with HLW resulted in coefficients of -0.86 (grain length, L:P 2 seeds for shorter, less prolate grains. Mutagenesis was carried out using EMS sulphonate (1.8 or 3.6%), sodium azide (2 or 20 mM) or X-rays (7.5 or 20 kR). 69 M 2 grains with altered shape were selected. Examination of the M 3 progeny confirmed 6 grain-shape mutants, most of them resulting from EMS treatment (Table). Two of the mutants showed TGW values significantly below the parental variety, but three mutants exhibited HLW and TGW values significantly greater than those of the parental variety. Microplot yield trails on selected M 3 lines are in progress. The influence of physical grain characteristics on HLW offers prospects for mechanical fractionation of large M 2 populations. The application of gravity separators (fractionation on the basis of grain density) and sieves (fractionation on the basis of grain length) in screening mutants possessing improved grain quality is being investigated

  12. The Swedish mutant barley collection

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1989-07-01

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

  13. The Swedish mutant barley collection

    International Nuclear Information System (INIS)

    1989-01-01

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

  14. Mutants of alfalfa mosaic virus

    International Nuclear Information System (INIS)

    Roosien, J.

    1983-01-01

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

  15. Mutant genes in pea breeding

    International Nuclear Information System (INIS)

    Swiecicki, W.K.

    1990-01-01

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

  16. Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2'-O-Methylation Mutant.

    Directory of Open Access Journals (Sweden)

    Bianca Schmid

    2015-12-01

    Full Text Available Dengue virus (DENV is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2'-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2'-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

  17. Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Akshay Bhinge

    2017-04-01

    Full Text Available Summary: Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS, it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. : In this article, Bhinge, Stanton, and colleagues use genome editing of patient-derived iPSCs to model ALS phenotypic defects in vitro. Transcriptomic analysis of disease MNs reveals activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs. Pharmacological screening uncovers activated ERK and JNK signaling as therapeutic targets in ALS. Keywords: ALS, SOD1, FUS, CRISPR-Cas9, p38, ERK, JNK, WNT, TP53, JUN

  18. Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

    Science.gov (United States)

    Wang, Aoli; Yan, Xiao-E; Wu, Hong; Wang, Wenchao; Hu, Chen; Chen, Cheng; Zhao, Zheng; Zhao, Peng; Li, Xixiang; Wang, Li; Wang, Beilei; Ye, Zi; Wang, Jinhua; Wang, Chu; Zhang, Wei; Gray, Nathanael S; Weisberg, Ellen L; Chen, Liang; Liu, Jing; Yun, Cai-Hong; Liu, Qingsong

    2016-10-25

    Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

  19. Development and characterization of mutant winter wheat (Triticum aestivum L.) accessions resistant to the herbicide quizalofop.

    Science.gov (United States)

    Ostlie, Michael; Haley, Scott D; Anderson, Victoria; Shaner, Dale; Manmathan, Harish; Beil, Craig; Westra, Phillip

    2015-02-01

    New herbicide resistance traits in wheat were produced through the use of induced mutagenesis. While herbicide-resistant crops have become common in many agricultural systems, wheat has seen few introductions of herbicide resistance traits. A population of Hatcher winter wheat treated with ethyl methanesulfonate was screened with quizalofop to identify herbicide-resistant plants. Initial testing identified plants that survived multiple quizalofop applications. A series of experiments were designed to characterize this trait. In greenhouse studies the mutants exhibited high levels of quizalofop resistance compared to non-mutant wheat. Sequencing ACC1 revealed a novel missense mutation causing an alanine to valine change at position 2004 (Alopecurus myosuroides reference sequence). Plants carrying single mutations in wheat's three genomes (A, B, D) were identified. Acetyl co-enzyme A carboxylase in resistant plants was 4- to 10-fold more tolerant to quizalofop. Populations of segregating backcross progenies were developed by crossing each of the three individual mutants with wild-type wheat. Experiments conducted with these populations confirmed largely normal segregation, with each mutant allele conferring an additive level of resistance. Further tests showed that the A genome mutation conferred the greatest resistance and the B genome mutation conferred the least resistance to quizalofop. The non-transgenic herbicide resistance trait identified will enhance weed control strategies in wheat.

  20. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

    Directory of Open Access Journals (Sweden)

    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

  1. Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

    Science.gov (United States)

    Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

    2012-01-01

    Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

  2. Identifying cognitive complexity factors affecting the complexity of procedural steps in emergency operating procedures of a nuclear power plant

    International Nuclear Information System (INIS)

    Park, Jinkyun; Jeong, Kwangsup; Jung, Wondea

    2005-01-01

    In complex systems such as a nuclear and chemical plant, it is well known that the provision of understandable procedures that allow operators to clarify what needs to be done and how to do it is one of the requisites to secure their safety. As a previous study in providing understandable procedures, the step complexity (SC) measure that can quantify the complexity of procedural steps in emergency operating procedures (EOPs) of a nuclear power plant (NPP) was suggested. However, the necessity of additional complexity factors that can consider a cognitive aspect in evaluating the complexity of procedural steps is raised. To this end, the comparisons between operators' performance data measured by the form of a step performance time with their behavior in carrying out the prescribed activities of procedural steps are conducted in this study. As a result, two kinds of complexity factors (the abstraction level of knowledge and the level of engineering decision) that could affect an operator's cognitive burden are identified. Although a well-designed experiment is indispensable for confirming the appropriateness of the additional complexity factors, it is strongly believed that the change of operators' performance data can be more authentically explained if the additional complexity factors are taken into consideration

  3. Identifying cognitive complexity factors affecting the complexity of procedural steps in emergency operating procedures of a nuclear power plant

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jinkyun [Integrated Safety Assessment Division, Korea Atomic Energy Research Institute, P.O. Box 105, Duckjin-Dong, Yusong-Ku, Taejon 305-600 (Korea, Republic of)]. E-mail: kshpjk@kaeri.re.kr; Jeong, Kwangsup [Integrated Safety Assessment Division, Korea Atomic Energy Research Institute, P.O. Box 105, Duckjin-Dong, Yusong-Ku, Taejon 305-600 (Korea, Republic of); Jung, Wondea [Integrated Safety Assessment Division, Korea Atomic Energy Research Institute, P.O. Box 105, Duckjin-Dong, Yusong-Ku, Taejon 305-600 (Korea, Republic of)

    2005-08-01

    In complex systems such as a nuclear and chemical plant, it is well known that the provision of understandable procedures that allow operators to clarify what needs to be done and how to do it is one of the requisites to secure their safety. As a previous study in providing understandable procedures, the step complexity (SC) measure that can quantify the complexity of procedural steps in emergency operating procedures (EOPs) of a nuclear power plant (NPP) was suggested. However, the necessity of additional complexity factors that can consider a cognitive aspect in evaluating the complexity of procedural steps is raised. To this end, the comparisons between operators' performance data measured by the form of a step performance time with their behavior in carrying out the prescribed activities of procedural steps are conducted in this study. As a result, two kinds of complexity factors (the abstraction level of knowledge and the level of engineering decision) that could affect an operator's cognitive burden are identified. Although a well-designed experiment is indispensable for confirming the appropriateness of the additional complexity factors, it is strongly believed that the change of operators' performance data can be more authentically explained if the additional complexity factors are taken into consideration.

  4. Identifying cognitive complexity factors affecting the complexity of procedural steps in emergency operating procedures of a nuclear power plant

    Energy Technology Data Exchange (ETDEWEB)

    Jinkyun Park; Kwangsup Jeong; Wondea Jung [Korea Atomic Energy Research Institute, Taejon (Korea). Integrated Safety Assessment Division

    2005-08-15

    In complex systems such as a nuclear and chemical plant, it is well known that the provision of understandable procedures that allow operators to clarify what needs to be done and how to do it is one of the requisites to secure their safety. As a previous study in providing understandable procedures, the step complexity (SC) measure that can quantify the complexity of procedural steps in emergency operating procedures (EOPs) of a nuclear power plant (NPP) was suggested. However, the necessity of additional complexity factors that can consider a cognitive aspect in evaluating the complexity of procedural steps is raised. To this end, the comparisons between operator' performance data measured by the form of a step performance time with their behavior in carrying out the prescribed activities of procedural steps are conducted in this study. As a result, two kinds of complexity factors (the abstraction level of knowledge and the level of engineering decision) that could affect an operator's cognitive burden are identified. Although a well-designed experiment is indispensable for confirming the appropriateness of the additional complexity factors, it is strongly believed that the change of operators' performance data can be more authentically explained if the additional complexity factors are taken into consideration. (author)

  5. Genetic fingerprinting of mutant rose cultivars

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-07-01

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

  6. Comparative analysis of the protein compositions between wild type and body color mutant of helicoverpa armigera adult

    International Nuclear Information System (INIS)

    He Lihua; Chen Jin'e; Liu Yan; Wang Yongqiang; Liu Peigang; Meng Zhiqi

    2012-01-01

    To gain an in-depth understanding of the fineness and regulation mechanism of body color mutant of Helicoverpa armigera Hbner, the protein composition differences between adult of dominant mutant, recessive mutant and wild type were studied using the SDS-PAGE combined with MALDI-TOF-TOF/MS and bioinformatics analysis. The results indicated that the protein composition of the dominant mutant and wild type had little difference. However, there were obvious differences between the recessive mutant and wild-type. Three specific stripe were chosen for mass spectrometry and bioinformatics analysis, and two types of proteins related to energy metabolism and cytoskeleton were identified. These findings suggested that the two types of proteins may be associated with occurrence and regulation of body color mutant traits of H. armigera. (authors)

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

    International Nuclear Information System (INIS)

    Nagatomi, Shigeki

    1996-01-01

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

  8. Evaluation of some garlic (Allium Sativum L.) mutants resistant to white rot disease by RAPD analysis

    International Nuclear Information System (INIS)

    Nabulsi, I.; Al-Safadi, B.; Mir ali, N.; Arabi, M.I.E.

    2002-01-01

    Random amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic diversity among eight garlic mutants resistant to white rot disease (Sclerotium cepivorum) and two controls. Twelve of 13 synthetic random primers were found to identify polymorphism in amplification products. Mutants characterised with moderate resistance to white rot were closely related to the control using cluster and correlation analyses. On the other hand, highly resistant mutants were quite distant from the control with low correlation coefficients. The banding patterns produced by primer OPB-15 (GGAAGGGTGTT) with highly resistant mutants may be used as genetic markers for early selection of resistant plants. (author)

  9. Purification and characterization of mutant miniPlasmin for thrombolytic therapy

    Directory of Open Access Journals (Sweden)

    Lin Xiaotao

    2013-01-01

    Full Text Available Abstract Background Previous animal studies by us and others have indicated that catheter-administered plasmin or its des-kringle derivatives may be more appropriate alternatives to plasminogen activators for treating thrombolytic diseases, since it has a very short serum half-life and therefore does not result in hemorrhaging. We have previously produced recombinant miniPlasmin (mPlasmin that was proven suitable for treating peripheral arterial occlusion in animal models. However, our previous results showed that non-specific cleavage at position K698 of mPlasmin during activation hindered the further development of this promising therapeutic candidate. In order to minimize or eliminate the non-specific cleavage problem, we performed saturation mutagenesis at the K698 position to develop a mutant form of mPlasmin for thrombolytic therapy. Methods We changed K698 to 16 other amino acids, with preferred E. coli codons. Each of these mutants were expressed in E. coli as inclusion bodies and then refolded, purified, and subsequently characterized by detailed kinetic assays/experiments/studies which identified highly active mutants devoid of non-specific cleavage. Results Activation studies indicated that at those conditions in which the wild type enzyme is cut at the non-specific position K698, the active mutants can be activated without being cleaved at this position. Conclusions From the above results, we selected two mutants, K698Q and K698N, as our lead candidates for further thrombolytic drug developments. The selected mutants are potentially better therapeutic candidates for thrombolytic therapy.

  10. DNA Misfolding Found to Cause Cancer in IDH-mutant Gliomas

    Science.gov (United States)

    Researchers studying IDH-mutant brain tumors have identified a previously unknown genetic mechanism that may contribute to cancer. A change in how DNA is arranged, or packaged, in the cell nucleus may inappropriately activate a gene associated with brain cancer.

  11. Synapsis-Defective Mutants Reveal a Correlation Between Chromosome Conformation and the Mode of Double-Strand Break Repair During Caenorhabditis elegans Meiosis

    OpenAIRE

    Smolikov, Sarit; Eizinger, Andreas; Hurlburt, Allison; Rogers, Eric; Villeneuve, Anne M.; Colaiácovo, Mónica P.

    2007-01-01

    SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspri...

  12. Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

    DEFF Research Database (Denmark)

    Welin, M.; Skovgaard, T.; Knecht, Wolfgang

    2005-01-01

    The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3...

  13. A QM-MD simulation approach to the analysis of FRET processes in (bio)molecular systems. A case study: complexes of E. coli purine nucleoside phosphorylase and its mutants with formycin A.

    Science.gov (United States)

    Sobieraj, M; Krzyśko, K A; Jarmuła, A; Kalinowski, M W; Lesyng, B; Prokopowicz, M; Cieśla, J; Gojdź, A; Kierdaszuk, B

    2015-04-01

    Predicting FRET pathways in proteins using computer simulation techniques is very important for reliable interpretation of experimental data. A novel and relatively simple methodology has been developed and applied to purine nucleoside phosphorylase (PNP) complexed with a fluorescent ligand - formycin A (FA). FRET occurs between an excited Tyr residue (D*) and FA (A). This study aims to interpret experimental data that, among others, suggests the absence of FRET for the PNPF159A mutant in complex with FA, based on novel theoretical methodology. MD simulations for the protein molecule containing D*, and complexed with A, are carried out. Interactions of D* with its molecular environment are accounted by including changes of the ESP charges in S1, compared to S0, and computed at the SCF-CI level. FRET probability W F depends on the inverse six-power of the D*-A distance, R da . The orientational factor 0 < k(2) < 4 between D* and A is computed and included in the analysis. Finally W F is time-averaged over the MD trajectories resulting in its mean value. The red-shift of the tyrosinate anion emission and thus lack of spectral overlap integral and thermal energy dissipation are the reasons for the FRET absence in the studied mutants at pH 7 and above. The presence of the tyrosinate anion results in a competitive energy dissipation channel and red-shifted emission, thus in consequence in the absence of FRET. These studies also indicate an important role of the phenyl ring of Phe159 for FRET in the wild-type PNP, which does not exist in the Ala159 mutant, and for the effective association of PNP with FA. In a more general context, our observations point out very interesting and biologically important properties of the tyrosine residue in its excited state, which may undergo spontaneous deprotonation in the biomolecular systems, resulting further in unexpected physical and/or biological phenomena. Until now, this observation has not been widely discussed in the

  14. Induction of Mutants in Durum Wheat

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  15. Rescue of the apoptotic-inducing function of mutant p53 by small molecule RITA.

    Science.gov (United States)

    Zhao, Carolyn Y; Grinkevich, Vera V; Nikulenkov, Fedor; Bao, Wenjie; Selivanova, Galina

    2010-05-01

    Expression of mutant p53 correlates with poor prognosis in many tumors, therefore strategies aimed at reactivation of mutant p53 are likely to provide important benefits for treatment of tumors that are resistant to chemotherapy and radiotherapy. We have previously identified and characterized a small molecule RITA which binds p53 and induces a conformational change which prevents the binding of p53 to several inhibitors, including its own destructor MDM2. In this way, RITA rescues the tumor suppression function of wild type p53. Here, we demonstrate that RITA suppressed the growth and induced apoptosis in human tumor cell lines of a diverse origin carrying mutant p53 proteins. RITA restored transcriptional transactivation and transrepression function of several hot spot p53 mutants. The ability of RITA to rescue the activity of different p53 mutants suggests its generic mechanism of action. Thus, RITA is a promising lead for the development of anti-cancer drugs that reactivate the tumor suppressor function of p53 in cancer cells irrespective whether they express mutant or wild type p53.

  16. Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

    Directory of Open Access Journals (Sweden)

    Andrey A Filippov

    Full Text Available BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

  17. Molecular mechanisms associated with leukemic transformation of MPL-mutant myeloproliferative neoplasms

    DEFF Research Database (Denmark)

    Beer, Philip A; Ortmann, Christina A; Stegelmann, Frank

    2010-01-01

    Somatic activating mutations in MPL, the thrombopoietin receptor, occur in the myeloproliferative neoplasms, although virtually nothing is known about their role in evolution to acute myeloid leukemia. In this study, the MPL T487A mutation, identified in de novo acute myeloid leukemia......, was not detected in 172 patients with a myeloproliferative neoplasm. In patients with a prior MPL W515L-mutant myeloproliferative neoplasm, leukemic transformation was accompanied by MPL-mutant leukemic blasts, was seen in the absence of prior cytoreductive therapy and often involved loss of wild-type MPL...

  18. Applying complexity theory: A primer for identifying and modeling firm anomalies

    Directory of Open Access Journals (Sweden)

    Arch G. Woodside

    2018-01-01

    Full Text Available This essay elaborates on the usefulness of embracing complexity theory, modeling outcomes rather than directionality, and modeling complex rather than simple outcomes in strategic management. Complexity theory includes the tenet that most antecedent conditions are neither sufficient nor necessary for the occurrence of a specific outcome. Identifying a firm by individual antecedents (i.e., non-innovative versus highly innovative, small versus large size in sales or number of employees, or serving local versus international markets provides shallow information in modeling specific outcomes (e.g., high sales growth or high profitability—even if directional analyses (e.g., regression analysis, including structural equation modeling indicates that the independent (main effects of the individual antecedents relate to outcomes directionally—because firm (case anomalies almost always occur to main effects. Examples: a number of highly innovative firms have low sales while others have high sales and a number of non-innovative firms have low sales while others have high sales. Breaking-away from the current dominant logic of directionality testing—null hypotheses statistic testing (NHST—to embrace somewhat precise outcome testing (SPOT is necessary for extracting highly useful information about the causes of anomalies—associations opposite to expected and “statistically significant” main effects. The study of anomalies extends to identifying the occurrences of four-corner strategy outcomes: firms doing well in favorable circumstances, firms doing badly in favorable circumstances, firms doing well in unfavorable circumstances, and firms doing badly in unfavorable circumstances. Models of four-corner strategy outcomes advances strategic management beyond the current dominant logic of directional modeling of single outcomes.

  19. RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

    Directory of Open Access Journals (Sweden)

    Simona Lamba

    2014-09-01

    Full Text Available KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

  20. Root hair mutants of barley

    International Nuclear Information System (INIS)

    Engvild, K.C.; Rasmussen, K.

    2005-01-01

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

  1. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    Science.gov (United States)

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Identifying a set of influential spreaders in complex networks

    Science.gov (United States)

    Zhang, Jian-Xiong; Chen, Duan-Bing; Dong, Qiang; Zhao, Zhi-Dan

    2016-06-01

    Identifying a set of influential spreaders in complex networks plays a crucial role in effective information spreading. A simple strategy is to choose top-r ranked nodes as spreaders according to influence ranking method such as PageRank, ClusterRank and k-shell decomposition. Besides, some heuristic methods such as hill-climbing, SPIN, degree discount and independent set based are also proposed. However, these approaches suffer from a possibility that some spreaders are so close together that they overlap sphere of influence or time consuming. In this report, we present a simply yet effectively iterative method named VoteRank to identify a set of decentralized spreaders with the best spreading ability. In this approach, all nodes vote in a spreader in each turn, and the voting ability of neighbors of elected spreader will be decreased in subsequent turn. Experimental results on four real networks show that under Susceptible-Infected-Recovered (SIR) and Susceptible-Infected (SI) models, VoteRank outperforms the traditional benchmark methods on both spreading rate and final affected scale. What’s more, VoteRank has superior computational efficiency.

