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Sample records for grla mutant mt5224c4

  1. Detection of grlA and gyrA mutations in 344 Staphylococcus aureus strains.

    Science.gov (United States)

    Wang, T; Tanaka, M; Sato, K

    1998-02-01

    Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC-->TTC or TAC (Ser-80-->Phe or Tyr) and the gyrA mutation of TCA-->TTA (Ser-84-->Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. The grlA point mutations of CAT-->CAC (His-77 [silent]), TCA-->CCA (Ser-81-->Pro), and ATA-->ATT (Ile-100 [silent]) were novel, as was the GAC-->GGC (Asp-73-->Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC > or = 3.13 microg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC50] = 50 microg/ml). Those with the Ser80-->Phe or Tyr alteration in grlA but wild-type gyrA showed a lower level of ciprofloxacin resistance (MIC50 < or = 12.5 microg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC < or = 6.25 microg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was < or = 6.25 microg/ml.

  2. Mutation in grlA and norA genes in Fluoroquinolone resistant strains of S. aureus

    Directory of Open Access Journals (Sweden)

    Leila Asadpour

    2017-03-01

    Full Text Available Background and Aim. Fluoroquinolones are one of the most commonly used broad spectrum classes of antibiotics with increasingly drug resistance. The present study was aimed to investigate mutation in grlA and norA genes in fluoroquinolone resistant strains of   S. aureus. Materials and methods: In this cross-sectional study, 85 strains of S. aureus was isolated from different clinical samples by biochemical and molecular tests. Susceptibility to fluoroquinolones in the isolates was tested by the disc diffusion method and broth macrodilution method was used to investigate ciprofloxacin minimum inhibitory concentration. Mutations in the quinolone resistance-determining regions (QRDR in chromosomal grlA gene and norA gen mutations was investigated in quinolone-resistant S. aureus isolates, by PCR amplification of grlA and norA genes and sequencing.   Results. Out of 85 tested bacteria, 17 (20% isolates recognized as fluoroquinolones resistant. Minimum inhibitory concentration of ciprofluxacin for resistant isolates ranged between 32 to 512 µg/ml. Two single point mutations S80F and P144S and one double mutation S80F + P144S were determined in grlA gene of Fluoroquinolone resistant strains of S. aureus  but none of resistant strains showed mutation in norA gene. Conclusion. The obtained results demonstrated that grlA gene mutation is one of the most important mechanisms of resistance to ciprofloxacin in clinical isolates of S. aureus in Rasht and most of resistant isolates acquired common mutation in codon 80. Key Words: Fluoroquinolone, S. aureus, grlA , norA

  3. 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.

  4. Secretos de Mutantes

    OpenAIRE

    Marín, Martha; Muñoz, Germán; Serrano, Rafael

    2017-01-01

    Apartándose de enfoques que consideran las culturas juveniles como ‘desviaciones sociales', ‘tribus urbanas' o ‘nuevos movimientos políticos', Secretos de mutantes bucea en culturas juveniles urbanas como la Skinhead, el Punk, el Metal, el Hardcore, el Grunge y el Hip Hop, explorándolas desde un punto de vista inédito: su dimensión de creación, para percibir los cruciales y casi desconocidos procesos que sus miembros llevan a cabo en estos vastos universos de experimentación. Esta obra se nut...

  5. ECB deacylase mutants

    Science.gov (United States)

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

    2002-01-01

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

  6. Nif- Hup- mutants of Rhizobium japonicum.

    OpenAIRE

    Moshiri, F; Stults, L; Novak, P.; Maier, R J

    1983-01-01

    Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains...

  7. Wild Accessions and Mutant Resources

    DEFF Research Database (Denmark)

    Kawaguchi, Masayoshi; Sandal, Niels Nørgaard

    2014-01-01

    Lotus japonicus, Lotus burttii, and Lotus filicaulis are species of Lotus genus that are utilized for molecular genetic analysis such as the construction of a linkage map and QTL analysis. Among them, a number of mutants have been isolated from two wild accessions: L. japonicus Gifu B-129...