  3. Spatial constraints govern competition of mutant clones in human epidermis.

    Science.gov (United States)

    Lynch, M D; Lynch, C N S; Craythorne, E; Liakath-Ali, K; Mallipeddi, R; Barker, J N; Watt, F M

    2017-10-24

    Deep sequencing can detect somatic DNA mutations in tissues permitting inference of clonal relationships. This has been applied to human epidermis, where sun exposure leads to the accumulation of mutations and an increased risk of skin cancer. However, previous studies have yielded conflicting conclusions about the relative importance of positive selection and neutral drift in clonal evolution. Here, we sequenced larger areas of skin than previously, focusing on cancer-prone skin spanning five decades of life. The mutant clones identified were too large to be accounted for solely by neutral drift. Rather, using mathematical modelling and computational lattice-based simulations, we show that observed clone size distributions can be explained by a combination of neutral drift and stochastic nucleation of mutations at the boundary of expanding mutant clones that have a competitive advantage. These findings demonstrate that spatial context and cell competition cooperate to determine the fate of a mutant stem cell.

  4. Molecular Imaging Of Metabolic Reprogramming In Mutant IDH Cells

    Directory of Open Access Journals (Sweden)

    Pavithra eViswanath

    2016-03-01

    Full Text Available Mutations in the metabolic enzyme isocitrate dehydrogenase (IDH have recently been identified as drivers in the development of several tumor types. Most notably, cytosolic IDH1 is mutated in 70-90% of low-grade gliomas and upgraded glioblastomas, and mitochondrial IDH2 is mutated in ~20% of acute myeloid leukemia cases. Wild-type IDH catalyzes the interconversion of isocitrate to α-ketoglutarate (α-KG. Mutations in the enzyme lead to loss of wild-type enzymatic activity and a neomorphic activity that converts α-KG to 2-hydroxyglutarate (2-HG. In turn, 2-HG, which has been termed an oncometabolite, inhibits key α-KG- dependent enzymes, resulting in alterations of the cellular epigenetic profile and, subsequently, inhibition of differentiation and initiation of tumorigenesis. In addition, it is now clear that the IDH mutation also induces a broad metabolic reprogramming that extends beyond 2-HG production, and this reprogramming often differs from what has been previously reported in other cancer types. In this review we will discuss in detail what is known to date about the metabolic reprogramming of mutant IDH cells and how this reprogramming has been investigated using molecular metabolic imaging. We will describe how metabolic imaging has helped shed light on the basic biology of mutant IDH cells and how this information can be leveraged to identify new therapeutic targets and to develop new clinically translatable imaging methods to detect and monitor mutant IDH tumors in vivo.

  5. Cooperativity of complex salt bridges

    OpenAIRE

    Gvritishvili, Anzor G.; Gribenko, Alexey V.; Makhatadze, George I.

    2008-01-01

    The energetic contribution of complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously, has been suggested to have importance for protein stability. Detailed analysis of the net energetics of complex salt bridge formation using double- and triple-mutant cycle analysis revealed conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative, i.e., the net strength of the complex salt bridge...

  6. Inducement and identification of an endosperm mutant in maize

    African Journals Online (AJOL)

    ajl yemi

    2011-11-30

    Nov 30, 2011 ... Drummond EP, Ausubel FM (2000). Three unique mutants of. Arabidopsis identify eds loci required for limiting growth of a biotrophic fungal pathogen. Plant J. 24(2): 205-218. Dinges JR, Colleoni C, Myers AM, James MG (2001). Molecular structure of three mutations at the maize sugary1 locus and their.

  7. Identification of a mutant α1 Na/K-ATPase that pumps but is defective in signal transduction.

    Science.gov (United States)

    Lai, Fangfang; Madan, Namrata; Ye, Qiqi; Duan, Qiming; Li, Zhichuan; Wang, Shaomeng; Si, Shuyi; Xie, Zijian

    2013-05-10

    It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. A420P mutant has normal pumping but not signaling function. Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.

  8. Isozyme differences in barley mutants

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  11. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

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

    Science.gov (United States)

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

    2010-12-01

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

  13. TOMATOMA Update: Phenotypic and Metabolite Information in the Micro-Tom Mutant Resource.

    Science.gov (United States)

    Shikata, Masahito; Hoshikawa, Ken; Ariizumi, Tohru; Fukuda, Naoya; Yamazaki, Yukiko; Ezura, Hiroshi

    2016-01-01

    TOMATOMA (http://tomatoma.nbrp.jp/) is a tomato mutant database providing visible phenotypic data of tomato mutant lines generated by ethylmethane sulfonate (EMS) treatment or γ-ray irradiation in the genetic background of Micro-Tom, a small and rapidly growing variety. To increase mutation efficiency further, mutagenized M3 seeds were subjected to a second round of EMS treatment; M3M1 populations were generated. These plants were self-pollinated, and 4,952 lines of M3M2 mutagenized seeds were generated. We checked for visible phenotypes in the M3M2 plants, and 618 mutant lines with 1,194 phenotypic categories were identified. In addition to the phenotypic information, we investigated Brix values and carotenoid contents in the fruits of individual mutants. Of 466 samples from 171 mutant lines, Brix values and carotenoid contents were between 3.2% and 11.6% and 6.9 and 37.3 µg g(-1) FW, respectively. This metabolite information concerning the mutant fruits would be useful in breeding programs as well as for the elucidation of metabolic regulation. Researchers are able to browse and search this phenotypic and metabolite information and order seeds of individual mutants via TOMATOMA. Our new Micro-Tom double-mutagenized populations and the metabolic information could provide a valuable genetic toolkit to accelerate tomato research and potential breeding programs. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. Probiotic features of Lactobacillus plantarum mutant strains.

    Science.gov (United States)

    Bove, Pasquale; Gallone, Anna; Russo, Pasquale; Capozzi, Vittorio; Albenzio, Marzia; Spano, Giuseppe; Fiocco, Daniela

    2012-10-01

    In this study, the probiotic potential of Lactobacillus plantarum wild-type and derivative mutant strains was investigated. Bacterial survival was evaluated in an in vitro system, simulating the transit along the human oro-gastro-intestinal tract. Interaction with human gut epithelial cells was studied by assessing bacterial adhesive ability to Caco-2 cells and induction of genes involved in innate immunity. L. plantarum strains were resistant to the combined stress at the various steps of the simulated gastrointestinal tract. Major decreases in the viability of L. plantarum cells were observed mainly under drastic acidic conditions (pH ≤ 2.0) of the gastric compartment. Abiotic stresses associated to small intestine poorly affected bacterial viability. All the bacterial strains significantly adhered to Caco-2 cells, with the ΔctsR mutant strain exhibiting the highest adhesion. Induction of immune-related genes resulted higher upon incubation with heat-inactivated bacteria rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with different L. plantarum strains, evidencing a possible role of the knocked out bacterial genes in the modulation of host cell response. In particular, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. Our study highlights the relevance of microbial genetic background in host-probiotic interaction and might contribute to identify candidate bacterial genes and molecules involved in probiosis.

  15. THO/TREX complex

    KAUST Repository

    Dö ll, Stefanie; Kuhlmann, Markus; Rutten, Twan; Mette, Michael F.; Scharfenberg, Sarah; Petridis, Antonios; Berreth, Dorothee-Carina; Mock, Hans-Peter

    2017-01-01

    Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low-temperature stress in Arabidopsis thaliana. A genetic screen for altered scopolin levels in A. thaliana revealed a mutant compromised in scopolin accumulation in response to stress; the lesion was present in a homologue of THO1 coding for a subunit of the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants defective in THO, AGO1, SDS3 and RDR6 were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing such as the trans-acting small interfering RNA pathway, which requires THO/TREX function.

  16. Isozyme differences in barley mutants

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-01-01

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

  17. Nonselective enrichment for yeast adenine mutants by flow cytometry

    Science.gov (United States)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  18. Identifying problems and generating recommendations for enhancing complex systems: applying the abstraction hierarchy framework as an analytical tool.

    Science.gov (United States)

    Xu, Wei

    2007-12-01

    This study adopts J. Rasmussen's (1985) abstraction hierarchy (AH) framework as an analytical tool to identify problems and pinpoint opportunities to enhance complex systems. The process of identifying problems and generating recommendations for complex systems using conventional methods is usually conducted based on incompletely defined work requirements. As the complexity of systems rises, the sheer mass of data generated from these methods becomes unwieldy to manage in a coherent, systematic form for analysis. There is little known work on adopting a broader perspective to fill these gaps. AH was used to analyze an aircraft-automation system in order to further identify breakdowns in pilot-automation interactions. Four steps follow: developing an AH model for the system, mapping the data generated by various methods onto the AH, identifying problems based on the mapped data, and presenting recommendations. The breakdowns lay primarily with automation operations that were more goal directed. Identified root causes include incomplete knowledge content and ineffective knowledge structure in pilots' mental models, lack of effective higher-order functional domain information displayed in the interface, and lack of sufficient automation procedures for pilots to effectively cope with unfamiliar situations. The AH is a valuable analytical tool to systematically identify problems and suggest opportunities for enhancing complex systems. It helps further examine the automation awareness problems and identify improvement areas from a work domain perspective. Applications include the identification of problems and generation of recommendations for complex systems as well as specific recommendations regarding pilot training, flight deck interfaces, and automation procedures.

  19. Prion propagation in cells expressing PrP glycosylation mutants.

    Science.gov (United States)

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

    2011-04-01

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

  20. Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants.

    Science.gov (United States)

    Wang, Peng; Grimm, Bernhard

    2016-11-01

    State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems. © 2016 American Society of Plant Biologists. All Rights Reserved.

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

    Science.gov (United States)

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

    2011-04-06

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

  2. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

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

  3. Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

    Science.gov (United States)

    McLenigan, Mary; Peat, Thomas S.; Frank, Ekaterina G.; McDonald, John P.; Gonzalez, Martín; Levine, Arthur S.; Hendrickson, Wayne A.; Woodgate, Roger

    1998-01-01

    Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′. PMID:9721309

  4. The GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Morgane eBatzenschlager

    2013-11-01

    Full Text Available During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs are nucleated from γ-Tubulin Complexes (γ-TuCs located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope are currently unknown. The γ-TuC Protein 3 (GCP3-Interacting Protein 1 (GIP1 is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects.In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of γ-TuC core proteins and MT fibre robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the nuclear envelope.These results provide the first evidence for the involvement of a γ-TuC component in both nuclear shaping and nuclear envelope organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

  5. A conserved endoplasmic reticulum membrane protein complex (EMC facilitates phospholipid transfer from the ER to mitochondria.

    Directory of Open Access Journals (Sweden)

    Sujoy Lahiri

    2014-10-01

    Full Text Available Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC has decreased phosphatidylserine (PS transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE. Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-01-07

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

  7. A systematic method for identifying vital areas at complex nuclear facilities.

    Energy Technology Data Exchange (ETDEWEB)

    Beck, David Franklin; Hockert, John

    2005-05-01

    Identifying the areas to be protected is an important part of the development of measures for physical protection against sabotage at complex nuclear facilities. In June 1999, the International Atomic Energy Agency published INFCIRC/225/Rev.4, 'The Physical Protection of Nuclear Material and Nuclear Facilities.' This guidance recommends that 'Safety specialists, in close cooperation with physical protection specialists, should evaluate the consequences of malevolent acts, considered in the context of the State's design basis threat, to identify nuclear material, or the minimum complement of equipment, systems or devices to be protected against sabotage.' This report presents a structured, transparent approach for identifying the areas that contain this minimum complement of equipment, systems, and devices to be protected against sabotage that is applicable to complex nuclear facilities. The method builds upon safety analyses to develop sabotage fault trees that reflect sabotage scenarios that could cause unacceptable radiological consequences. The sabotage actions represented in the fault trees are linked to the areas from which they can be accomplished. The fault tree is then transformed (by negation) into its dual, the protection location tree, which reflects the sabotage actions that must be prevented in order to prevent unacceptable radiological consequences. The minimum path sets of this fault tree dual yield, through the area linkage, sets of areas, each of which contains nuclear material, or a minimum complement of equipment, systems or devices that, if protected, will prevent sabotage. This method also provides guidance for the selection of the minimum path set that permits optimization of the trade-offs among physical protection effectiveness, safety impact, cost and operational impact.

  8. Dwarf mutant of rice variety Seratus Malam

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  9. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

    Science.gov (United States)

    Rahme, Laurence G.; Tan, Man-Wah; Le, Long; Wong, Sandy M.; Tompkins, Ronald G.; Calderwood, Stephen B.; Ausubel, Frederick M.

    1997-01-01

    We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis. PMID:9371831

  10. Characterization of a Thermo-Inducible Chlorophyll-Deficient Mutant in Barley

    Directory of Open Access Journals (Sweden)

    Rong Wang

    2017-11-01

    Full Text Available Leaf color is an important trait for not only controlling crop yield but also monitoring plant status under temperature stress. In this study, a thermo-inducible chlorophyll-deficient mutant, named V-V-Y, was identified from a gamma-radiated population of the barley variety Vlamingh. The leaves of the mutant were green under normal growing temperature but turned yellowish under high temperature in the glasshouse experiment. The ratio of chlorophyll a and chlorophyll b in the mutant declined much faster in the first 7–9 days under heat treatment. The leaves of V-V-Y turned yellowish but took longer to senesce under heat stress in the field experiment. Genetic analysis indicated that a single nuclear gene controlled the mutant trait. The mutant gene (vvy was mapped to the long arm of chromosome 4H between SNP markers 1_0269 and 1_1531 with a genetic distance of 2.2 cM and a physical interval of 9.85 Mb. A QTL for grain yield was mapped to the same interval and explained 10.4% of the yield variation with a LOD score of 4. This QTL is coincident with the vvy gene interval that is responsible for the thermo-inducible chlorophyll-deficient trait. Fine mapping, based on the barley reference genome sequence, further narrowed the vvy gene to a physical interval of 0.428 Mb with 11 annotated genes. This is the first report of fine mapping a thermo-inducible chlorophyll-deficient gene in barley.

  11. Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes.

    Science.gov (United States)

    Lee, Kenneth K; Sardiu, Mihaela E; Swanson, Selene K; Gilmore, Joshua M; Torok, Michael; Grant, Patrick A; Florens, Laurence; Workman, Jerry L; Washburn, Michael P

    2011-07-05

    Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt-Ada-Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.

  12. Mig6 Puts the Brakes on Mutant EGFR-Driven Lung Cancer | Center for Cancer Research

    Science.gov (United States)

    Lung cancer is the most common cause of cancer-related death worldwide. These cancers are often induced by mutations in the epidermal growth factor receptor (EGFR), resulting in constitutive activation of the protein’s tyrosine kinase domain. Lung cancers expressing these EGFR mutants are initially sensitive to tyrosine kinase inhibitors (TKIs), such as erlotinib, but often become resistant by developing compensatory mutations in EGFR or other growth-promoting pathways. To better understand how mutant EGFR initiates and maintains tumor growth in the hopes of identifying novel targets for drug development, Udayan Guha, M.D., Ph.D., of CCR’s Thoracic and Gastrointestinal Oncology Branch, and his colleagues examined the landscape of proteins phosphorylated in EGFR wild type and mutant cells. One protein hyper-phosphorylated in mutant EGFR cells was Mig6, a putative tumor suppressor.

  13. Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly.

    Directory of Open Access Journals (Sweden)

    Hanqian Mao

    2016-09-01

    Full Text Available The exon junction complex (EJC is an RNA binding complex comprised of the core components Magoh, Rbm8a, and Eif4a3. Human mutations in EJC components cause neurodevelopmental pathologies. Further, mice heterozygous for either Magoh or Rbm8a exhibit aberrant neurogenesis and microcephaly. Yet despite the requirement of these genes for neurodevelopment, the pathogenic mechanisms linking EJC dysfunction to microcephaly remain poorly understood. Here we employ mouse genetics, transcriptomic and proteomic analyses to demonstrate that haploinsufficiency for each of the 3 core EJC components causes microcephaly via converging regulation of p53 signaling. Using a new conditional allele, we first show that Eif4a3 haploinsufficiency phenocopies aberrant neurogenesis and microcephaly of Magoh and Rbm8a mutant mice. Transcriptomic and proteomic analyses of embryonic brains at the onset of neurogenesis identifies common pathways altered in each of the 3 EJC mutants, including ribosome, proteasome, and p53 signaling components. We further demonstrate all 3 mutants exhibit defective splicing of RNA regulatory proteins, implying an EJC dependent RNA regulatory network that fine-tunes gene expression. Finally, we show that genetic ablation of one downstream pathway, p53, significantly rescues microcephaly of all 3 EJC mutants. This implicates p53 activation as a major node of neurodevelopmental pathogenesis following EJC impairment. Altogether our study reveals new mechanisms to help explain how EJC mutations influence neurogenesis and underlie neurodevelopmental disease.

  14. Proteostasis and ageing: insights from long-lived mutant mice.

    Science.gov (United States)

    Sands, William A; Page, Melissa M; Selman, Colin

    2017-10-15

    The global increase in life expectancy is creating significant medical, social and economic challenges to current and future generations. Consequently, there is a need to identify the fundamental mechanisms underlying the ageing process. This knowledge should help develop realistic interventions capable of combatting age-related disease, and thus improving late-life health and vitality. While several mechanisms have been proposed as conserved lifespan determinants, the loss of proteostasis - where proteostasis is defined here as the maintenance of the proteome - appears highly relevant to both ageing and disease. Several studies have shown that multiple proteostatic mechanisms, including the endoplasmic reticulum (ER)-induced unfolded protein response (UPR), the ubiquitin-proteasome system (UPS) and autophagy, appear indispensable for longevity in many long-lived invertebrate mutants. Similarly, interspecific comparisons suggest that proteostasis may be an important lifespan determinant in vertebrates. Over the last 20 years a number of long-lived mouse mutants have been described, many of which carry single-gene mutations within the growth-hormone, insulin/IGF-1 or mTOR signalling pathways. However, we still do not know how these mutations act mechanistically to increase lifespan and healthspan, and accordingly whether mechanistic commonality occurs between different mutants. Recent evidence supports the premise that the successful maintenance of the proteome during ageing may be linked to the increased lifespan and healthspan of long-lived mouse mutants. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

  15. A bio-inspired methodology of identifying influential nodes in complex networks.

    Directory of Open Access Journals (Sweden)

    Cai Gao

    Full Text Available How to identify influential nodes is a key issue in complex networks. The degree centrality is simple, but is incapable to reflect the global characteristics of networks. Betweenness centrality and closeness centrality do not consider the location of nodes in the networks, and semi-local centrality, leaderRank and pageRank approaches can be only applied in unweighted networks. In this paper, a bio-inspired centrality measure model is proposed, which combines the Physarum centrality with the K-shell index obtained by K-shell decomposition analysis, to identify influential nodes in weighted networks. Then, we use the Susceptible-Infected (SI model to evaluate the performance. Examples and applications are given to demonstrate the adaptivity and efficiency of the proposed method. In addition, the results are compared with existing methods.