  8. Bacterial mutants for enhanced succinate production

    NARCIS (Netherlands)

    Baart, G.J.E.; Beauprez, J.J.R.; Foulquie, M.M.R.; Heijnen, J.J.; Maertens, J.

    2010-01-01

    The present invention relates to a method for obtaining enhanced metabolite production in micro-organisms, and to mutants and/or transformants obtained with said method. More particularly, it relates to bacterial mutants and/or transformants for enhanced succinate production, especially mutants and/

  9. 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…

  10. Nif- Hup- mutants of Rhizobium japonicum.

    Science.gov (United States)

    Moshiri, F; Stults, L; Novak, P; Maier, R J

    1983-01-01

    Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity. Images PMID:6874648

  11. Identification of a Long Rice Spikelet Mutant

    Institute of Scientific and Technical Information of China (English)

    WU Xian-jun; WANG Bin; HAN Zan-ping; XIE Zhao-hui; MOU Chun-hong; WANG Xu-dong

    2004-01-01

    A spontaneously occurring rice (Oryza sativa L. ) mutant, characterized by homeotic conversion in glumes and stamens, was found in the progeny of a cross. The mutant showed long glumes and glumaceous lodicules and morphological transformation of stamens into pistils. Mutant florets consisted of 1 to 3 completely developed pistils, some pistilloid stamens with filaments, but tipped by bulged tissue and 0 to 3 stigmas. It seens that the mutant phenotype of the homeotic conversions in glumes and stamens is similar to that of the B loss-of-function mutants in Arabidopsis and Antirrhinum. The mutant is controlled by a single recessive gene as a segregation ratio of 3:1 (wild type to mutant plants) was observed in the F2 generation.

  12. Biochemical and histological characterization of tomato mutants

    Directory of Open Access Journals (Sweden)

    Carolina C. Monteiro

    2012-06-01

    Full Text Available Biochemical responses inherent to antioxidant systems as well morphological and anatomical properties of photomorphogenic, hormonal and developmental tomato mutants were investigated. Compared to the non-mutant Micro-Tom (MT, we observed that the malondialdehyde (MDA content was enhanced in the diageotropica (dgt and lutescent (l mutants, whilst the highest levels of hydrogen peroxide (H2O2 were observed in high pigment 1 (hp1 and aurea (au mutants. The analyses of antioxidant enzymes revealed that all mutants exhibited reduced catalase (CAT activity when compared to MT. Guaiacol peroxidase (GPOX was enhanced in both sitiens (sit and notabilis (not mutants, whereas in not mutant there was an increase in ascorbate peroxidase (APX. Based on PAGE analysis, the activities of glutathione reductase (GR isoforms III, IV, V and VI were increased in l leaves, while the activity of superoxide dismutase (SOD isoform III was reduced in leaves of sit, epi, Never ripe (Nr and green flesh (gf mutants. Microscopic analyses revealed that hp1 and au showed an increase in leaf intercellular spaces, whereas sit exhibited a decrease. The au and hp1 mutants also exhibited a decreased in the number of leaf trichomes. The characterization of these mutants is essential for their future use in plant development and ecophysiology studies, such as abiotic and biotic stresses on the oxidative metabolism.Neste trabalho, analisamos as respostas bioquímicas inerentes ao sistema antioxidante, assim como propriedades morfológicas e anatômicas de mutantes fotomorfogenéticos e hormonais de tomateiro. Comparados ao não mutante Micro-Tom (MT, observamos que o conteúdo de malondialdeído (MDA aumentou nos mutantes diageotropica (dgt e lutescent (l, enquanto os maiores níveis de H2O2 foram encontrados nos mutantes high pigment 1 (hp1 e aurea (au. Análises de enzimas antioxidantes mostraram que todos os mutantes reduziram a atividade de catalase (CAT quando comparado a MT. A

  13. Study on culturing Trichodema mutants

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-ai; WANG Wei-ming

    2004-01-01

    @@ Trichodema mutants strains T5, T0803, T1010, T1003were cultured in different conditions and media, also in the presence of fungicides at 40 mg/kg (CK or procymidone + chlorothalonil, or maneb or phosethyl-Al) . The pH values of media were 5, 6, 7 and 8 and hyphae were grown at temperatures of 15, 20, 25 and 30 ℃. After being cultured for 3, 4, 5, or 6 days, the strains were transferred at a lower temperature to sporulate (20℃) Obtained data were analyzed statistically, with the orthogonal array and ranges (R) differing dependes on the treatments (R = 40.0,42.4, 48.0, 62.8,107.0). The results indicated that the most important factor was the nature of the strain (R =107.0), while the change in temperature and time of cultivation produced the lowest effect (R =40.0). Each factor variance was significant and A3B4C2D1E3 was the optimum combined condition, in which strain T1010 grew more quickly and sporulated most.

  14. CMPD: cancer mutant proteome database.

    Science.gov (United States)

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes.

  15. Muscle development in mdx mutant mice.

    Science.gov (United States)

    Dangain, J; Vrbova, G

    1984-01-01

    Mechanical and contractile properties of tibialis anterior (TA) muscles from X-linked muscular dystrophic (mdx) mutant mice at different stages of development are compared to those of muscles from normal control animals. There is no difference between the tension output, speeds of contraction and relaxation, and weight of TA muscles from mutant adults and normal control animals. However, it is found that in 3-4-week-old mutant animals, tension output and muscle weight are very much reduced, and half relaxation time is prolonged. Thus, during this stage of development, muscles from mdx mice do not function properly. Histological examination of these muscles provides further evidence that, in these animals, rapid muscle destruction occurs at a particular time of development and that it is followed by complete recovery. This new mutant therefore presents an interesting case of muscle destruction and rapid regeneration. However, it is not an adequate model for Duchenne muscular dystrophy.

  16. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1-Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1-Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...mutations found in breast cancer using both structural and cell based assays. We have now have evidence for the effects of the most recurrent

  17. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    Introduction Approximately 70% of ER+ breast cancers harbor expression of the estrogen receptor and are dependent upon its activity for various aspects of the...resistance to current FDA approved ER antagonists, but that more potent and selective estrogen receptor antagonists will be sufficiently active to...antagonists and their potency against ER mutants both in vitro and in vivo . Targeting ESR1-Mutant Breast Cancer W81XWH-14-1-0359 9 4. Impact A) Impact

  18. Detoxification of Aflatoxin B1 by Saccharomyces cerevisiae Mutants of Anti-Oxidative Relating Genes%酿酒酵母抗氧化相关基因突变体对黄曲霉毒素B1的清除作用

    Institute of Scientific and Technical Information of China (English)