  16. Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-yl carbamate.

    Science.gov (United States)

    Li, M; Zhang, H Y; Liang, B

    2013-01-01

    Twelve-low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe-the molecular lesion likely to be responsible-for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at-amino acid 168, having Phe (TTC) instead of Ser (TCC)', was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5'-flanking regions in 12 LR mutants of T. harzianum.

  17. Evaluation of Mungbean Mutant Lines to Drought Stress and Their Genetic Relationships Using SSR Markers

    Directory of Open Access Journals (Sweden)

    Yuliasti

    2015-12-01

    Full Text Available Development of mungbean cultivarstolerant to drought stress through mutation breeding approach would enable us to anticipate the crop yield-reducing effects of climate changes. The objective of this research was to evaluate the yield performance of mungbean mutant lines that showed tolerance to drought stress, and to analyze their genetic diversity and relationship among mutant lines using SSR markers. The study was conducted during the dry season of 2012 in the Muneng experimental farm, Probolinggo, East Java. The experiment was laid out in a randomized block design with four replications. Five mutant lines and two parental lines as control were tested for evaluation of yield and drought tolerance under twoenvironments of two irrigation systems as treatment. The two environmental conditions consisted of optimal irrigation (at least three times: at planting, flowering and during pod filling and suboptimal irrigation (two times at planting and flowering. To evaluate genetic variation among selected mutant lines and their discrimination from parental lines in molecular level, a cluster analysis was performed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA in the NTSYS software. The results showed that three mutant lines, including PsJ30, PsJ31, PsJ32 produced the highest grain yields of 1.17, 1.01, and 1.04 ton/ha, respectively, compared to the other mutant lines and the parents Gelatik (0.85 ton/ha and Perkutut (0.87 ton/ha as control check. Of those mutant lines, PSJ31 was the most tolerant to drought with sensitivity index value of 0.47. The PSJ31 has now been officially released as a new variety ( 2013, named as Muri which was identified to have high yield and tolerant to drought. Based on 23 SSR markers used for clustering analysis of those 3 selected mutant lines,9SSR markers (MBSS R033; satt137; MBSSR008; MBSSR203; MBSSR013; MBSSR021; MBSSR016; MBSSR136; and DMBSSR013 were successfully identified the three mungbean mutant

  18. High linolenic acid mutant in soybean induced by X-ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Takagi, Y. [Saga Univ. (Japan); Hossain, A. B.M.M.; Yanagita, T.; Kusaba, S.

    1989-12-15

    Soybean [Glycine max (L.) Merr. cv. Bay] seeds were irradiated with X-rays (25kR) and the M{sub 2} progeny was screened for changes in the fatty acid composition of seed oil. X-ray irradiation remarkably increased the variability of the fatty acid composition in the oil of the Bay cultivar. A mutant in which linolenic acid accounted for 18.4 per cent of the total oil cornpared with 9.4 per cent in the Bay cultivar was identified among 2006 M{sub 2} plants. The M{sub 3} generation of the mutant also showed a linolenic acid content approximately two times higher than that of the original variety.

  19. High linolenic acid mutant in soybean induced by X-ray irradiation

    International Nuclear Information System (INIS)

    Takagi, Y.; Hossain, A.B.M.M.; Yanagita, T.; Kusaba, S.

    1989-01-01

    Soybean [Glycine max (L.) Merr. cv. Bay] seeds were irradiated with X-rays (25kR) and the M 2 progeny was screened for changes in the fatty acid composition of seed oil. X-ray irradiation remarkably increased the variability of the fatty acid composition in the oil of the Bay cultivar. A mutant in which linolenic acid accounted for 18.4 per cent of the total oil cornpared with 9.4 per cent in the Bay cultivar was identified among 2006 M 2 plants. The M 3 generation of the mutant also showed a linolenic acid content approximately two times higher than that of the original variety

  20. Genetic Analysis and Molecular Mapping of a Novel Chlorophyll-Deficit Mutant Gene in Rice

    Directory of Open Access Journals (Sweden)

    Xiao-qun HUANG

    2008-03-01

    Full Text Available A rice etiolation mutant 824ys featured with chlorophyll deficiency was identified from a normal green rice variety 824B. It showed whole green-yellow plant from the seedling stage, reduced number of tillers and longer growth duration. The contents of chlorophyll, chlorophyll a, chlorophyll b and net photosynthetic rate in leaves of the mutant obviously decreased, as well as the number of spikelets per panicle, seed setting rate and 1000-grain weight compared with its wild-type parent. Genetic analyses on F1 and F2 generations of 824ys crossed with three normal green varieties showed that the chlorophyll-deficit mutant character was controlled by a pair of recessive nuclear gene. Genetic mapping of the mutant gene was conducted by using microsatellite markers and F2 mapping population of 495R/824ys, and the mutant gene of 824ys was mapped on the short arm of rice chromosome 3. The genetic distances from the target gene to the markers RM218, RM282 and RM6959 were 25.6 cM, 5.2 cM and 21.8 cM, respectively. It was considered to be a new chlorophyll-deficit mutant gene and tentatively named as chl11(t.

  1. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    International Nuclear Information System (INIS)

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-01-01

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials

  2. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Bernhard [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany); Lecker, Laura S. M.; Zoltner, Martin [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Jaenicke, Elmar [Johannes Gutenberg-Universität, Jakob Welder Weg 26, 55128 Mainz (Germany); Schnell, Robert [Karolinska Institutet, 17 177 Stockholm (Sweden); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 4EH, Scotland (United Kingdom); Brenk, Ruth, E-mail: w.n.hunter@dundee.ac.uk [Johannes Gutenberg-Universität, Staudinger Weg 5, 55128 Mainz (Germany)

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  3. A Natural Mutation Involving both Pathogenicity and Perithecium Formation in the Fusarium graminearum Species Complex

    Directory of Open Access Journals (Sweden)

    Haruhisha Suga

    2016-12-01

    Full Text Available Members of the Fusarium graminearum species complex (Fg complex or FGSC are the primary pathogens causing Fusarium head blight in wheat and barley worldwide. A natural pathogenicity mutant (strain 0225022 was found in a sample of the Fg complex collected in Japan. The mutant strain did not induce symptoms in wheat spikes beyond the point of inoculation, and did not form perithecia. No segregation of phenotypic deficiencies occurred in the progenies of a cross between the mutant and a fully pathogenic wild-type strain, which suggested that a single genetic locus controlled both traits. The locus was mapped to chromosome 2 by using sequence-tagged markers; and a deletion of ∼3 kb was detected in the mapped region of the mutant strain. The wild-type strain contains the FGSG_02810 gene, encoding a putative glycosylphosphatidylinositol anchor protein, in this region. The contribution of FGSG_02810 to pathogenicity and perithecium formation was confirmed by complementation in the mutant strain using gene transfer, and by gene disruption in the wild-type strain.

  4. Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.

    Science.gov (United States)

    Singh, Upinder; Brewer, Jeremy L; Boothroyd, John C

    2002-05-01

    Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

  5. Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

    Science.gov (United States)

    Fornero, Christy; Suo, Bangxia; Zahde, Mais; Juveland, Katelyn; Kirik, Viktor

    2017-11-01

    Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

  6. The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies.

    Science.gov (United States)

    Li, Guotian; Jain, Rashmi; Chern, Mawsheng; Pham, Nikki T; Martin, Joel A; Wei, Tong; Schackwitz, Wendy S; Lipzen, Anna M; Duong, Phat Q; Jones, Kyle C; Jiang, Liangrong; Ruan, Deling; Bauer, Diane; Peng, Yi; Barry, Kerrie W; Schmutz, Jeremy; Ronald, Pamela C

    2017-06-01

    The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake ( Oryza sativa ssp japonica ), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. © 2017 American Society of Plant Biologists. All rights reserved.

  7. Water-deficit tolerant classification in mutant lines of indica rice

    Directory of Open Access Journals (Sweden)

    Suriyan Cha-um

    2012-04-01

    Full Text Available Water shortage is a major abiotic stress for crop production worldwide, limiting the productivity of crop species, especially in dry-land agricultural areas. This investigation aimed to classify the water-deficit tolerance in mutant rice (Oryza sativa L. spp. indica genotypes during the reproductive stage. Proline content in the flag leaf of mutant lines increased when plants were subjected to water deficit. Relative water content (RWC in the flag leaf of different mutant lines dropped in relation to water deficit stress. A decrease RWC was positively related to chlorophyll a degradation. Chlorophyll a , chlorophyll b , total chlorophyll , total carotenoids , maximum quantum yield of PSII , stomatal conductance , transpiration rate and water use efficiency in mutant lines grown under water deficit conditions declined in comparison to the well-watered, leading to a reduction in net-photosynthetic rate. In addition, when exposed to water deficit, panicle traits, including panicle length and fertile grains were dropped. The biochemical and physiological data were subjected to classify the water deficit tolerance. NSG19 (positive control and DD14 were identified as water deficit tolerant, and AA11, AA12, AA16, BB13, BB16, CC12, CC15, EE12, FF15, FF17, G11 and IR20 (negative control as water deficit sensitive, using Ward's method.

  8. Characterization and protective property of Brucella abortus cydC and looP mutants.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Hahn, Tae-Wook

    2014-11-01

    Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Genomic data reveal Toxoplasma gondii differentiation mutants are also impaired with respect to switching into a novel extracellular tachyzoite state.

    Directory of Open Access Journals (Sweden)

    Pamela J Lescault

    2010-12-01

    Full Text Available Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites, replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. We present the analysis of seven novel T. gondii insertional mutants that do not undergo normal differentiation to bradyzoites. Microarray quantification of the variation in genome-wide RNA levels for each parasite line and times after induction allowed us to describe states in the normal differentiation process, to analyze mutant lines in the context of these states, and to identify genes that may have roles in initiating the transition from tachyzoite to bradyzoite. Gene expression patterns in wild-type parasites undergoing differentiation suggest a novel extracellular state within the tachyzoite stage. All mutant lines exhibit aberrant regulation of bradyzoite gene expression and notably some of the mutant lines appear to exhibit high proportions of the intracellular tachyzoite state regardless of whether they are intracellular or extracellular. In addition to the genes identified by the insertional mutagenesis screen, mixture model analysis allowed us to identify a small number of genes, in mutants, for which expression patterns could not be accounted for using the three parasite states--genes that may play a mechanistic role in switching from the tachyzoite to bradyzoite stage.

  10. Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants1[OPEN

    Science.gov (United States)

    Wang, Peng

    2016-01-01

    State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems. PMID:27663408

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

    Directory of Open Access Journals (Sweden)

    Houchard Kimberly R

    2005-02-01

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

  12. High pressure near infrared study of the mutated light-harvesting complex LH2

    Directory of Open Access Journals (Sweden)

    Braun P.

    2005-01-01

    Full Text Available The pressure sensitivities of the near infrared spectra of the light-harvesting (LH2 complex and a mutant complex with a simplified BChl-B850 binding pocket were compared. In the mutant an abrupt change in the spectral properties occurred at 250 MPa, which was not observed with the native sample. Increased disorder due to collapse of the chromophore pocket is suggested.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-03-01

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

  14. Semi-dwarf mutants for rice improvement

    International Nuclear Information System (INIS)

    Othman, Ramli; Osman, Mohammad; Ibrahim, Rusli

    1990-01-01

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

  15. Phenotypic characterization and inheritance of two foliar mutants in pea (Pisum Sativum L.): 'Reduced leaf size' and 'Orange leaf'

    International Nuclear Information System (INIS)

    Naidenova, N.; Vassilevska-Ivanova, R.; Tcekova, Z.

    2003-01-01

    Two foliar pea (Pisum sativum L.) mutants characterized by reduced leaf size (2/978) and orange leaf (2/1409 M) were established. Both mutants were described morphologically and their productivity potential , pollen viability and inheritance of the mutant traits were evaluated. The mutant 2/978 was identified after irradiation of dry seeds from cv Borek with 15 Gy fast neutrons and was related to the leaf mutation 'rogue'. Reciprocal crosses between mutant 2/978 and cv Borel were executed, and F 1 and F 2 generations were analyzed. The altered leaf trait was presented in all F 1 plants suggesting a dominant character. F 2 segregation data indicated that the trait was controlled by a single dominant gene. The mutant 2/1409M originated from the mutant 2/978 after irradiation with 50 Gy γ-rays. The main mutant's phenotypic characteristic was the orange-yellow coloration of leaves and plants. After of series of crosses it was established that induced chlorophyll mutation is monogenic, recessive and both mutant traits are independently inherited. Two mutants could be used as appropriate plant material for genetic and biological investigations

  16. Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

    Science.gov (United States)

    Kumar, Jitendra; Gunapati, Samatha; Alok, Anshu; Lalit, Adarsh; Gadre, Rekha; Sharma, Naresh C; Roy, Joy K; Singh, Sudhir P

    2015-05-01

    Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

  17. Evaluation of tall rice mutant

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  18. Cox4i2, Ifit2, and Prdm11 Mutant Mice

    DEFF Research Database (Denmark)

    Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as imm...

  19. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

    Science.gov (United States)

    Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

    2015-12-01

    Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. © 2015. Published by The Company of

  20. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    Directory of Open Access Journals (Sweden)

    Andrew Parker

    2015-12-01

    Full Text Available Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182 in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  1. Evaluation of Some Chemical Characteristics of barley Mutants induced by Gamma Irradiation

    International Nuclear Information System (INIS)

    Abdeldaiem, M.H.; Ali, H.G.M.

    2011-01-01

    This study aims to evaluate the antioxidant activity of acetonic extract from some barley mutations (P1, P2 and P3 varieties) induced by gamma irradiation as compared with local barley variety (Hordeum vulgare L.) as control. Barley samples were obtained from Plant Breeding Unit, Plant Research Department, Nuclear Research Centre, Atomic Energy Authority, Egypt. The measurements of the antioxidant activity using a radical scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ?-carotene bleaching assay were assessed in the barley acetonic extract. Furthermore, amino acids composition of barley mutant samples was determined. The results indicated that the acetonic extract of barley varieties under investigation possess marked antioxidant and anti radical capacities. The data showed that the acetonic extract of barley mutant P1 possessed the higher antioxidant activity as compared with the antioxidant activities of acetonic extract from control and other barley mutant samples. Meanwhile, the flour of barley mutations under investigation contained trace elements of iron, copper and manganese. GC and mass analyses were used to identify the active compound of extract of control and mutant barley samples. The results illustrated that the main components of the control sample of barely extract was pentane, 3 methyl (47.73%) while gamma irradiation caused noticeable change in the relative percentage of some components of acetonic extract from barley mutant samples. Moreover, the results presented that changes were disappeared, and some compounds of the acetonic extract from mutant barley samples were appeared. Furthermore, the results exhibited that barley flour supplemented with wheat flour at 30% level produced acceptable cookies. Accordingly, the phenolic constituents of barley acetonic extract induced by gamma irradiation, especially samples of P1 mutant, may have a future role as ingredients in the development of functional foods.

  2. A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation.

    Science.gov (United States)

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

    2012-08-01

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

  3. TransDetect Identifies a New Regulatory Module Controlling Phosphate Accumulation.

    Science.gov (United States)

    Pal, Sikander; Kisko, Mushtak; Dubos, Christian; Lacombe, Benoit; Berthomieu, Pierre; Krouk, Gabriel; Rouached, Hatem

    2017-10-01

    Identifying transcription factor (TFs) cooperation controlling target gene expression is still an arduous challenge. The accuracy of current methods at genome scale significantly drops with the increase in number of genes, which limits their applicability to more complex genomes, like animals and plants. Here, we developed an algorithm, TransDetect, able to predict TF combinations controlling the expression level of a given gene. TransDetect was used to identify novel TF modules regulating the expression of Arabidopsis ( Arabidopsis thaliana ) phosphate transporter PHO1;H3 comprising MYB15, MYB84, bHLH35, and ICE1. These TFs were confirmed to interact between themselves and with the PHO1;H3 promoter. Phenotypic and genetic analyses of TF mutants enable the organization of these four TFs and PHO1;H3 in a new gene regulatory network controlling phosphate accumulation in zinc-dependent manner. This demonstrates the potential of TransDetect to extract directionality in nondynamic transcriptomes and to provide a blueprint to identify gene regulatory network involved in a given biological process. © 2017 American Society of Plant Biologists. All Rights Reserved.

  4. Prion Propagation in Cells Expressing PrP Glycosylation Mutants

    Science.gov (United States)

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

    2011-01-01

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

  5. Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

    Science.gov (United States)

    Nakamura, Masayoshi; Yagi, Noriyoshi; Kato, Takehide; Fujita, Satoshi; Kawashima, Noriyuki; Ehrhardt, David W; Hashimoto, Takashi

    2012-07-01

    Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  6. Studies on reduced height mutants in rice

    International Nuclear Information System (INIS)

    Narahari, P.; Bhagwat, S.G.

    1984-01-01

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

  7. Identification of Vitis vinifera L. grape berry skin color mutants and polyphenolic profile.

    Science.gov (United States)

    Ferreira, Vanessa; Fernandes, Fátima; Pinto-Carnide, Olinda; Valentão, Patrícia; Falco, Virgílio; Martín, Juan Pedro; Ortiz, Jesús María; Arroyo-García, Rosa; Andrade, Paula B; Castro, Isaura

    2016-03-01

    A germplasm set of twenty-five grapevine accessions, forming eleven groups of possible berry skin color mutants, were genotyped with twelve microsatellite loci, being eleven of them identified as true color mutants. The polyphenolic profiling of the confirmed mutant cultivars revealed a total of twenty-four polyphenols, comprising non-colored compounds (phenolic acids, flavan-3-ols, flavonols and a stilbene) and anthocyanins. Results showed differences in the contribution of malvidin-3-O-glucoside to the characteristic Pinot Noir anthocyanins profile. Regarding the two Pique-Poul colored variants, the lighter variant was richer than the darker one in all classes of compounds, excepting anthocyanins. In Moscatel Galego Roxo the F3'H pathway seems to be more active than F3'5'H, resulting in higher amounts of cyanidin, precursor of the cyanidin derivatives. As far as we are aware, this is the first time that a relationship between the content of polyphenolic compounds is established in groups of grape berry skin color mutant cultivars. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

    Science.gov (United States)

    Nagata, Mariko; Ishino, Sonoko; Yamagami, Takeshi; Ogino, Hiromi; Simons, Jan-Robert; Kanai, Tamotsu; Atomi, Haruyuki; Ishino, Yoshizumi

    2017-10-13

    The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose

    DEFF Research Database (Denmark)

    Hong, Kuk-Ki; Nielsen, Jens

    2013-01-01

    the molecular mechanisms. In this study, adaptively evolved yeast mutants with improved galactose utilization ability showed impaired glucose utilization. The molecular genetic basis of this trade-off was investigated using a systems biology approach. Transcriptional and metabolic changes resulting from...... the improvement of galactose utilization were found maintained during growth on glucose. Moreover, glucose repression related genes showed conserved expression patterns during growth on both sugars. Mutations in the RAS2 gene that were identified as beneficial for galactose utilization in evolved mutants...

  10. Dwarf mutant of Papaver somniferum with high morphine content

    International Nuclear Information System (INIS)

    Chauhan, S.P.; Patra, N.K.; Srivastava, H.K.