    史锋; 黄宇啸; 李永富

    2012-01-01

    黄曲霉毒素(AF)是粮食作物和饲料原料中容易污染的一种强毒性和强致癌性物质,酿酒酵母具有毒素清除功能.利用HPLC分析了酿酒酵母野生菌BY4742及三株关键的抗氧化相关基因缺失茵zwf1△、sod2△、glr1△对黄曲霉毒素B1的清除能力.结果表明,在PBS缓冲液中存活和死亡的细胞对AFB1的清除率分别为74%~76%和71%~73%,说明酵母细胞对AFB1的清除以生物吸附作用为主.在培养基中,3种突变菌活细胞对AFB1的清除率发生不同程度的降低,其中glr1△的AFB1清除能力下降最明显,其次是sod2△,而zwf1△下降最少,说明这些关键的抗氧化基因的缺失会影响细胞在生长状态下对AFB1的清除作用.%Aflatoxins are a group of mycotoxins with strong mutagenic and carcinogenic properties. Various commodities including crop and feed materials are easy to be contaminated with aflatoxin. Saccharomyces cerevisiae have been reported to bind or degrade aflatoxin. Here, detoxification of aflatoxin B1 (AFB1 ) by wild-type strain of S. cerevisiae (BY4742) and three mutants of anti-oxidative relating genes (zw/l△, sod2△and girl △) were determined by HPLC. In PBS buffer, AFBi binding abilities of viable and dead cells were 74% -76% and 71% — 73%, respectively, indicating AFB1 was removed by yeast cells mainly through cell adsorption. In YPD medium, clearance of AFB1 by three mutant viable cells reduced, while that by wild-type BY4742 remaining high. AFB1 binding ability of g/rlA decreased most seriously, then was that of sod2△ and zwfl△. Thus, the deletion of critical anti-oxidative relating genes would decrease the AFB1 binding ability of S. cerevisiae growing cells.

  19. Phanerochaete mutants with enhanced ligninolytic activity

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-06-01

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

  20. Enhanced cellulase production in mutants of Thermomonospora

    Energy Technology Data Exchange (ETDEWEB)

    Fennington, G.; Lupo, D.; Stutzenberger, F.

    1982-01-01

    Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta, 1-4-glucanase (EG) when grown on cellulose-mineral salts liquid medium. The EG activity (measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differed in their kinetic properties. Exposure of T. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plants containing 2-deoxyglucose or glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures. (Refs. 40).

  1. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

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

  2. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; Clark, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  3. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  4. Aging Kit mutant mice develop cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Lei Ye

    Full Text Available Both bone marrow (BM and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+ cells counts and ii. the stability of left ventricular (LV contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography in two groups of Kit mutant (W/Wv and W41/W42 and in wild type (WT mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF and LV fractional shortening rates (FS were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

  5. Behavioral characterization of system xc- mutant mice.

    Science.gov (United States)

    McCullagh, Elizabeth A; Featherstone, David E

    2014-05-15

    The slc7a11 gene encodes xCT, an essential component of 'system xc-', a plasma membrane exchanger that imports cystine and exports glutamate. Slc7a11 is expressed primarily in the brain, but its role there is not clear. We performed behavioral tests on two different strains of homozygous slc7a11 mutant mice ('sut' and 'xCT'), as well as heteroallelic offspring of these two strains ('xCT/sut') and their associated genetic backgrounds. Homozygous sut mutant males showed reduced spontaneous alternation in spontaneous alternation tasks as well as reduced movement in an open field maze, but xCT and xCT/sut strains did not show significant changes in these tasks compared to appropriate controls. Neither xCT nor sut mutants showed differences from controls in rotarod tests. Female behavioral phenotypes were independent of estrus cycle stage. To ensure that homozygous xCT, sut, and xCT/sut strains all represent protein null alleles, we measured whole brain xCT protein levels using immunoblots. xCT, sut and xCT/sut strains showed no detectable xCT protein expression, confirming them as null alleles. Previously published microdialysis experiments showed reduced striatal glutamate in xCT mutants. Using the same methods, we measured reduced interstitial glutamate levels in the striatum but not cerebellum of sut mutants. However, we detected no glutamate change in the striatum or cerebellum of sut/xCT mice. We detected no changes in whole brain EAAT-1, -2, or -3 expression. We conclude that the behavioral and chemical differences exist between slc7a11 mutant strains, but we were unable to definitively attribute any of these differences to loss of system xc-.

  6. Targeting ESR1-Mutant Breast Cancer

    Science.gov (United States)

    2015-09-01

    cell line, biochemical and structural biology techniques to uncover the best candidate drugs for the clinical targeting of these mutants. Targeting...ESR1-­‐Mutant  Breast  Cancer   W81XWH-­‐14-­‐1-­‐0360   4   2. Keywords Estrogen Receptor Acquired Drug Resistance Metastatic Breast...preparations for publication: 1) “ESR1 Somatic Mutations Y537S and D538G Confer Breast Cancer Endocrine Resistance by Stabilizing the Active AF-2 conformation

  7. Rapid Antibiotic Resistance Evolution of GASP Mutants

    Science.gov (United States)

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

    2012-02-01

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

  8. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    deleterious effects on living organisms (Shigaeva et al.,. 1994); they are also ... It was cultured at 25±0.5°C with a fluorescent light intensity of approximately 6 ... mutants) resulted in three new colonies characterized by different green colors ...

  9. A dominant semi dwarf mutant in rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ In the winter of 1997, a semi dwarf mutant was found in the F6 population of M9056/ R8018 xuan in Hainan Province. In the spring of 1998, the seeds were sown in Hefei, Anhui Province and the plant height of the population was measured at maturity.

  10. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    Science.gov (United States)

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  11. Mutant PTEN in Cancer : Worse Than Nothing

    NARCIS (Netherlands)

    Leslie, Nick R; den Hertog, Jeroen

    2014-01-01

    Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant

  12. Colored HOMFLY polynomials can distinguish mutant knots

    CERN Document Server

    Nawata, Satoshi; Singh, Vivek Kumar

    2015-01-01

    We illustrate from the viewpoint of braiding operations on WZNW conformal blocks how colored HOMFLY polynomials with multiplicity structure can detect mutations. As an example, we explicitly evaluate the (2,1)-colored HOMFLY polynomials that distinguish a famous mutant pair, Kinoshita-Terasaka and Conway knot.

  13. GAMPMS: Genetic algorithm managed peptide mutant screening.

    Science.gov (United States)

    Long, Thomas; McDougal, Owen M; Andersen, Tim

    2015-06-30

    The prominence of endogenous peptide ligands targeted to receptors makes peptides with the desired binding activity good molecular scaffolds for drug development. Minor modifications to a peptide's primary sequence can significantly alter its binding properties with a receptor, and screening collections of peptide mutants is a useful technique for probing the receptor-ligand binding domain. Unfortunately, the combinatorial growth of such collections can limit the number of mutations which can be explored using structure-based molecular docking techniques. Genetic algorithm managed peptide mutant screening (GAMPMS) uses a genetic algorithm to conduct a heuristic search of the peptide's mutation space for peptides with optimal binding activity, significantly reducing the computational requirements of the virtual screening. The GAMPMS procedure was implemented and used to explore the binding domain of the nicotinic acetylcholine receptor (nAChR) α3β2-isoform with a library of 64,000 α-conotoxin (α-CTx) MII peptide mutants. To assess GAMPMS's performance, it was compared with a virtual screening procedure that used AutoDock to predict the binding affinity of each of the α-CTx MII peptide mutants with the α3β2-nAChR. The GAMPMS implementation performed AutoDock simulations for as few as 1140 of the 64,000 α-CTx MII peptide mutants and could consistently identify a set of 10 peptides with an aggregated binding energy that was at least 98% of the aggregated binding energy of the 10 top peptides from the exhaustive AutoDock screening.

  14. Phanerochaete mutants with enhanced ligninolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Kakar, S.N.; Perez, A.; Gonzales, J. (Argonne National Lab., IL (United States))

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. 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, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated, or are hyperproducers or supersectors of key enzymes under enriched conditions. Through UV-light and [gamma]-ray mutagenesis, the authors 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. 17 refs., 1 fig., 3 tabs.

  15. Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

    Science.gov (United States)

    Fedorenko, Inna V; Fang, Bin; Koomen, John M; Gibney, Geoffrey T; Smalley, Keiran S M

    2014-10-01

    Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

  16. Characterization of a Legionella micdadei mip mutant

    DEFF Research Database (Denmark)

    O'Connell, W A; Bangsborg, Jette Marie; Cianciotto, N P

    1995-01-01

    The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human m...... into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei....