    1987-01-01

    Opium poppy, Papaver somniferum L. is an important medicinal plant known for its morphine, codeine, and thebaine alkaloids. This Institute had earlier released two latex opium yielding poppy varieties, Shyama and Shweta, which are now cultivated by the farmers under the supervision of the Narcotic Department of the Government of India. However, both these varieties became susceptible to downy mildew (Peronospora arborescens). Lodging due to heavy capsule weight is another problem affecting latex yield. With these problems in mind, we undertook mutation breeding on the above mentioned two varieties employing gamma rays (5 kR, 15 kR, 20 kR) and EMS (0.2%, 0.4%, 0.6%) and combined mutagens (5 kR + 0.2% EMS, 5 kR + 0.4% EMS and 5 kR + 0.6% EMS). M 1 from the treated seeds (405 plants) was raised in winter 1984-85. M 2 generation of 13,500 plants (i.e. 270 M 1 progenies x 50 plants) was raised in winter 1985/86. A dwarf mutant with high morphine content was identified in M 2 from the variety Shweta treated with 5 kR + 0.4% EMS. The mutant differs by its dwarf stature, compact leaf arrangements, multilocular capsules, increased capsule number, and small capsule size. The mutant is under testing for its superior morphine production. It may be used as dwarf gene source in hybridization for improving lodging resistance. This mutant is a novel type, which was not available in our germplasm collection

  11. A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

    International Nuclear Information System (INIS)

    Liu Qingmei; Yuan Hang; Wang Jun; Gong Guohong; Zhou Wei; Fan Yonghong; Wang Li; Yao Jianming; Yu Zengliang

    2006-01-01

    In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope (SEM). The optimum condition of ions implantation, 20 keV of energy and 2.6x10 15 N + /cm 2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum (ESI/MS) and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain

  12. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    DEFF Research Database (Denmark)

    Costa, Sara; Marek, Magdalena; Axelsen, Kristian Buhl

    2016-01-01

    and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues......) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications...

  13. Identifying deterministic signals in simulated gravitational wave data: algorithmic complexity and the surrogate data method

    International Nuclear Information System (INIS)

    Zhao Yi; Small, Michael; Coward, David; Howell, Eric; Zhao Chunnong; Ju Li; Blair, David

    2006-01-01

    We describe the application of complexity estimation and the surrogate data method to identify deterministic dynamics in simulated gravitational wave (GW) data contaminated with white and coloured noises. The surrogate method uses algorithmic complexity as a discriminating statistic to decide if noisy data contain a statistically significant level of deterministic dynamics (the GW signal). The results illustrate that the complexity method is sensitive to a small amplitude simulated GW background (SNR down to 0.08 for white noise and 0.05 for coloured noise) and is also more robust than commonly used linear methods (autocorrelation or Fourier analysis)

  14. Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

    Science.gov (United States)

    Ren, Zhao-Yu; Xu, Xiao-Ming; Wang, Shui-Cai; Xin, Yue-Yong; He, Jun-Fang; Hou, Xun

    2003-10-01

    A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wild-type rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wild-type. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.

  15. Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

    Science.gov (United States)

    2012-01-01

    Background Hepatitis B virus (HBV), because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients. Results Among the core, preS, and basic core promoter (BCP) deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023). In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007). Particularly, preS2 deletions were associated with the usage of nucleos(t)ide analog therapy (Fisher exact test, P = 0.023). Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that preS2 deletions alone

  16. Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines

    Directory of Open Access Journals (Sweden)

    Robert Silas Allen

    2013-09-01

    Full Text Available Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognise in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP residing in HASTY, a previously characterised gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.

  17. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants.

    Science.gov (United States)

    Chen, Longzheng; Li, Wei; Katin-Grazzini, Lorenzo; Ding, Jing; Gu, Xianbin; Li, Yanjun; Gu, Tingting; Wang, Ren; Lin, Xinchun; Deng, Ziniu; McAvoy, Richard J; Gmitter, Frederick G; Deng, Zhanao; Zhao, Yunde; Li, Yi

    2018-01-01

    Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium -mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase ( PDS ) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

  18. Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

    Science.gov (United States)

    Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

    2017-06-01

    Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

    Science.gov (United States)

    Schär, Primo; Herrmann, Gernot; Daly, Graham; Lindahl, Tomas

    1997-01-01

    Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115

  20. Molecular analysis of waxy mutants in rice

    International Nuclear Information System (INIS)

    Yatou, O.; Amano, E.

    1990-01-01

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

  1. Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

    Energy Technology Data Exchange (ETDEWEB)

    Chu, K.-W. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chan, Shirley K.W. [Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chow, King L. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) and Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)]. E-mail: bokchow@ust.hk

    2005-09-30

    Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

  2. Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

    International Nuclear Information System (INIS)

    Chu, K.-W.; Chan, Shirley K.W.; Chow, King L.

    2005-01-01

    Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring

  3. Phenotypic, fermentation characterization, and resistance mechanism analysis of bacteriophage-resistant mutants of Lactobacillus delbrueckii ssp. bulgaricus isolated from traditional Chinese dairy products.

    Science.gov (United States)

    Deng, Kaibo; Fang, Wei; Zheng, Baodong; Miao, Song; Huo, Guicheng

    2018-03-01

    Bacteriophage infection is a large factor in dairy industrial production failure on the basis of pure inoculation fermentation, and developing good commercial starter cultures from wild dairy products and improving the environmental vigor of starter cultures by enhancing their phage resistance are still the most effective solutions. Here we used a spontaneous isolation method to obtain bacteriophage-resistant mutants of Lactobacillus delbrueckii ssp. bulgaricus strains that are used in traditional Chinese fermented dairy products. We analyzed their phenotypes, fermentation characteristics, and resistance mechanisms. The results showed that bacteriophage-insensitive mutants (BIM) BIM8 and BIM12 had high bacteriophage resistance while exhibiting fermentation and coagulation attributes that were as satisfying as those of their respective parent strains KLDS1.1016 and KLDS1.1028. According to the attachment receptor detection, mutants BIM8 and BIM12 exhibited reduced absorption to bacteriophage phiLdb compared with their respective bacteriophage-sensitive parent strains because of changes to the polysaccharides or teichoic acids connected to their peptidoglycan layer. Additionally, genes, including HSDR, HSDM, and HSDS, encoding 3 subunits of a type I restriction-modification system were identified in their respective parent strains. We also discovered that HSDR and HSDM were highly conserved but that HSDS was variable because it is responsible for the DNA specificity of the complex. The late lysis that occurred only in strain KLDS1.1016 and not in strain KLDS1.1028 suggests that the former and its mutant BIM8 also may have an activatable restriction-modification mechanism. We conclude that the L. bulgaricus BIM8 and BIM12 mutants have great potential in the dairy industry as starter cultures, and their phage-resistance mechanism was effective mainly due to the adsorption interference and restriction-modification system. Copyright © 2018 American Dairy Science

  4. Random T-DNA mutagenesis identifies a Cu-Zn-superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum

    Science.gov (United States)

    Agrobacterium-mediated transformation (AMT) was used to identify potential virulence factors in Sclerotinia sclerotiorum. Screening AMT transformants identified two mutants showing significantly reduced virulence. The mutants showed similar growth rate, colony morphology, and sclerotial and oxalate ...

  5. X-rays sensitive mammalian cell mutant

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi

    1982-01-01

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

  6. Cln6 mutants associated with neuronal ceroid lipofuscinosis are degraded in a proteasome-dependent manner.

    Science.gov (United States)

    Oresic, Kristina; Mueller, Britta; Tortorella, Domenico

    2009-06-01

    NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.

  7. Radiation induced mutants in cassava (Manihot esculenta Crantz)

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  8. Erythrocyte pyruvate kinase deficiency in the Ohio Amish: origin and characterization of the mutant enzyme.

    OpenAIRE

    Muir, W A; Beutler, E; Wasson, C

    1984-01-01

    We have identified eight individuals in an Amish population in Geauga County, Ohio, who have a congenital hemolytic anemia and red cell pyruvate kinase (PK) deficiency. The mutant enzyme is a low Km phosphoenolpyruvate (PEP) variant associated with a slower (77.5% of normal) electrophoretic mobility in starch gel. Because of the high consanguinity in this population, we assume the affected individuals are homozygous for the mutant gene. Genealogical records allow us to trace all eight cases b...

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

    Directory of Open Access Journals (Sweden)

    Sara eAmorim-Vaz

    2015-05-01

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

  10. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

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

    Science.gov (United States)

    Butler, Jeffrey A.; Mishur, Robert J.; Bhaskaran, Shylesh; Rea, Shane L.

    2012-01-01

    SUMMARY Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent upon concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer’s disease. When the expression of DLD in wild type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened but, at higher doses, it was significantly lengthened, suggesting DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. PMID:23173729

  12. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    Science.gov (United States)

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  13. Officially released mutant varieties in China

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  14. Ten-a affects the fusion of central complex primordia in Drosophila.

    Directory of Open Access Journals (Sweden)

    Xuebo Cheng

    Full Text Available The central complex of Drosophila melanogaster plays important functions in various behaviors, such as visual and olfactory memory, visual orientation, sleep, and movement control. However little is known about the genes regulating the development of the central complex. Here we report that a mutant gene affecting central complex morphology, cbd (central brain defect, was mapped to ten-a, a type II trans-membrane protein coding gene. Down-regulation of ten-a in pan-neural cells contributed to abnormal morphology of central complex. Over-expression of ten-a by C767-Gal4 was able to partially restore the abnormal central complex morphology in the cbd mutant. Tracking the development of FB primordia revealed that C767-Gal4 labeled interhemispheric junction that separated fan-shaped body precursors at larval stage withdrew to allow the fusion of the precursors. While the C767-Gal4 labeled structure did not withdraw properly and detached from FB primordia, the two fan-shaped body precursors failed to fuse in the cbd mutant. We propose that the withdrawal of C767-Gal4 labeled structure is related to the formation of the fan-shaped body. Our result revealed the function of ten-a in central brain development, and possible cellular mechanism underlying Drosophila fan-shaped body formation.

  15. Temperature sensitive riboflavin mutants of Penicillium vermiculatum Dangeard

    International Nuclear Information System (INIS)

    Mitra, J.; Chaudhari, K.L.

    1974-01-01

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

  16. Synapsis-defective mutants reveal a correlation between chromosome conformation and the mode of double-strand break repair during Caenorhabditis elegans meiosis.

    Science.gov (United States)

    Smolikov, Sarit; Eizinger, Andreas; Hurlburt, Allison; Rogers, Eric; Villeneuve, Anne M; Colaiácovo, Mónica P

    2007-08-01

    SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.

  17. High yielding mutants of blackgram variety 'PH-25'

    International Nuclear Information System (INIS)

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

    2001-01-01

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

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

    International Nuclear Information System (INIS)

    Vaitkuniene, V.

    1995-01-01

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

  19. Drastic anthocyanin increase in response to PAP1 overexpression in fls1 knockout mutant confers enhanced osmotic stress tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Lee, Won Je; Jeong, Chan Young; Kwon, Jaeyoung; Van Kien, Vu; Lee, Dongho; Hong, Suk-Whan; Lee, Hojoung

    2016-11-01

    KEY MESSAGE : pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress. Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

  20. A large-scale mutant panel in wheat developed using heavy-ion beam mutagenesis and its application to genetic research

    Energy Technology Data Exchange (ETDEWEB)

    Murai, Koji, E-mail: murai@fpu.ac.jp [Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195 (Japan); Nishiura, Aiko [Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195 (Japan); Kazama, Yusuke [RIKEN, Innovation Center, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Abe, Tomoko [RIKEN, Innovation Center, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); RIKEN, Nishina Center, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2013-11-01

    Mutation analysis is a powerful tool for studying gene function. Heavy-ion beam mutagenesis is a comparatively new approach to inducing mutations in plants and is particularly efficient because of its high linear energy transfer (LET). High LET radiation induces a higher rate of DNA double-strand breaks than other mutagenic methods. Over the last 12 years, we have constructed a large-scale mutant panel in diploid einkorn wheat (Triticum monococcum) using heavy-ion beam mutagenesis. Einkorn wheat seeds were exposed to a heavy-ion beam and then sown in the field. Selfed seeds from each spike of M{sub 1} plants were used to generate M{sub 2} lines. Every year, we obtained approximately 1000 M{sub 2} lines and eventually developed a mutant panel with 10,000 M{sub 2} lines in total. This mutant panel is being systematically screened for mutations affecting reproductive growth, and especially for flowering-time mutants. To date, we have identified several flowering-time mutants of great interest: non-flowering mutants (mvp: maintained vegetative phase), late-flowering mutants, and early-flowering mutants. These novel mutations will be of value for investigations of the genetic mechanism of flowering in wheat.

  1. Selection and genetic relationship of salt tolerant rice mutants by in vitro mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jae Young; Kim, Dong Sub; Lee, Kyung Jun; Kim, Jin Baek; Kim, Sang Hoon; Kang, Si Yong [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Lee, Myung Chul [National Academy of Agriculture and Science, Suwon (Korea, Republic of); Yun, Song Joong [Chonbuk National University, Jeonju (Korea, Republic of)

    2010-12-15

    Plants have evolved physiological, biochemical and metabolic mechanisms to increase their survival under the adverse conditions. This present study has been performed to select salt tolerant rice mutant lines through in vivo and in vitro mutagenesis with gamma-rays. For the selection of the salt-tolerant rice mutants, we conducted three times of selection procedure using 1,500 gamma ray mutant lines resulted from an embryo culture of the original rice cv. Dongan (wild-type, WT): first, selection in the a nutrient solution with 171 mM NaCI: second, selection under in vitro condition with 171 mM NaCI: and third, selection in a reclaimed saline land. Based on a growth comparison of the entries, out of the mutant lines, two putative 2 salt tolerant (ST) rice mutant lines, ST-87 and ST-301, were finally selected. The survival rate of the WT, ST-87 and ST-301 were 36.6%, 60% and 66.3% after 7 days in 171 mM NaCI treatment, respectively. The WT and two salt tolerant mutant lines were used to analyze their genetic variations. A total of 21 EcoRI and Msel primer combinations were used to analyze the genetic relationship of among the two salt tolerant lines and the WT using the ABI3130 capillary electrophoresis system. In the AFLP analysis, a total of 1469 bands were produced by the 21 primer combinations, and 700 (47.6%) of them were identified as having polymorphism. The genetic similarity coefficients were ranged from 0.52 between the ST-87 and WT to 0.24 between the ST-301 and the WT. These rice mutant lines will be used as a control plot for physiological analysis and genetic research on salt tolerance.

  2. Towards the Identification of New Genes Involved in ABA-Dependent Abiotic Stresses Using Arabidopsis Suppressor Mutants of abh1 Hypersensitivity to ABA during Seed Germination

    Directory of Open Access Journals (Sweden)

    Iwona Szarejko

    2013-06-01

    Full Text Available Abscisic acid plays a pivotal role in the abiotic stress response in plants. Although great progress has been achieved explaining the complexity of the stress and ABA signaling cascade, there are still many questions to answer. Mutants are a valuable tool in the identification of new genes or new alleles of already known genes and in elucidating their role in signaling pathways. We applied a suppressor mutation approach in order to find new components of ABA and abiotic stress signaling in Arabidopsis. Using the abh1 (ABA hypersensitive 1 insertional mutant as a parental line for EMS mutagenesis, we selected several mutants with suppressed hypersensitivity to ABA during seed germination. Here, we present the response to ABA and a wide range of abiotic stresses during the seed germination and young seedling development of two suppressor mutants—soa2 (suppressor of abh1 hypersensitivity to ABA 2 and soa3 (suppressor of abh1 hypersensitivity to ABA 3. Generally, both mutants displayed a suppression of the hypersensitivity of abh1 to ABA, NaCl and mannitol during germination. Both mutants showed a higher level of tolerance than Columbia-0 (Col-0—the parental line of abh1 in high concentrations of glucose. Additionally, soa2 exhibited better root growth than Col-0 in the presence of high ABA concentrations. soa2 and soa3 were drought tolerant and both had about 50% fewer stomata per mm2 than the wild-type but the same number as their parental line—abh1. Taking into account that suppressor mutants had the same genetic background as their parental line—abh1, it was necessary to backcross abh1 with Landsberg erecta four times for the map-based cloning approach. Mapping populations, derived from the cross of abh1 in the Landsberg erecta background with each suppressor mutant, were created. Map based cloning in order to identify the suppressor genes is in progress.

  3. Induction of drought tolerant mutants of rice

    International Nuclear Information System (INIS)

    El-Hissewy, A.A.; Abd Allah, A.

    2001-01-01

    The ultimate goal of crop breeding is to develop varieties with a high yield potential and desirable agronomic characteristics. In Egypt, the most important qualities sought by breeders have been high yield potential, resistance to major diseases and insects, and improved grain and eating quality. However, breeding efforts should concentrate on varieties with the potential to minimize yield losses under unfavorable conditions such as drought, and to maximize yields when conditions are favorable. Rice (Oryza sativa L.) in Egypt is completely irrigated and a significant portion of the rice cultivated area is subject to water deficit resulting from an inadequate or insufficient irrigation supply. Drought tolerance is a complex trait in that it results from the interaction of histological and physiological characters of plant with environmental factors, both above-ground and under-ground. Accordingly, root characters are closely related to drought tolerance. Little attention has been paid in Egyptian breeding programs to root characters and their relation to shoot characters. Furthermore, induced mutations are considered as one of the most important methods to induce useful mutants, especially with improved root characters, to overcome the drought problem. The present investigation aimed to study the effect of different doses of gamma rays on several characters of three Egyptian rice varieties, i.e. 'Giza 171', 'Giza 175' and 'Giza 176' and to induce one or more mutants possessing drought tolerance

  4. Productive mutants of niger

    International Nuclear Information System (INIS)

    Misra, R.C.

    2001-01-01

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

  5. Isolation and characterization of MMS-sensitive mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Prakash, L.; Prakash, S.

    1977-01-01

    We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to uv or x rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to uv and x rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups

  6. Efficient Identification of Causal Mutations through Sequencing of Bulked F2 from Two Allelic Bloomless Mutants of Sorghum bicolor

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    Yinping Jiao

    2018-01-01

    Full Text Available Sorghum (Sorghum bicolor Moench, L. plant accumulates copious layers of epi-cuticular wax (EW on its aerial surfaces, to a greater extent than most other crops. EW provides a vapor barrier that reduces water loss, and is therefore considered to be a major determinant of sorghum's drought tolerance. However, little is known about the genes responsible for wax accumulation in sorghum. We isolated two allelic mutants, bloomless40-1 (bm40-1 and bm40-2, from a mutant library constructed from ethyl methane sulfonate (EMS treated seeds of an inbred, BTx623. Both mutants were nearly devoid of the EW layer. Each bm mutant was crossed to the un-mutated BTx623 to generated F2 populations that segregated for the bm phenotype. Genomic DNA from 20 bm F2 plants from each population was bulked for whole genome sequencing. A single gene, Sobic.001G228100, encoding a GDSL-like lipase/acylhydrolase, had unique homozygous mutations in each bulked F2 population. Mutant bm40-1 harbored a missense mutation in the gene, whereas bm40-2 had a splice donor site mutation. Our findings thus provide strong evidence that mutation in this GDSL-like lipase gene causes the bm phenotype, and further demonstrate that this approach of sequencing two independent allelic mutant populations is an efficient method for identifying causal mutations. Combined with allelic mutants, MutMap provides powerful method to identify all causal genes for the large collection of bm mutants in sorghum, which will provide insight into how sorghum plants accumulate such abundant EW on their aerial surface. This knowledge may facilitate the development of tools for engineering drought-tolerant crops with reduced water loss.

  7. Isoenzymes performance of some rice varieties and their mutants

    International Nuclear Information System (INIS)

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

    1992-01-01

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

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  9. Isolation of transformation-defective, replication-nondefective early region 1B mutants of adenovirus 12

    International Nuclear Information System (INIS)

    Fukui, Y.; Saito, I.; Shiroki, K.; Shimojo, H.

    1984-01-01

    The authors isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation

  10. Greater than the sum of its parts: single-nucleus sequencing identifies convergent evolution of independent EGFR mutants in GBM.

    Science.gov (United States)

    Gini, Beatrice; Mischel, Paul S

    2014-08-01

    Single-cell sequencing approaches are needed to characterize the genomic diversity of complex tumors, shedding light on their evolutionary paths and potentially suggesting more effective therapies. In this issue of Cancer Discovery, Francis and colleagues develop a novel integrative approach to identify distinct tumor subpopulations based on joint detection of clonal and subclonal events from bulk tumor and single-nucleus whole-genome sequencing, allowing them to infer a subclonal architecture. Surprisingly, the authors identify convergent evolution of multiple, mutually exclusive, independent EGFR gain-of-function variants in a single tumor. This study demonstrates the value of integrative single-cell genomics and highlights the biologic primacy of EGFR as an actionable target in glioblastoma. ©2014 American Association for Cancer Research.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

    Brokaw, C J; Luck, D J

    1985-01-01

    Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

  13. Analysis on expression of gene for flower shape in Dendrobium sonia mutants using differential display technique

    International Nuclear Information System (INIS)

    Affrida Abu Hassan; Ahmad Syazni Kamarudin; Nurul Nadia Aminuddin; Mohd Nazir Basiran

    2004-01-01

    In vitro mutagenesis on Dendrobium Sonia in MINT has produced mutants with wide range of flower form and colour variations. Among the mutants are plants with different flower size and shape. These changes could be caused by alterations to the expression level of the genes responsible for the characteristics. In this studies, Differential Display technique was used to identify and analyse altered gene expression at the mRNA level. Total RNA of the control and mutants were reversed transcribed using three anchored oligo-d T primers. Subsequently, these cDNAs were Pcr amplified in combination with 16 arbitrary primers. The amplified products were electrophoresed side by side on agarose gel. Differentially expressed bands are isolated for further analysis. (Author)

  14. Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects.

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    Chris Jopling

    2007-12-01

    Full Text Available Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.

  15. [Genetic control of the sensitivity of Aspergillus nidulans to mutagenic factors. VII. Inheritance of cross-sensitivity to different mutagenic factors by uvs-mutants].

    Science.gov (United States)

    Evseeva, G V; Kameneva, S V

    1977-01-01

    To study the inheritance of the sensitivity to UV, X-rays, methylmethanesulphonate (MMS), nitrosoguanidine (NG) and nitrous acid (NA) in five uvs mutants of Aspergillus nidulans, having multiple sensitivity to these factors, the sensitivity of recombinants obtained from crossing uvs mutants with uvs+ strain, resistant to all the factors analysed, and uvs leads to uvs+ revertants is investigated. Four uvs mutants (15, 17, 19 and 26) are found to have a nomogenic control of sensitivity to different mutagens. In one mutant (uvs11) the sensitivity to five factors is controlled by two non-linked mutations, one of them determining the sensitivity to UV, NG, NA, and the other--to X-rays and MMC. Phenotypic manifestations of uvs mutations is modified by cell genotype, both chromosomal and cytoplasmic factors being responsible for the modification. Phenotypic modification of uvs mutation results in the change to some (but not to all) mutagenic factors. It suggests, that not the product of uvs gene, but some other components of the reparation complex are modified. Otherwise, reparation of different DNA damages can be carried out by a single enzyme acting in different reparation complexes.

  16. [Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

    Science.gov (United States)

    Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

    2008-10-01

    To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

  17. The endosomal sorting complex required for transport (ESCRT) is required for the sensitivity of yeast cells to nickel ions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Luo, Chong; Cao, Chunlei; Jiang, Linghuo

    2016-05-01

    Nickel is one of the toxic environment metal pollutants and is linked to various human diseases. In this study, through a functional genomics approach we have identified 16 nickel-sensitive and 22 nickel-tolerant diploid deletion mutants of budding yeast genes, many of which are novel players in the regulation of nickel homeostasis. The 16 nickel-sensitive mutants are of genes mainly involved in the protein folding, modification and destination and the cellular transport processes, while the 22 nickel-tolerant mutants are of genes encoding components of ESCRT complexes as well as protein factors involved in both the cell wall integrity maintenance and the vacuolar protein sorting process. In consistence with their phenotypes, most of these nickel-sensitive mutants show reduced intracellular nickel contents, while the majority of these nickel-tolerant mutants show elevated intracellular nickel contents, as compared to the wild type in response to nickel stress. Our data provides a basis for our understanding the regulation of nickel homeostasis and molecular mechanisms of nickel-induced human pathogenesis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Multilocation trial of potential selected mutant lines of groundnut (arachis hypogaea) at 3 location in Peninsular Malaysia

    International Nuclear Information System (INIS)

    Abdul Rahim Harun; Rusli Ibrahim; Khairuddin Abdul Rahim; Shuhaimi Shamsuddin

    2002-01-01

    Two fixed mutant lines of groundnut derived from cultivar Matjan were selected for their yield potential at M 1 0 generation. Multilocation trial of these mutants (MJ40/42 and MJ20/165-5) was carried out to evaluate genotype stability at different climate and soil types in Peninsular Malaysia. The mutant lines were planted and compared with their parent (Matjan) and control variety (MKT1). The identified locations were in Taiping (Perak), Machang (Kelantan), and Air Hitam (Johor). The soils at the locations were of the Serdang, Bungor and Rengam series, respectively. The trial was carried out simultaneously in the same year at each location. Mutant MJ20/165-5 showed stable performance at all location compared to other genotypes tested. Its yield was higher than the parent in Kelantan and Johor trial and showed similar performance in Perak. This mutant also showed better yield performance than the control varieties in the Kelantan trial. Meanwhile, mutant line MJ40/42 gave better yield in Kelantan and Johor but did not perform well in Perak as compared to its parent and control varieties. (Author)

  19. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    Directory of Open Access Journals (Sweden)

    Eliton da Silva Vasconcelos

    2013-12-01

    Full Text Available Clavulanic acid (CA is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064. The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  20. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

    Science.gov (United States)

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-12-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  1. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    Science.gov (United States)

    Epsztejn-Litman, Silvina; Cohen-Hadad, Yaara; Aharoni, Shira; Altarescu, Gheona; Renbaum, Paul; Levy-Lahad, Ephrat; Schonberger, Oshrat; Eldar-Geva, Talia; Zeligson, Sharon; Eiges, Rachel

    2015-01-01

    We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

  2. Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis.

    Directory of Open Access Journals (Sweden)

    Silvina Epsztejn-Litman

    Full Text Available We report on the derivation of a diploid 46(XX human embryonic stem cell (HESC line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19, monitoring the expression of two parentally imprinted genes (SNRPN and H19 and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.

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

    Science.gov (United States)

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

    1993-03-01

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

  4. Identifying influential spreaders in complex networks through local effective spreading paths

    Science.gov (United States)

    Wang, Xiaojie; Zhang, Xue; Yi, Dongyun; Zhao, Chengli

    2017-05-01

    How to effectively identify a set of influential spreaders in complex networks is of great theoretical and practical value, which can help to inhibit the rapid spread of epidemics, promote the sales of products by word-of-mouth advertising, and so on. A naive strategy is to select the top ranked nodes as identified by some centrality indices, and other strategies are mainly based on greedy methods and heuristic methods. However, most of those approaches did not concern the connections between nodes. Usually, the distances between the selected spreaders are very close, leading to a serious overlapping of their influence. As a consequence, the global influence of the spreaders in networks will be greatly reduced, which largely restricts the performance of those methods. In this paper, a simple and efficient method is proposed to identify a set of discrete yet influential spreaders. By analyzing the spreading paths in the network, we present the concept of effective spreading paths and measure the influence of nodes via expectation calculation. The numerical analysis in undirected and directed networks all show that our proposed method outperforms many other centrality-based and heuristic benchmarks, especially in large-scale networks. Besides, experimental results on different spreading models and parameters demonstrates the stability and wide applicability of our method.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  6. Fitness ranking of individual mutants drives patterns of epistatic interactions in HIV-1.

    Directory of Open Access Journals (Sweden)

    Javier P Martínez

    Full Text Available Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1 resistance to azidothymidine (AZT requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41-215 cluster of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions.

  7. Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams.

    Science.gov (United States)

    Takano, Nao; Takahashi, Yuko; Yamamoto, Mitsuru; Teranishi, Mika; Yamaguchi, Hiroko; Sakamoto, Ayako N; Hase, Yoshihiro; Fujisawa, Hiroko; Wu, Jianzhong; Matsumoto, Takashi; Toki, Seiichi; Hidema, Jun

    2013-07-01

    UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UV Tolerant Rice 319), was isolated from a mutagenized population derived from 2500 M1 seeds (of the UVB-resistant cultivar 'Sasanishiki') that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined.

  8. Isolation of a novel UVB-tolerant rice mutant obtained by exposure to carbon-ion beams

    International Nuclear Information System (INIS)

    Takano, Nao; Takahashi, Yuko; Yamamoto, Mitsuru; Teranishi, Mika; Yamaguchi, Hiroko; Sakamoto, Ayako N.; Hase, Yoshihiro; Fujisawa, Hiroko; Wu, Jianzhong; Matsumoto, Takashi; Toki, Seiichi; Hidema, Jun

    2013-01-01

    UVB radiation suppresses photosynthesis and protein biosynthesis in plants, which in turn decreases growth and productivity. Here, an ultraviolet-B (UVB)-tolerant rice mutant, utr319 (UVTolerantRice319), was isolated from a mutagenized population derived from 2500 M 1 seeds (of the UVB-resistant cultivar ‘Sasanishiki’) that were exposed to carbon ions. The utr319 mutant was more tolerant to UVB than the wild type. Neither the levels of UVB-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) pyrimidine-pyrimidone photodimers [(6-4) photoproducts], nor the repair of CPDs or (6-4) photoproducts, was altered in the utr319 mutant. Thus, the utr319 mutant may be impaired in the production of a previously unidentified factor that confers UVB tolerance. To identify the mutated region in the utr319 mutant, microarray-based comparative genomic hybridization analysis was performed. Two adjacent genes on chromosome 7, Os07g0264900 and Os07g0265100, were predicted to represent the mutant allele. Sequence analysis of the chromosome region in utr319 revealed a deletion of 45 419 bp. RNAi analysis indicated that Os07g0265100 is most likely the mutated gene. Database analysis indicated that the Os07g0265100 gene, UTR319, encodes a putative protein with unknown characteristics or function. In addition, the homologs of UTR319 are conserved only among land plants. Therefore, utr319 is a novel UVB-tolerant rice mutant and UTR319 may be crucial for the determination of UVB sensitivity in rice, although the function of UTR319 has not yet been determined

  9. HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors.

    Science.gov (United States)

    Zhang, Weibin; Miley, Natasha; Zastrow, Michael S; MacQueen, Amy J; Sato, Aya; Nabeshima, Kentaro; Martinez-Perez, Enrique; Mlynarczyk-Evans, Susanna; Carlton, Peter M; Villeneuve, Anne M

    2012-01-01

    During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification.

  10. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

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    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  11. Bacterio-opsin mutants of Halobacterium halobium

    Science.gov (United States)

    Betlach, Mary; Pfeifer, Felicitas; Friedman, James; Boyer, Herbert W.

    1983-01-01

    The bacterio-opsin (bop) gene of Halobacterium halobium R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of H. halobium. A 5.1-kilobase Pst I fragment containing the bop gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the bop gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the bop gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5′ end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the bop mRNA—i.e., it has a 5′ leader sequence of three ribonucleotides—these observations indicate that the bop mRNA might be processed from a large mRNA transcript. Images PMID:16593291

  12. A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) mutant with enhanced pathogen-induced cell death and altered receptor processing.

    Science.gov (United States)

    Petutschnig, Elena K; Stolze, Marnie; Lipka, Ulrike; Kopischke, Michaela; Horlacher, Juliane; Valerius, Oliver; Rozhon, Wilfried; Gust, Andrea A; Kemmerling, Birgit; Poppenberger, Brigitte; Braus, Gerhard H; Nürnberger, Thorsten; Lipka, Volker

    2014-12-01

    Plants detect pathogens by sensing microbe-associated molecular patterns (MAMPs) through pattern recognition receptors. Pattern recognition receptor complexes also have roles in cell death control, but the underlying mechanisms are poorly understood. Here, we report isolation of cerk1-4, a novel mutant allele of the Arabidopsis chitin receptor CERK1 with enhanced defense responses. We identified cerk1-4 in a forward genetic screen with barley powdery mildew and consequently characterized it by pathogen assays, mutant crosses and analysis of defense pathways. CERK1 and CERK1-4 proteins were analyzed biochemically. The cerk1-4 mutation causes an amino acid exchange in the CERK1 ectodomain. Mutant plants maintain chitin signaling capacity but exhibit hyper-inducible salicylic acid concentrations and deregulated cell death upon pathogen challenge. In contrast to chitin signaling, the cerk1-4 phenotype does not require kinase activity and is conferred by the N-terminal part of the receptor. CERK1 undergoes ectodomain shedding, a well-known process in animal cell surface proteins. Wild-type plants contain the full-length CERK1 receptor protein as well as a soluble form of the CERK1 ectodomain, whereas cerk1-4 plants lack the N-terminal shedding product. Our work suggests that CERK1 may have a chitin-independent role in cell death control and is the first report of ectodomain shedding in plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  13. A novel mouse Fgfr2 mutant, hobbyhorse (hob, exhibits complete XY gonadal sex reversal.

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    Pam Siggers

    Full Text Available The secreted molecule fibroblast growth factor 9 (FGF9 plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob, which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6 genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected.

  14. High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

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    Ehud Goldin

    Full Text Available Gaucher disease (GD, the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase. Previously, wildtype GCase was used for high throughput screening (HTS of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

  15. The Caenorhabditis elegans Elongator complex regulates neuronal alpha-tubulin acetylation.

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    Jachen A Solinger

    2010-01-01

    Full Text Available Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3 and in a scaffold subunit (Elp1 have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

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

    Science.gov (United States)

    Butler, Jeffrey A; Mishur, Robert J; Bhaskaran, Shylesh; Rea, Shane L

    2013-02-01

    Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild-type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

  17. Elevated expression of ribosomal protein genes L37, RPP-1, and S2 in the presence of mutant p53.

    Science.gov (United States)

    Loging, W T; Reisman, D

    1999-11-01

    The wild-type p53 protein is a DNA-binding transcription factor that activates genes such as p21, MDM2, GADD45, and Bax that are required for the regulation of cell cycle progression or apoptosis in response to DNA damage. Mutant forms of p53, which are transforming oncogenes and are expressed at high levels in tumor cells, generally have a reduced binding affinity for the consensus DNA sequence. Interestingly, some p53 mutants that are no longer effective at binding to the consensus DNA sequence and transactivating promoters containing this target site have acquired the ability to transform cells in culture, in part through their ability to transactivate promoters of a number of genes that are not targets of the wild-type protein. Certain p53 mutants are therefore considered to be gain-of-function mutants and appear to be promoting proliferation or transforming cells through their ability to alter the expression of novel sets of genes. Our goal is to identify genes that have altered expression in the presence of a specific mutant p53 (Arg to Trp mutation at codon 248) protein. Through examining differential gene expression in cells devoid of p53 expression and in cells that express high levels of mutant p53 protein, we have identified three ribosomal protein genes that have elevated expression in response to mutant p53. Consistent with these findings, the overexpression of a number of ribosomal protein genes in human tumors and evidence for their contribution to oncogenic transformation have been reported previously, although the mechanism leading to this overexpression has remained elusive. We show results that indicate that expression of these specific ribosomal protein genes is increased in the presence of the R248W p53 mutant, which provides a mechanism for their overexpression in human tumors.

  18. The application of shortened upper leaf mutant in barley breeding

    International Nuclear Information System (INIS)

    Jin Hua

    2004-01-01

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

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

    International Nuclear Information System (INIS)

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

    1975-01-01

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

  20. Navigating the Complexities at an LGTTQQI-Identified Charter School: An Ethnography of C/Overt Narratives

    Science.gov (United States)

    Goodrich, Kristopher M.; Luke, Melissa

    2016-01-01

    The authors describe ethnographic research exploring the experiences of school stakeholders at a lesbian, gay, bisexual, transgender, queer, questioning, and intersex (LGBTQQI)-identified charter school. Participants evidenced use of an overt and covert narrative that appeared to reflect how they navigated the complexities at the…

  1. Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages.

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    Matthew Dale Woolard

    2013-02-01

    Full Text Available Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS induces macrophages to synthesize prostaglandin E2 (PGE2. Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain Francisella tularensis subspecies novicida U112 (U112 two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI. Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used

  2. Altered metabolism of growth hormone receptor mutant mice: a combined NMR metabonomics and microarray study.

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    Horst Joachim Schirra

    Full Text Available BACKGROUND: Growth hormone is an important regulator of post-natal growth and metabolism. We have investigated the metabolic consequences of altered growth hormone signalling in mutant mice that have truncations at position 569 and 391 of the intracellular domain of the growth hormone receptor, and thus exhibit either low (around 30% maximum or no growth hormone-dependent STAT5 signalling respectively. These mutations result in altered liver metabolism, obesity and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: The analysis of metabolic changes was performed using microarray analysis of liver tissue and NMR metabonomics of urine and liver tissue. Data were analyzed using multivariate statistics and Gene Ontology tools. The metabolic profiles characteristic for each of the two mutant groups and wild-type mice were identified with NMR metabonomics. We found decreased urinary levels of taurine, citrate and 2-oxoglutarate, and increased levels of trimethylamine, creatine and creatinine when compared to wild-type mice. These results indicate significant changes in lipid and choline metabolism, and were coupled with increased fat deposition, leading to obesity. The microarray analysis identified changes in expression of metabolic enzymes correlating with alterations in metabolite concentration both in urine and liver. Similarity of mutant 569 to the wild-type was seen in young mice, but the pattern of metabolites shifted to that of the 391 mutant as the 569 mice became obese after six months age. CONCLUSIONS/SIGNIFICANCE: The metabonomic observations were consistent with the parallel analysis of gene expression and pathway mapping using microarray data, identifying metabolites and gene transcripts involved in hepatic metabolism, especially for taurine, choline and creatinine metabolism. The systems biology approach applied in this study provides a coherent picture of metabolic changes resulting from impaired STAT5 signalling by the growth hormone

  3. Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora.

    Science.gov (United States)

    Frey, Stefan; Lahmann, Yasmine; Hartmann, Thomas; Seiler, Stephan; Pöggeler, Stefanie

    2015-08-01

    The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility. © 2015 John Wiley & Sons Ltd.

  4. Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

    Science.gov (United States)

    Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming

    2012-05-01

    Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  5. Dictyostelium discoideum: mutants in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides

    International Nuclear Information System (INIS)

    Freeze, H.; Willies, L.; Hamilton, S.

    1986-01-01

    The lysosomal enzymes of Dictyostelium discoideum share highly immunogenic oligosaccharides which contain multiple Man-6-SO 4 residues. Two mutant strains which lack the shared antigenic determinant were analyzed in an attempt to identify the primary defect in each. [ 3 H]Man labelled N-linked oligosaccharides of secreted glycoproteins were released by Endo/PNGaseF digestion and analyzed. Both of the mutant strains produced smaller, less sulfated oligosaccharides compared to the wild-type, yet both still contained considerable amounts of Man-6-SO 4 . The size of the precursor lipid-linked oligosaccharide from the wild-type is consistent with a Glc 3 Man 9 GlcNAc 2 structure, while those from both of the mutants have an oligosaccharide the size of Man 5 GlcNAc 2 . The authors conclude that both of the mutants are defective in the biosynthesis of the precursor oligosaccharide. Both oligosaccharides from the mutants contain a tri-mannosyl core and are not glucosylated. Two of the five Man residues are released by a 1,2 specific α mannosidase. Based on the size and mannosidase digestions the authors conclude that 4/5 of the Man residues on the α1,6 branch of the β-linked Man residues are missing. Thus, these residues must be required to define the shared antigenic determinant

  6. Semi-dwarf mutants in triticale and wheat breeding

    International Nuclear Information System (INIS)

    Driscoll, C.J.

    1984-01-01

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

  7. A study of eukaryotic response mechanisms to atmospheric pressure cold plasma by using Saccharomyces cerevisiae single gene mutants

    International Nuclear Information System (INIS)

    Feng Hongqing; Wang Ruixue; Sun Peng; Wu Haiyan; Liu Qi; Li Fangting; Fang Jing; Zhang Jue; Zhu Weidong

    2010-01-01

    The mechanisms of eukaryotic cell response to cold plasma are studied. A series of single gene mutants of eukaryotic model organism Saccharomyces cerevisiae are used to compare their sensitivity to plasma treatment with the wild type. We examined 12 mutants in the oxidative stress pathway and the cell cycle pathway, in which 8 are found to be hypersensitive to plasma processing. The mutated genes' roles in the two pathways are analyzed to understand the biological response mechanisms of plasma treatment. The results demonstrate that genes from both pathways are needed for the eukaryotic cells to survive the complex plasma treatment.

  8. Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

    Science.gov (United States)

    Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B.; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C.; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

    2012-01-01

    Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies. PMID:22542184

  9. Genetic analysis of the induced mutants of rice resistant to bacterial leaf blight

    International Nuclear Information System (INIS)

    Nakai, H.

    1990-01-01

    Full text: Seeds of the rice cultivar 'Harebare', which is susceptible to bacterial leaf blight (BLB), were treated with thermal neutrons, gamma-rays, ethyleneimine and ethylmethane-sulfonate. In the M2, plants with better resistance to BLB were identified through inoculation at the seedling and the flag leaf stages with an isolate (T7174) of the Japanese differential race I. Several mutant lines resistant to BLB were selected through tests of the M 3 or M 4 lines derived from selected resistant M 2 plants. The frequency of resistant mutants was significantly higher after the thermal neutron treatment than after treatments with other mutagens. Two mutants, which originated from the neutron treatment, showing a highly quantitative resistance to multiple BLB races were analysed for gene(s) for resistance. The resistance of one of them (M41) to the Japanese races I, II, III, IV, and V was found to be conditioned by a single recessive gene. Three other recessive genes for resistance are known, but their reaction to differential races is different. Therefore, this gene was thought to be new and was tentatively designated as xa-nm(t). The resistance of another mutant (M57) was found to be polygenically inherited. (author)

  10. Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

    Science.gov (United States)

    Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

    2016-01-01

    The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

  11. A rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by direct analysis in real-time mass spectrometry

    Directory of Open Access Journals (Sweden)

    Jang Young

    2011-06-01

    Full Text Available Abstract Background Efficient high throughput screening systems of useful mutants are prerequisite for study of plant functional genomics and lots of application fields. Advance in such screening tools, thanks to the development of analytic instruments. Direct analysis in real-time (DART-mass spectrometry (MS by ionization of complex materials at atmospheric pressure is a rapid, simple, high-resolution analytical technique. Here we describe a rapid, simple method for the genetic discrimination of intact Arabidopsis thaliana mutant seeds using metabolic profiling by DART-MS. Results To determine whether this DART-MS combined by multivariate analysis can perform genetic discrimination based on global metabolic profiling, intact Arabidopsis thaliana mutant seeds were subjected to DART-MS without any sample preparation. Partial least squares-discriminant analysis (PLS-DA of DART-MS spectral data from intact seeds classified 14 different lines of seeds into two distinct groups: Columbia (Col-0 and Landsberg erecta (Ler ecotype backgrounds. A hierarchical dendrogram based on partial least squares-discriminant analysis (PLS-DA subdivided the Col-0 ecotype into two groups: mutant lines harboring defects in the phenylpropanoid biosynthetic pathway and mutants without these defects. These results indicated that metabolic profiling with DART-MS could discriminate intact Arabidopsis seeds at least ecotype level and metabolic pathway level within same ecotype. Conclusion The described DART-MS combined by multivariate analysis allows for rapid screening and metabolic characterization of lots of Arabidopsis mutant seeds without complex metabolic preparation steps. Moreover, potential novel metabolic markers can be detected and used to clarify the genetic relationship between Arabidopsis cultivars. Furthermore this technique can be applied to predict the novel gene function of metabolic mutants regardless of morphological phenotypes.

  12. A probabilistic framework for identifying biosignatures using Pathway Complexity

    Science.gov (United States)

    Marshall, Stuart M.; Murray, Alastair R. G.; Cronin, Leroy

    2017-11-01

    One thing that discriminates living things from inanimate matter is their ability to generate similarly complex or non-random structures in a large abundance. From DNA sequences to folded protein structures, living cells, microbial communities and multicellular structures, the material configurations in biology can easily be distinguished from non-living material assemblies. Many complex artefacts, from ordinary bioproducts to human tools, though they are not living things, are ultimately produced by biological processes-whether those processes occur at the scale of cells or societies, they are the consequences of living systems. While these objects are not living, they cannot randomly form, as they are the product of a biological organism and hence are either technological or cultural biosignatures. A generalized approach that aims to evaluate complex objects as possible biosignatures could be useful to explore the cosmos for new life forms. However, it is not obvious how it might be possible to create such a self-contained approach. This would require us to prove rigorously that a given artefact is too complex to have formed by chance. In this paper, we present a new type of complexity measure, which we call `Pathway Complexity', that allows us not only to threshold the abiotic-biotic divide, but also to demonstrate a probabilistic approach based on object abundance and complexity which can be used to unambiguously assign complex objects as biosignatures. We hope that this approach will not only open up the search for biosignatures beyond the Earth, but also allow us to explore the Earth for new types of biology, and to determine when a complex chemical system discovered in the laboratory could be considered alive. This article is part of the themed issue 'Reconceptualizing the origins of life'.

  13. Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

    Science.gov (United States)

    Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

    2011-09-01

    The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

  14. Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes.

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    Xiaoyun Wu

    Full Text Available The retinoblastoma (Rb tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.

  15. Cytogenetic characteristics of soft wheat mutants under x-irradiation

    International Nuclear Information System (INIS)

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

    1981-01-01

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

  16. Genetics of Ustilago violacea. I. Carotenoid mutants and carotenogenesis

    International Nuclear Information System (INIS)

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

    1975-01-01

    Wild-type strains of Ustilago violacea produce pink colonies on laboratory medium and yield white, orange, pumpkin, and yellow colonies after uv mutagenesis. The wild-type strains contain neurosporene and lycopene; one orange mutant, γ-carotene; and one yellow mutant, β-carotene. One white mutant had no detectable carotenoids. Diploid colonies heterozygous for wild type and orange, pumpkin, yellow, or white are phenotypically wild type. Diploid colonies heterozygous for yellow and orange are also phenotypically wild type. Diploid colonies heterozygous for white and orange; white and yellow; and white, yellow, and orange are phenotypically light orange, light yellow, and orange-yellow, respectively. The white mutants give a circular complementation map; the color mutants fit a linear complementation map. We propose a multienzyme of four identical dehydrogenases and one or two identical cyclases for carotenogenesis in this species. The white and color mutants represent structural mutations altering the conformation of the dehydrogenase or cyclase, respectively. Furthermore, cyclases may or may not aggregate in association with the dehydrogenase aggregate to form the multienzyme aggregate responsible for the color mutants

  17. Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.

    Science.gov (United States)

    Bonawitz, Nicholas D; Kim, Jeong Im; Tobimatsu, Yuki; Ciesielski, Peter N; Anderson, Nickolas A; Ximenes, Eduardo; Maeda, Junko; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

    2014-05-15

    Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

  18. Analysis of the albino-locus region of the mouse. II. Mosaic mutants

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    Among 119 mutations involving the c locus that were recovered in the course of mouse specific-locus experiments with external radiations, 16 were found in mosaic, or fractional, mutants. The number of additional c-locus fractionals that could have occurred in these experiments and, for a variety of reasons, might not have been clearly identified, probably does not exceed the present number. There was no evidence for radiation induction of the fractionals, and even those occurring in the irradiated groups may thus be assumed to be of spontaneous origin. Since only two mutations in the control groups were found in whole-body mutants, it appears that the bulk of spontaneous c-locus mutations are fractionals. None of the mutations recovered in fractional mutants was homozygous lethal; 25% were viable intermediate alleles, and the remainder were albino-like mutants, all viable except for one subvital and one not tested. Genetic tests of the fractionals indicated no major selection against the new mutations, either gametically or in the progeny. For the group of fractionals as a whole, about one-half of the germinal tissue carried the mutation, indicating that the fractionals came from an overall blastomere population that was one-half mutant. Such a population could result from mutation in one strand of the gamete DNA, in a daughter chromosome derived from pronuclear DNA synthesis of the zygote, or in one of the first two blastomeres prior to replication. Since the mouse embryo does not stem from all of the cleavage products of the zygote, the frequency of fractionals observeed underestimates the frequency of mutational events that result in two types of blastomeres

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

    Science.gov (United States)

    Barrington, Chloe L.; Katsanis, Nicholas

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sarah C Goetz

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

  1. Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.

    Directory of Open Access Journals (Sweden)

    Adrian W R Serohijos

    2008-02-01

    Full Text Available The absence of a functional ATP Binding Cassette (ABC protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR from apical membranes of epithelial cells is responsible for cystic fibrosis (CF. Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1. Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of

  2. Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.

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    Mark P DeAndrade

    Full Text Available Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS, a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

  3. Enhanced hippocampal long-term potentiation and fear memory in Btbd9 mutant mice.

    Science.gov (United States)

    DeAndrade, Mark P; Zhang, Li; Doroodchi, Atbin; Yokoi, Fumiaki; Cheetham, Chad C; Chen, Huan-Xin; Roper, Steven N; Sweatt, J David; Li, Yuqing

    2012-01-01

    Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS.

  4. Spectrum of induced floral mutants in Petunia

    International Nuclear Information System (INIS)

    Padmaja, V.; Sudhakar, P.

    1987-01-01

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

  5. A genome-scale RNA-interference screen identifies RRAS signaling as a pathologic feature of Huntington's disease.

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    John P Miller

    Full Text Available A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.

  6. The rapid isolation of mutants of some Gram-positive bacteria

    International Nuclear Information System (INIS)

    Dijkhuizen, L.; Keijer, L.

    1981-01-01

    In this communication the authors describe a method for isolating at high frequency independent mutants of a number of Gram-positive bacteria. The method was originally developed for use with an Arthrobacter sp. and appears to work best with this and other coryneform bacteria. All the bacteria used were from the culture collections maintained at the University of Warwick or the Centre for Applied Microbiological Research. For mutagenesis using UV light, cells were grown in complex media and used while still in the logarithmic phase of growth. Details of the irradiation procedure are given in the text. (Auth.)

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Promising semi-dwarf mutant in wheat variety K68

    Energy Technology Data Exchange (ETDEWEB)

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

    1977-04-01

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

  9. Spectrum of mutant characters utilized in developing improved cultivars

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  10. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  11. Circulation of Pneumocystis dihydropteroate synthase mutants in France.

    Science.gov (United States)

    Le Gal, Solène; Damiani, Céline; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Quinio, Dorothée; Moalic, Elodie; Saliou, Philippe; Berthou, Christian; Le Meur, Yann; Totet, Anne; Nevez, Gilles

    2012-10-01

    Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

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    Mark Merchant

    Full Text Available Mitogen-activated protein kinase (MAPK pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and

  13. Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

    OpenAIRE

    Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

    2003-01-01

    Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, r...

  14. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

    Science.gov (United States)

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

    2013-12-01

    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.

  15. Mutant number distribution in an exponentially growing population

    Science.gov (United States)

    Keller, Peter; Antal, Tibor

    2015-01-01

    We present an explicit solution to a classic model of cell-population growth introduced by Luria and Delbrück (1943 Genetics 28 491-511) 70 years ago to study the emergence of mutations in bacterial populations. In this model a wild-type population is assumed to grow exponentially in a deterministic fashion. Proportional to the wild-type population size, mutants arrive randomly and initiate new sub-populations of mutants that grow stochastically according to a supercritical birth and death process. We give an exact expression for the generating function of the total number of mutants at a given wild-type population size. We present a simple expression for the probability of finding no mutants, and a recursion formula for the probability of finding a given number of mutants. In the ‘large population-small mutation’ limit we recover recent results of Kessler and Levine (2014 J. Stat. Phys. doi:10.1007/s10955-014-1143-3) for a fully stochastic version of the process.

  16. Characteristics of mutant lines of sweet potato flour

    International Nuclear Information System (INIS)

    Aryanti

    2012-01-01

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

  17. Identification of a BET Family Bromodomain/Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function

    Directory of Open Access Journals (Sweden)

    Hyun-Soo Kim

    2014-03-01

    Full Text Available Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02, casein kinase II (CKII, and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.

  18. Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function.

    Science.gov (United States)

    Kim, Hyun-Soo; Mukhopadhyay, Rituparna; Rothbart, Scott B; Silva, Andrea C; Vanoosthuyse, Vincent; Radovani, Ernest; Kislinger, Thomas; Roguev, Assen; Ryan, Colm J; Xu, Jiewei; Jahari, Harlizawati; Hardwick, Kevin G; Greenblatt, Jack F; Krogan, Nevan J; Fillingham, Jeffrey S; Strahl, Brian D; Bouhassira, Eric E; Edelmann, Winfried; Keogh, Michael-Christopher

    2014-03-13

    Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. X-ray-induced mutants resistant to 8-azaguanine

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  20. Biofilm formation in Escherichia coli cra mutants is impaired due to down-regulation of curli biosynthesis.

    Science.gov (United States)

    Reshamwala, Shamlan M S; Noronha, Santosh B

    2011-10-01

    Cra is a pleiotropic regulatory protein that controls carbon and energy flux in enteric bacteria. Recent studies have shown that Cra also regulates other cell processes and influences biofilm formation. The purpose of the present study was to investigate the role of Cra in biofilm formation in Escherichia coli. Congo red-binding studies suggested that curli biosynthesis is impaired in cra mutants. Microarray analysis of wild-type and mutant E. coli cultivated in conditions promoting biofilm formation revealed that the curli biosynthesis genes, csgBAC and csgDEFG, are poorly expressed in the mutant, suggesting that transcription of genes required for curli production is regulated by Cra. Four putative Cra-binding sites were identified in the curli intergenic region, which were experimentally validated by performing electromobility shift assays. Site-directed mutagenesis of three Cra-binding sites in the promoter region of the csgDEFG operon suggests that Cra activates transcription of this operon upon binding to operator regions both downstream and upstream of the transcription start site. Based on the Cra-binding sites identified in this and other studies, the Cra consensus sequence is refined.

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

    Science.gov (United States)

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

    2012-01-01

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

  2. Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

    Science.gov (United States)

    Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon; Yim, Moon B

    2011-05-15

    Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. Published by Elsevier Inc.

  3. The research progress on plant mutant germplasm resources in China

    International Nuclear Information System (INIS)

    He Cexi; Ji Linzhen; Zhao Shirong

    1991-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Edson Jiovany Ramírez-Nava

    2017-10-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD and G6PD Nefza (Leu323Pro, and the double mutant G6PD A− (Asn126Asp + Leu323Pro. The mutants showed lower residual activity (≤50% of WT G6PD and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

  6. Phanerochaete mutants with enhanced ligninolytic activity

    International Nuclear Information System (INIS)

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

    1994-01-01

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

  7. Detection of genetic variability in Basmati and non-Basmati rice varieties and their radiation induced mutants through random amplified polymorphic DNA (RAPD)

    Energy Technology Data Exchange (ETDEWEB)

    Farooq, S; Iqbal, N; Arif, M [Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad (Pakistan)

    1998-03-01

    Random Amplified Polymorphic DNA (RAPDs) markers were utilized to detect polymorphism between pure lines and commercially available Basmati rice varieties to assess variation which may be helpful in quality control and varietal identification (Basmati-370 and derived radiation induced mutants), differentiation of mutants and parents, and identification of RAPD markers co-segregating with important agronomic traits including plant height, days to flower and grain quality. Basmati varieties were distinguished from non-Basmati varieties with the help of five diagnostic markers which will be useful for detecting mixing of non-Basmati and Basmati rices, currently a serious marketing problem. Different Basmati cultivars were identified with the help of diagnostic RAPD markers which can be used in quality control as well as for ``fingerprinting`` of cultivars. Different radiation induced mutants were also successfully distinguished from the parents on the basis of variety specific and mutant specific markers which will be useful for varietal identification. In addition to this, other markers were also identified which can differentiate mutants from each other and are being, used for the fingerprinting of different mutants, particularly the dwarf mutants having similar appearance but different parentage. For identification of RAPD markers co-segregating with plant height and days to flower, 50 F{sub 2} plants and four F{sub 3} families were studied from a reciprocal cross made between Kashmir Basmati (tall and early) and Basmati-198 (dwarf and late). Segregating bands were observed within these populations, and indicating the possible use of RAPD markers for tagging gene(s) of agronomic importance in rice. (author). 38 refs, 6 figs, 3 tabs.

  8. Detection of genetic variability in Basmati and non-Basmati rice varieties and their radiation induced mutants through random amplified polymorphic DNA (RAPD)

    International Nuclear Information System (INIS)

    Farooq, S.; Iqbal, N.; Arif, M.

    1998-01-01

    Random Amplified Polymorphic DNA (RAPDs) markers were utilized to detect polymorphism between pure lines and commercially available Basmati rice varieties to assess variation which may be helpful in quality control and varietal identification (Basmati-370 and derived radiation induced mutants), differentiation of mutants and parents, and identification of RAPD markers co-segregating with important agronomic traits including plant height, days to flower and grain quality. Basmati varieties were distinguished from non-Basmati varieties with the help of five diagnostic markers which will be useful for detecting mixing of non-Basmati and Basmati rices, currently a serious marketing problem. Different Basmati cultivars were identified with the help of diagnostic RAPD markers which can be used in quality control as well as for ''fingerprinting'' of cultivars. Different radiation induced mutants were also successfully distinguished from the parents on the basis of variety specific and mutant specific markers which will be useful for varietal identification. In addition to this, other markers were also identified which can differentiate mutants from each other and are being, used for the fingerprinting of different mutants, particularly the dwarf mutants having similar appearance but different parentage. For identification of RAPD markers co-segregating with plant height and days to flower, 50 F 2 plants and four F 3 families were studied from a reciprocal cross made between Kashmir Basmati (tall and early) and Basmati-198 (dwarf and late). Segregating bands were observed within these populations, and indicating the possible use of RAPD markers for tagging gene(s) of agronomic importance in rice. (author)

  9. Isolation of New Gravitropic Mutants under Hypergravity Conditions.

    Science.gov (United States)

    Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T

    2016-01-01

    Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 ( eal1 ) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene ( enhancer of eal1 ) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis .

  10. Inactivation of carbenicillin by some radioresistant mutant strains

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  11. Agronomic performance of old soybean variety 'Altona' derived mutants

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  12. The Impact of Collisions on the Ability to Detect Rare Mutant Alleles Using Barcode-Type Next-Generation Sequencing Techniques

    Directory of Open Access Journals (Sweden)

    Jenna VanLiere Canzoniero

    2017-07-01

    Full Text Available Barcoding techniques are used to reduce error from next-generation sequencing, with applications ranging from understanding tumor subclone populations to detecting circulating tumor DNA. Collisions occur when more than one sample molecule is tagged by the same unique identifier (UID and can result in failure to detect very-low-frequency mutations and error in estimating mutation frequency. Here, we created computer models of barcoding technique, with and without amplification bias introduced by the UID, and analyzed the effect of collisions for a range of mutant allele frequencies (1e−6 to 0.2, number of sample molecules (10 000 to 1e7, and number of UIDs (4 10 -4 14 . Inability to detect rare mutant alleles occurred in 0% to 100% of simulations, depending on collisions and number of mutant molecules. Collisions also introduced error in estimating mutant allele frequency resulting in underestimation of minor allele frequency. Incorporating an understanding of the effect of collisions into experimental design can allow for optimization of the number of sample molecules and number of UIDs to minimize the negative impact on rare mutant detection and mutant frequency estimation.

  13. Biochemical characteristics of mutant lines of currant tomato

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  14. Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum

    Science.gov (United States)

    Thatcher, Louise F.; Cevik, Volkan; Grant, Murray; Zhai, Bing; Jones, Jonathan D.G.; Manners, John M.; Kazan, Kemal

    2016-01-01

    In Arabidopsis, jasmonate (JA)-signaling plays a key role in mediating Fusarium oxysporum disease outcome. However, the roles of JASMONATE ZIM-domain (JAZ) proteins that repress JA-signaling have not been characterized in host resistance or susceptibility to this pathogen. Here, we found most JAZ genes are induced following F. oxysporum challenge, and screening T-DNA insertion lines in Arabidopsis JAZ family members identified a highly disease-susceptible JAZ7 mutant (jaz7-1D). This mutant exhibited constitutive JAZ7 expression and conferred increased JA-sensitivity, suggesting activation of JA-signaling. Unlike jaz7 loss-of-function alleles, jaz7-1D also had enhanced JA-responsive gene expression, altered development and increased susceptibility to the bacterial pathogen Pst DC3000 that also disrupts host JA-responses. We also demonstrate that JAZ7 interacts with transcription factors functioning as activators (MYC3, MYC4) or repressors (JAM1) of JA-signaling and contains a functional EAR repressor motif mediating transcriptional repression via the co-repressor TOPLESS (TPL). We propose through direct TPL recruitment, in wild-type plants JAZ7 functions as a repressor within the JA-response network and that in jaz7-1D plants, misregulated ectopic JAZ7 expression hyper-activates JA-signaling in part by disturbing finely-tuned COI1-JAZ-TPL-TF complexes. PMID:26896849

  15. Molecular mechanism of action of pharmacoperone rescue of misrouted GPCR mutants: the GnRH receptor.

    Science.gov (United States)

    Janovick, Jo Ann; Patny, Akshay; Mosley, Ralph; Goulet, Mark T; Altman, Michael D; Rush, Thomas S; Cornea, Anda; Conn, P Michael

    2009-02-01

    The human GnRH receptor (hGnRHR), a G protein-coupled receptor, is a useful model for studying pharmacological chaperones (pharmacoperones), drugs that rescue misfolded and misrouted protein mutants and restore them to function. This technique forms the basis of a therapeutic approach of rescuing mutants associated with human disease and restoring them to function. The present study relies on computational modeling, followed by site-directed mutagenesis, assessment of ligand binding, effector activation, and confocal microscopy. Our results show that two different chemical classes of pharmacoperones act to stabilize hGnRHR mutants by bridging residues D(98) and K(121). This ligand-mediated bridge serves as a surrogate for a naturally occurring and highly conserved salt bridge (E(90)-K(121)) that stabilizes the relation between transmembranes 2 and 3, which is required for passage of the receptor through the cellular quality control system and to the plasma membrane. Our model was used to reveal important pharmacophoric features, and then identify a novel chemical ligand, which was able to rescue a D(98) mutant of the hGnRHR that could not be rescued as effectively by previously known pharmacoperones.

  16. Allele-specific Gene Silencing of Mutant mRNA Restores Cellular Function in Ullrich Congenital Muscular Dystrophy Fibroblasts

    Directory of Open Access Journals (Sweden)

    Satoru Noguchi

    2014-01-01

    Full Text Available Ullrich congenital muscular dystrophy (UCMD is an inherited muscle disorder characterized clinically by muscle weakness, distal joint hyperlaxity, and proximal joint contractures. Sporadic and recessive mutations in the three collagen VI genes, COL6A1, COL6A2, and COL6A3, are reported to be causative. In the sporadic forms, a heterozygous point mutation causing glycine substitution in the triple helical domain has been identified in higher rate. In this study, we examined the efficacy of siRNAs, which target point mutation site, on specific knockdown toward transcripts from mutant allele and evaluated consequent cellular phenotype of UCMD fibroblasts. We evaluated the effect of siRNAs targeted to silence-specific COL6A1 alleles in UCMD fibroblasts, where simultaneous expression of both wild-type and mutant collagen VI resulted in defective collagen localization. Addition of mutant-specific siRNAs allowed normal extracellular localization of collagen VI surrounding fibroblasts, suggesting selective inhibition of mutant collagen VI. Targeting the single-nucleotide COL6A1 c.850G>A (p.G284R mutation responsible a sporadic autosomal dominant form of UCMD can potently and selectively block expression of mutant collagen VI. These results suggest that allele-specific knockdown of the mutant mRNA can potentially be considered as a therapeutic procedure in UCMD due to COL6A1 point mutations.

  17. Generation of gamma irradiation and EMS-induced mutant lines of the H7996 tomato (Solanum lycopersicum L.)

    International Nuclear Information System (INIS)

    Canama, Alma O.; Galvez, Hayde F.; Tongson, Eden Jane U.; Quilloy, Reynaldo B.; Hautea, Desiree M.

    2010-01-01

    Tomato (L.) is one of the most important vegetable crops grown worldwide for the fresh vegetable market and food processing industry. With the completion of the genome-sequencing projects in various crops, the major challenge will be determine the gene function. One approach is to generate and to analyze mutant phenotypes. The paper reports the generation of gamma-irradiated and ethy methane sulfonate (EMS)-treated mutant populations, identification and phenotypic characterization of dominant and visible mutations in tomato mutant lines. Mutant populations of tomato H7996 were created using physical (cobalt 60 gamma ray) and chemical EMS mutagens. Generally, based on high-throughput phenotypic characterization, mutations were observed on the plant habit, size, morphology, leaf and flower color and morphology and fruit characteristics. Specifically, the most common dominant and visible mutations noted in the M 1 generation were monopodial, compact, short internodes, multi-branch plant type, light yellow and ghost leaf coloration, tiny and long pedicel leaf morphology and small or short plant size. In the M2 generation, homogeneous and segregating M 2 families were selected to constitute the core set of visible tomato mutants. Initial bacterial wilt resistance (BWR) gene knockouts were also identified. The mutant lines will be used as a rich source of genetic materials for breeding and functional genomics of tomato. (author)

  18. Genetic analyses of nonfluorescent root mutants induced by mutagenesis in soybean

    International Nuclear Information System (INIS)

    Sawada, S.; Palmer, R.G.

    1987-01-01

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

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

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  20. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans.

    Science.gov (United States)

    Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease.

  1. Development of high yielding mutants in lentil

    International Nuclear Information System (INIS)

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

    2001-01-01

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

  2. Methods of producing protoporphyrin IX and bacterial mutants therefor

    Science.gov (United States)

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  3. [Predicting individual risk of high healthcare cost to identify complex chronic patients].

    Science.gov (United States)

    Coderch, Jordi; Sánchez-Pérez, Inma; Ibern, Pere; Carreras, Marc; Pérez-Berruezo, Xavier; Inoriza, José M

    2014-01-01

    To develop a predictive model for the risk of high consumption of healthcare resources, and assess the ability of the model to identify complex chronic patients. A cross-sectional study was performed within a healthcare management organization by using individual data from 2 consecutive years (88,795 people). The dependent variable consisted of healthcare costs above the 95th percentile (P95), including all services provided by the organization and pharmaceutical consumption outside of the institution. The predictive variables were age, sex, morbidity-based on clinical risk groups (CRG)-and selected data from previous utilization (use of hospitalization, use of high-cost drugs in ambulatory care, pharmaceutical expenditure). A univariate descriptive analysis was performed. We constructed a logistic regression model with a 95% confidence level and analyzed sensitivity, specificity, positive predictive values (PPV), and the area under the ROC curve (AUC). Individuals incurring costs >P95 accumulated 44% of total healthcare costs and were concentrated in ACRG3 (aggregated CRG level 3) categories related to multiple chronic diseases. All variables were statistically significant except for sex. The model had a sensitivity of 48.4% (CI: 46.9%-49.8%), specificity of 97.2% (CI: 97.0%-97.3%), PPV of 46.5% (CI: 45.0%-47.9%), and an AUC of 0.897 (CI: 0.892 to 0.902). High consumption of healthcare resources is associated with complex chronic morbidity. A model based on age, morbidity, and prior utilization is able to predict high-cost risk and identify a target population requiring proactive care. Copyright © 2013 SESPAS. Published by Elsevier Espana. All rights reserved.

  4. Genetic studies with morphological mutants of Aspergillus niger

    International Nuclear Information System (INIS)

    Roy, Ponty; Das, Arati

    1979-01-01

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

  5. CloudNeo: a cloud pipeline for identifying patient-specific tumor neoantigens.

    Science.gov (United States)

    Bais, Preeti; Namburi, Sandeep; Gatti, Daniel M; Zhang, Xinyu; Chuang, Jeffrey H

    2017-10-01

    We present CloudNeo, a cloud-based computational workflow for identifying patient-specific tumor neoantigens from next generation sequencing data. Tumor-specific mutant peptides can be detected by the immune system through their interactions with the human leukocyte antigen complex, and neoantigen presence has recently been shown to correlate with anti T-cell immunity and efficacy of checkpoint inhibitor therapy. However computing capabilities to identify neoantigens from genomic sequencing data are a limiting factor for understanding their role. This challenge has grown as cancer datasets become increasingly abundant, making them cumbersome to store and analyze on local servers. Our cloud-based pipeline provides scalable computation capabilities for neoantigen identification while eliminating the need to invest in local infrastructure for data transfer, storage or compute. The pipeline is a Common Workflow Language (CWL) implementation of human leukocyte antigen (HLA) typing using Polysolver or HLAminer combined with custom scripts for mutant peptide identification and NetMHCpan for neoantigen prediction. We have demonstrated the efficacy of these pipelines on Amazon cloud instances through the Seven Bridges Genomics implementation of the NCI Cancer Genomics Cloud, which provides graphical interfaces for running and editing, infrastructure for workflow sharing and version tracking, and access to TCGA data. The CWL implementation is at: https://github.com/TheJacksonLaboratory/CloudNeo. For users who have obtained licenses for all internal software, integrated versions in CWL and on the Seven Bridges Cancer Genomics Cloud platform (https://cgc.sbgenomics.com/, recommended version) can be obtained by contacting the authors. jeff.chuang@jax.org. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

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

    Science.gov (United States)

    Hannan, M A; Duck, P; Nasim, A

    1976-08-01

    The induction of lethal sectoring and pure mutant clones by ultraviolet light has been studied in a homogeneous G1 population of Saccharomyces cerevisiae grown in a normal growth medium. At the lowest UV dose of 250 ergs, which corresponds to a shoulder in the survival curve, all mutants appeared as pure clones. At higher doses the frequency of mosaic mutants progressively increased. These results indicate a relationship between the highest frequency of complete mutants and the maximum repair activity. In addition, the frequency of lethal sectoring at all doses tested was too low to account for the origin of pure mutant clones.

  7. Gene expression profile analysis of Ligon lintless-1 (Li1) mutant reveals important genes and pathways in cotton leaf and fiber development.

    Science.gov (United States)

    Ding, Mingquan; Jiang, Yurong; Cao, Yuefen; Lin, Lifeng; He, Shae; Zhou, Wei; Rong, Junkang

    2014-02-10

    Ligon lintless-1 (Li1) is a monogenic dominant mutant of Gossypium hirsutum (upland cotton) with a phenotype of impaired vegetative growth and short lint fibers. Despite years of research involving genetic mapping and gene expression profile analysis of Li1 mutant ovule tissues, the gene remains uncloned and the underlying pathway of cotton fiber elongation is still unclear. In this study, we report the whole genome-level deep-sequencing analysis of leaf tissues of the Li1 mutant. Differentially expressed genes in leaf tissues of mutant versus wild-type (WT) plants are identified, and the underlying pathways and potential genes that control leaf and fiber development are inferred. The results show that transcription factors AS2, YABBY5, and KANDI-like are significantly differentially expressed in mutant tissues compared with WT ones. Interestingly, several fiber development-related genes are found in the downregulated gene list of the mutant leaf transcriptome. These genes include heat shock protein family, cytoskeleton arrangement, cell wall synthesis, energy, H2O2 metabolism-related genes, and WRKY transcription factors. This finding suggests that the genes are involved in leaf morphology determination and fiber elongation. The expression data are also compared with the previously published microarray data of Li1 ovule tissues. Comparative analysis of the ovule transcriptomes of Li1 and WT reveals that a number of pathways important for fiber elongation are enriched in the downregulated gene list at different fiber development stages (0, 6, 9, 12, 15, 18dpa). Differentially expressed genes identified in both leaf and fiber samples are aligned with cotton whole genome sequences and combined with the genetic fine mapping results to identify a list of candidate genes for Li1. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Pollen irradiation method to obtain mutants in cucumber

    International Nuclear Information System (INIS)

    Iida, S.; Amano, E.

    1988-01-01

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

  9. Mutant number distribution in an exponentially growing population

    International Nuclear Information System (INIS)

    Keller, Peter; Antal, Tibor

    2015-01-01

    We present an explicit solution to a classic model of cell-population growth introduced by Luria and Delbrück (1943 Genetics 28 491–511) 70 years ago to study the emergence of mutations in bacterial populations. In this model a wild-type population is assumed to grow exponentially in a deterministic fashion. Proportional to the wild-type population size, mutants arrive randomly and initiate new sub-populations of mutants that grow stochastically according to a supercritical birth and death process. We give an exact expression for the generating function of the total number of mutants at a given wild-type population size. We present a simple expression for the probability of finding no mutants, and a recursion formula for the probability of finding a given number of mutants. In the ‘large population-small mutation’ limit we recover recent results of Kessler and Levine (2014 J. Stat. Phys. doi:10.1007/s10955-014-1143-3) for a fully stochastic version of the process. (paper)

  10. A wheat cold resistance mutant derived from space mutagenesis

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  11. A wheat cold resistance mutant derived from space mutagenesis

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  12. Selection and agronomic evaluation of induced mutant lines of sesame

    International Nuclear Information System (INIS)

    Hoballah, A.A.

    2001-01-01

    Station yield trial: Three high yielding mutants (8, 48, and EFM92) with better and stable performance were developed in our breeding programme and submitted for registration to the Agricultural Research Center (ARC), Egyptian Ministry of Agriculture and Land Reclamation. Multi-location yield trials indicated that mutant line EFM92 ranked first in all locations; significant yield increases recorded for it ranged from 14.7 to 74.0% over the check variety. Moreover, it was 15-20 days earlier than the check and/or other mutants. Mutant lines 8 and 48 produced higher seed yields than the check at two different locations. These mutants can probably be grown and produce more yield than the check variety at the low yielding environments. Seed quality assay: During 1996 and 1997, 15 promising lines of sesame including mutants and hybrid populations as well as the local variety were evaluated for seed protein, oil content and fatty acid composition. The protein content varied from 20.6 to 26.7%; hybrid population EXM90 gave the highest value. About 85% of the total fatty acids in the oil are unsaturated (oleic and linoleic) and 15% saturated, mainly palmitic and stearic. Linoleic acid ranged from 41.8 to 47.9%. Mutant lines 6, 9, and EFM92, which gave high oil content (54-55.5%) together with high linoleic acid values (45.2-47.8%), are recommended for breeding for seed oil quality. Heterosis, combining ability and type of gene action in sesame: A half diallel set of crosses involving seven parents was used to study heterosis and combining ability in the F 1 generation as well as the nature of gene action controlling seed yield and its contributing traits in both F 1 and F 2 in order to identify the most efficient breeding methods leading to rapid genetic improvement. The expressions of heterosis varied with the crosses and characters investigated. The maximal significant positive useful heterosis was observed for branches/plant (52.9%) followed by seed yield/plant (38

  13. Isolation of new gravitropic mutants under hypergravity conditions

    Directory of Open Access Journals (Sweden)

    Akiko Mori

    2016-09-01

    Full Text Available Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upwards. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes. In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using Next-Generation Sequencing (NGS and Single Nucleotide Polymorphism (SNP-based markers. Using the endodermal-amyloplast less 1 (eal1 mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1 mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis.

  14. Categories and inheritance of resistance to Nilaparvata lugens (Hemiptera: Delphacidae) in mutants of indica rice 'IR64'.

    Science.gov (United States)

    Sangha, Jatinder Singh; Chen, Yolanda H; Palchamy, Kadirvel; Jahn, Gary C; Maheswaran, M; Adalla, Candida B; Leung, Hei

    2008-04-01

    Varietal mutants can be useful for developing durable resistance, understanding categories of resistance, and identifying candidate genes involved in defense responses. We used mutants of rice 'IR64' to isolate new sources of resistance to the planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). We compared two mutants that showed a gain and loss of resistance to N. lugens, to determine the categories of resistance to this pest. Under choice tests, female planthoppers avoided settling and laid fewer eggs on the resistant mutant 'D518' than on the susceptible mutant D1131, susceptible check 'TN1', and wild-type IR64, indicating that antixenosis was the resistance category. Similarly, under no-choice conditions, planthoppers laid 29% fewer eggs in D518 than in IR64, but they oviposited more in 'D1131' and TN1. Honeydew excretion was greater on D1131 seedlings but slightly lower on D518 than on IR64. Nymphal survival and adult female weight did not differ among rice cultivars. D518 showed higher tolerance of N. lugens infestations than IR64. Genetic analysis of the F1, F2, and F3 populations derived from D518 x IR64 revealed that resistance in D518 is dominant and controlled by a single gene. Despite the variation in resistance to N. lugens, both mutants and IR64 performed similarly in the field. The mutant D518 is a new source of durable resistance to N. lugens, mainly due to enhanced antixenosis to female hoppers for settling and oviposition.

  15. Improved yields of daunomycinone glycosides in developmental mutants of Streptomyces coeruleorubidus

    International Nuclear Information System (INIS)

    Blumauerova, M.; Pokorny, V.; Stastna, J.; Hostalek, Z.; Vanek, Z.

    1978-01-01

    When improving Streptomyces coeruleorubidus JA 10092, a producer of antibiotics of the daunomycinone complex, the most active variants were found among isolates of morphological types bld-1 (with a suppressed production of the aerial mycelium on organic media containing glucose) and whi (with an asporogenic aerial mycelium on glucose media and with the bald phenotype on media containing starch). Submerged cultures of the whi mutants produced increased quantities of daunomycinone glycosides in the antibiotic complex, the amount of free anthracyclinones being simultaneously decreased. The whi strains differed from the wild type also in higher demands for aeration, concentration of glucose and in an increased production capacity in starch media. The overall antibiotic activity increased more than 40 times after a six-step selection (application of UV light, γ-radiation, nitrous acid and natural spreads) combined with an altered fermentation technology. (author)

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

    Directory of Open Access Journals (Sweden)

    Biddle Kelly D

    2008-10-01

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

  17. Identifying technical aliases in SELDI mass spectra of complex mixtures of proteins

    Science.gov (United States)

    2013-01-01

    Background Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only. Results In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification. Conclusions This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation. PMID:24010718

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

    International Nuclear Information System (INIS)

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

    1983-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Da-Long Guo

    2016-01-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    International Nuclear Information System (INIS)

    Hao Zhongna; Zhang Hongzhi; Tao Rongixang

    2007-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-01

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

  3. Variation in Yield and Physicochemical Quality Traits among Mutants of Japonica Rice Cultivar Wuyujing 3

    Directory of Open Access Journals (Sweden)

    Jose Daniel Abacar

    2016-01-01

    Full Text Available To select elite germplasms, 112 mutants derived from japonica rice cultivar Wuyujing 3 were evaluated. The yield components such as panicle number per square meter, grain number per panicle, and grain weight were measured. The quality traits such as percentage of chalky grains (PCG, brown rice yield (BRY, milled rice yield (MRY, degree of milling (DM, amylose content (AC, protein content (PC, and relationships among traits were inverstigated. Results showed that grain yield ranged from 2.15 to 12.49 t/hm2 with a mean of 6.4 t/hm2 and number of grains per square meter contributed for 94.64% in grain yield variation. For quality traits, all rice mutants had short size (grain length ≤ 5.5 mm and bold shape (grain length to width ratio = 1.10–2.00. Most of rice mutants (87.5% had PCG values below 20%. All mutants had MRY values above 50%, AC values below 20%, and PC values below 10%. Percentage of chalky grains was significantly negatively correlated with MRY and positively correlated with DM. BRY and MRY were significantly negatively correlated with DM. PC was significantly and positively correlated with MRY and negatively correlated with DM, while AC had no significant correlation with these quality traits. It was concluded that there were 25 rice mutants which fulfilled the major requirements of Jiangsu standard japonica rice such as low percentage of chalky grains, low amylose content, optimal protein content, and which could be used as elite germplasms. Thus the mutants identified may lead to significant progress in improvement of rice quality.

  4. Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery

    Directory of Open Access Journals (Sweden)

    Edith eCoronado

    2014-11-01

    Full Text Available The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested, which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

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

    International Nuclear Information System (INIS)

    Molkhova, E.; Vasileva, M.

    1977-01-01

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

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

    NARCIS (Netherlands)

    Lolkema, J.S.; Poolman, B.

    1995-01-01

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

  7. Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

    DEFF Research Database (Denmark)

    Roszko, Kelly L; Bi, Ruiye; Gorvin, Caroline M

    2017-01-01

    in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated...... human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11...

  8. The agronomic characters of a high protein rice mutant

    International Nuclear Information System (INIS)

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

    1975-01-01

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

  9. Induction and characterization of Arabidopsis mutants by Ion beam

    International Nuclear Information System (INIS)

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

    2008-03-01

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

  10. Induction and characterization of Arabidopsis mutants by Ion beam

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-03-15

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

  11. Multivariate analysis for selecting apple mutants

    International Nuclear Information System (INIS)

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

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hassan Abdel-Latif A. Mohamed

    2006-06-01

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

  13. Characterization of MMS-sensitive mutants of Neurospora crassa

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-01-01

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

  14. Productive mutants in lemongrass induced by gamma rays

    International Nuclear Information System (INIS)

    Gopinathan Nair, V.

    1980-01-01

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

  15. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

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

  16. A multienzyme complex channels substrates and electrons through acetyl-CoA and methane biosynthesis pathways in Methanosarcina.

    Directory of Open Access Journals (Sweden)

    Dillon J Lieber

    Full Text Available Multienzyme complexes catalyze important metabolic reactions in many organisms, but little is known about the complexes involved in biological methane production (methanogenesis. A crosslinking-mass spectrometry (XL-MS strategy was employed to identify proteins associated with coenzyme M-coenzyme B heterodisulfide reductase (Hdr, an essential enzyme in all methane-producing archaea (methanogens. In Methanosarcina acetivorans, Hdr forms a multienzyme complex with acetyl-CoA decarbonylase synthase (ACDS, and F420-dependent methylene-H4MPT reductase (Mer. ACDS is essential for production of acetyl-CoA during growth on methanol, or for methanogenesis from acetate, whereas Mer is essential for methanogenesis from all substrates. Existence of a Hdr:ACDS:Mer complex is consistent with growth phenotypes of ACDS and Mer mutant strains in which the complex samples the redox status of electron carriers and directs carbon flux to acetyl-CoA or methanogenesis. We propose the Hdr:ACDS:Mer complex comprises a special class of multienzyme redox complex which functions as a "biological router" that physically links methanogenesis and acetyl-CoA biosynthesis pathways.

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

    Science.gov (United States)

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

    2013-06-01

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

  18. Study on ionizing radiosensitivity of respiratory deficiency yeast mutants

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  19. Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis

    International Nuclear Information System (INIS)

    Shirley, B.W.; Kubasek, W.L.; Storz, G.; Bruggemann, E.; Koornneef, M.; Ausubel, F.M.; Goodman, H.M.

    1995-01-01

    Eleven loci that play a role in the synthesis of flavonoids in Arabidopsis are described. Mutations at these loci, collectively named transparent testa (tt), disrupt the synthesis of brown pigments in the seed coat (testa). Several of these loci (tt3, tt4, tt5 and ttg) are also required for the accumulation of purple anthocyanins in leaves and stems and one locus (ttg) plays additional roles in trichome and root hair development. Specific functions were previously assigned to tt1-7 and ttg. Here, the results of additional genetic, biochemical and molecular analyses of these mutants are described. Genetic map positions were determined for tt8, tt9 and tt10. Thin-layer chromatography identified tissue- and locus-specific differences in the flavonols and anthocyanidins synthesized by mutant and wild-type plants. It was found that UV light reveals distinct differences in the floral tissues of tt3, tt4, tt5, tt6 and ttg, even though these tissues are indistinguishable under visible light. Evidence was also uncovered that tt8 and ttg specifically affect dihydroflavonol reductase gene expression. A summary of these and previously published results are incorporated into an overview of the genetics of flavonoid biosynthesis in Arabidopsis

  20. Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes

    Science.gov (United States)

    Kim, Kevin; Lee, Young Sik; Carthew, Richard W.

    2007-01-01

    In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA. PMID:17123955

  1. Transcriptional regulation of male-sterility in 7B-1 male-sterile tomato mutant.

    Directory of Open Access Journals (Sweden)

    Vahid Omidvar

    Full Text Available The 7B-1 tomato (Solanum lycopersicum L. cv Rutgers is a male-sterile mutant with enhanced tolerance to abiotic stress, which makes it a potential candidate for hybrid seed breeding and stress engineering. To underline the molecular mechanism regulating the male-sterility in 7B-1, transcriptomic profiles of the 7B-1 male-sterile and wild type (WT anthers were studied using mRNA sequencing (RNA-Seq. In total, 768 differentially expressed genes (DEGs were identified, including 132 up-regulated and 636 down-regulated transcripts. Gene ontology (GO enrichment analysis of DEGs suggested a general impact of the 7B-1 mutation on metabolic processes, such as proteolysis and carbohydrate catabolic process. Sixteen candidates with key roles in regulation of anther development were subjected to further analysis using qRT-PCR and in situ hybridization. Cytological studies showed several defects associated with anther development in the 7B-1 mutant, including unsynchronized anther maturation, dysfunctional meiosis, arrested microspores, defect in callose degradation and abnormal tapetum development. TUNEL assay showed a defect in programmed cell death (PCD of tapetal cells in 7B-1 anthers. The present study provides insights into the transcriptome of the 7B-1 mutant. We identified several genes with altered expression level in 7B-1 (including beta-1,3 glucanase, GA2oxs, cystatin, cysteine protease, pectinesterase, TA29, and actin that could potentially regulate anther developmental processes, such as meiosis, tapetum development, and cell-wall formation/degradation.

  2. Bioactive compounds of fourth generation gamma-irradiated Typhoniumflagelliforme Lodd. mutants based on gas chromatography-mass spectrometry

    Science.gov (United States)

    Sianipar, N. F.; Purnamaningsih, R.; Rosaria

    2016-08-01

    Rodent tuber (Typhonium flagelliforme Lodd.) is an Indonesian anticancer medicinal plant. The natural genetic diversity of rodent tuber is low due to vegetative propagation. Plant's genetic diversity has to be increased for obtaining clones which contain a high amount of anticancer compounds. In vitro calli were irradiated with 6 Gy of gamma ray to produce in vitro mutant plantlets. Mutant plantlets were acclimated and propagated in a greenhouse. This research was aimed to identify the chemical compounds in the leaves and tubers ofthe fourth generation of rodent tuber's vegetative mutant clones (MV4) and control plantsby using GC- MS method. Leaves and tubers of MV4 each contained 2 and 5 anticancer compounds which quantities were higher compared to control plants. MV4 leaves contained 5 new anticancer compounds while its tubers contained 3 new anticancer compounds which were not found in control. The new anticancer compounds in leaves were hexadecanoic acid, stigmast-5-en-3-ol, ergost-5-en-3-ol, farnesol isomer a, and oleic acid while the new anticancer compounds in tubers were alpha tocopherol, ergost-5-en-3-ol, and beta-elemene. Rodent tuber mutant clones are very potential to be developed into anticancer drugs.

  3. Engineered disulfide bonds restore chaperone-like function of DJ-1 mutants linked to familial Parkinson's disease.

    Science.gov (United States)

    Logan, Todd; Clark, Lindsay; Ray, Soumya S

    2010-07-13

    Loss-of-function mutations such as L166P, A104T, and M26I in the DJ-1 gene (PARK7) have been linked to autosomal-recessive early onset Parkinson's disease (PD). Cellular and structural studies of the familial mutants suggest that these mutations may destabilize the dimeric structure. To look for common dynamical signatures among the DJ-1 mutants, short MD simulations of up to 1000 ps were conducted to identify the weakest region of the protein (residues 38-70). In an attempt to stabilize the protein, we mutated residue Val 51 to cysteine (V51C) to make a symmetry-related disulfide bridge with the preexisting Cys 53 on the opposite subunit. We found that the introduction of this disulfide linkage stabilized the mutants A104T and M26I against thermal denaturation, improved their ability to scavenge reactive oxygen species (ROS), and restored a chaperone-like function of blocking alpha-synuclein aggregation. The L166P mutant was far too unstable to be rescued by introduction of the V51C mutation. The results presented here point to the possible development of pharmacological chaperones, which may eventually lead to PD therapeutics.

  4. Fine Mapping of a Degenerated Abdominal Legs Mutant (Edl in Silkworm, Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Honglei Wang

    Full Text Available In insects, abdominal appendages, also called prolegs, vary due to adaptive evolution. Mutations on prolegs within species provide insights to better understand the mechanisms underlying appendage development and diversity. In silkworm Bombyx mori, extra-crescents and degenerated abdominal legs (Edl mutant, belonging to the E pseudoallele group, is a spontaneous mutation that adds crescents and degenerates prolegs on the third abdominal segment (A3. This mutation may be a homeotic transformation of A3 to A2. In this study, the Edl locus was mapped within approximately a 211 Kb region that is 10 Kb upstream of Bmabdominal-A (Bmabd-A. RT-quantitative PCR (RT-qPCR and Western blot analysis of Bmabd-A expression showed a slight but significant decrease, while the expression of BmUltrabithorax (BmUbx was up-regulated in the Edl mutant compared to wildtype (Dazao. Moreover, we also found that BmDistal-less (BmDll, which regulated the development of distal proleg structures, was missing at the tips of the A3 prolegs in the Edl mutant compared to BmDll expression in normally developed prolegs in both the wildtype and mutant. Collectively, we identified approximately a 211 Kb region in the Edl locus that regulates BmUbx and Bmabd-A expression and found that changes in BmUbx and Bmabd-A expression may lead to the loss of distal proleg structures in B. mori.

  5. Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krueppel

    International Nuclear Information System (INIS)

    Bedian, V.; Summers, M.C.; Kauffman, S.A.

    1988-01-01

    We have identified early embryo proteins related to the segmentation gene Krueppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins

  6. Development of Database Software with Plant Mutant Resources

    International Nuclear Information System (INIS)

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

    2007-03-01

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

  7. Construindo Marcas Mutantes

    Directory of Open Access Journals (Sweden)

    Elizete De Azevedo Kreutz

    2012-09-01

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

  8. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Science.gov (United States)

    Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

    2015-11-01

    There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  9. FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

    Directory of Open Access Journals (Sweden)

    David Bednar

    2015-11-01

    Full Text Available There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

  10. A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

    Science.gov (United States)

    Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

    2009-05-15

    In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

  11. Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

    Science.gov (United States)

    Karita, M; Etterbeek, M L; Forsyth, M H; Tummuru, M K; Blaser, M J

    1997-10-01

    Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate. When wild-type H. pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation. Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H. pylori despite evidence that insertional mutagenesis had occurred. The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies. The data imply that the H. pylori dapE mutant may be useful as a conditionally lethal vaccine.

  12. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  13. Identifying designatable units for intraspecific conservation prioritization: a hierarchical approach applied to the lake whitefish species complex (Coregonus spp.).

    Science.gov (United States)

    Mee, Jonathan A; Bernatchez, Louis; Reist, Jim D; Rogers, Sean M; Taylor, Eric B

    2015-06-01

    The concept of the designatable unit (DU) affords a practical approach to identifying diversity below the species level for conservation prioritization. However, its suitability for defining conservation units in ecologically diverse, geographically widespread and taxonomically challenging species complexes has not been broadly evaluated. The lake whitefish species complex (Coregonus spp.) is geographically widespread in the Northern Hemisphere, and it contains a great deal of variability in ecology and evolutionary legacy within and among populations, as well as a great deal of taxonomic ambiguity. Here, we employ a set of hierarchical criteria to identify DUs within the Canadian distribution of the lake whitefish species complex. We identified 36 DUs based on (i) reproductive isolation, (ii) phylogeographic groupings, (iii) local adaptation and (iv) biogeographic regions. The identification of DUs is required for clear discussion regarding the conservation prioritization of lake whitefish populations. We suggest conservation priorities among lake whitefish DUs based on biological consequences of extinction, risk of extinction and distinctiveness. Our results exemplify the need for extensive genetic and biogeographic analyses for any species with broad geographic distributions and the need for detailed evaluation of evolutionary history and adaptive ecological divergence when defining intraspecific conservation units.

  14. Activation of DAF-16/FOXO by reactive oxygen species contributes to longevity in long-lived mitochondrial mutants in Caenorhabditis elegans.

    Science.gov (United States)

    Senchuk, Megan M; Dues, Dylan J; Schaar, Claire E; Johnson, Benjamin K; Madaj, Zachary B; Bowman, Megan J; Winn, Mary E; Van Raamsdonk, Jeremy M

    2018-03-01

    Mild deficits in mitochondrial function have been shown to increase lifespan in multiple species including worms, flies and mice. Here, we study three C. elegans mitochondrial mutants (clk-1, isp-1 and nuo-6) to identify overlapping genetic pathways that contribute to their longevity. We find that genes regulated by the FOXO transcription factor DAF-16 are upregulated in all three strains, and that the transcriptional changes present in these worms overlap significantly with the long-lived insulin-IGF1 signaling pathway mutant daf-2. We show that DAF-16 and multiple DAF-16 interacting proteins (MATH-33, IMB-2, CST-1/2, BAR-1) are required for the full longevity of all three mitochondrial mutants. Our results suggest that the activation of DAF-16 in these mutants results from elevated levels of reactive oxygen species. Overall, this work reveals an overlapping genetic pathway required for longevity in three mitochondrial mutants, and, combined with previous work, demonstrates that DAF-16 is a downstream mediator of lifespan extension in multiple pathways of longevity.

  15. The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection1[C][W

    Science.gov (United States)

    Cazzaniga, Stefano; Li, Zhirong; Niyogi, Krishna K.; Bassi, Roberto; Dall’Osto, Luca

    2012-01-01

    Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII. PMID:23029671

  16. Identifying influential spreaders in complex networks based on kshell hybrid method

    Science.gov (United States)

    Namtirtha, Amrita; Dutta, Animesh; Dutta, Biswanath

    2018-06-01

    Influential spreaders are the key players in maximizing or controlling the spreading in a complex network. Identifying the influential spreaders using kshell decomposition method has become very popular in the recent time. In the literature, the core nodes i.e. with the largest kshell index of a network are considered as the most influential spreaders. We have studied the kshell method and spreading dynamics of nodes using Susceptible-Infected-Recovered (SIR) epidemic model to understand the behavior of influential spreaders in terms of its topological location in the network. From the study, we have found that every node in the core area is not the most influential spreader. Even a strategically placed lower shell node can also be a most influential spreader. Moreover, the core area can also be situated at the periphery of the network. The existing indexing methods are only designed to identify the most influential spreaders from core nodes and not from lower shells. In this work, we propose a kshell hybrid method to identify highly influential spreaders not only from the core but also from lower shells. The proposed method comprises the parameters such as kshell power, node's degree, contact distance, and many levels of neighbors' influence potential. The proposed method is evaluated using nine real world network datasets. In terms of the spreading dynamics, the experimental results show the superiority of the proposed method over the other existing indexing methods such as the kshell method, the neighborhood coreness centrality, the mixed degree decomposition, etc. Furthermore, the proposed method can also be applied to large-scale networks by considering the three levels of neighbors' influence potential.

  17. High-Protein Soybean Mutants by Using Irradiation Technique

    International Nuclear Information System (INIS)

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

    2009-07-01

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

  18. Gamma ray induced male sterility mutant in lentil

    International Nuclear Information System (INIS)

    Srivastava, A.; Yadav, A.K.

    2001-01-01

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

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

    International Nuclear Information System (INIS)

    Koment, R.W.; Rapp, F.

    1975-01-01

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

  20. Semi-dwarf mutant lines of hexaploid triticale

    International Nuclear Information System (INIS)

    Pidra, M.

    1989-01-01

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