  17. Some Mutant Forms of Quantum Mechanics

    CERN Document Server

    Takeuchi, Tatsu; Lewis, Zachary; Minic, Djordje

    2013-01-01

    We construct a `mutant' form of quantum mechanics on a vector space over the finite Galois field GF(q). We find that the correlations in our model do not violate the Clauser-Horne-Shimony-Holt (CHSH) version of Bell's inequality, despite the fact that the predictions of this discretized quantum mechanics cannot be reproduced with any hidden variable theory. An alternative `mutation' is also suggested.

  18. Spontaneous Nif- mutants of Rhodopseudomonas capsulata.

    OpenAIRE

    Wall, J D; Love, J.; Quinn, S P

    1984-01-01

    Revertible, spontaneous Nif- mutants of Rhodopseudomonas capsulata have been shown to accumulate in cultures growing photosynthetically with an amino acid as the nitrogen source such that H2 is maximally produced. The majority of such strains carry mutations which are clustered in a short region of the chromosome, probably representing one or two genes. Because this cluster includes temperature-sensitive mutations, it is also likely that it identifies the structural gene of a polypeptide. The...

  19. Mutant chaperonin proteins: new tools for nanotechnology

    Energy Technology Data Exchange (ETDEWEB)

    Li, Y [SETI Institute, 515 N Whisman Road, Mountain View, CA 94043 (United States); Paavola, C D [NASA Ames Research Center, Bioengineering Branch, Mail Stop 239-15, Moffett Field, CA 94035 (United States); Kagawa, H [SETI Institute, 515 N Whisman Road, Mountain View, CA 94043 (United States); Chan, S L [SETI Institute, 515 N Whisman Road, Mountain View, CA 94043 (United States); Trent, J D [NASA Ames Research Center, Bioengineering Branch, Mail Stop 239-15, Moffett Field, CA 94035 (United States)

    2007-11-14

    Much effort has gone into finding peptides that bind potentially useful nanoparticles, but relatively little effort has focused on the scaffolds that organize these peptides into useful nanostructures. Chaperonins are protein complexes with 14-18 protein subunits that self-assemble into double-ring complexes and function as scaffolds for peptides or amino acids that bind metallic and semiconductor quantum dots. The utility of chaperonins as scaffolds depends on their structure and their ability to self-assemble into double-rings and higher-order structures, such as filaments and two-dimensional arrays. To better understand the structure of chaperonins, we constructed a model of a group II chaperonin and, based on this model, genetically constructed five mutant subunits with significant deletions. We expressed these mutants as recombinant proteins and observed by native polyacrylamide gel electrophoresis (PAGE) and transmission electron microscopy (TEM) that they all self-assembled into double rings. Our model predicted and TEM confirmed that these deletions did not significantly change the 17 nm diameter of the wild-type double rings, but decreased their height and opened their central cavities. Four of the five mutants formed higher-order structures: chains of rings, bundles of chains or filaments, and two-dimensional arrays, which we suggest can be useful nanostructures.

  20. Isolation of a novel mutant from Bacillus subtilis natto.

    Science.gov (United States)

    Yoshida, Kazuo

    2006-01-01

    For the construction of strains with full probiotics function in intestines, deoxycholate resistant mutants were isolated from Bacillus subtilis natto. The partial characterization of the mutants was carried out and described.

  1. Inducement and identification of an endosperm mutant in maize

    African Journals Online (AJOL)

    ajl yemi

    2011-11-30

    Nov 30, 2011 ... “super sweet” phenotype were derived from the mutated offspring. ... characteristics and distinguished molecular mechanism to the previous mutants of gene sh2, these three mutant lines are ...... Physical association of starch biosynthetic ... reduced seedling mutant in oilseed rape, Brassica napus, for.

  2. Mutant prevention concentration and mutant selection window for 10 antimicrobial agents against Rhodococcus equi.

    Science.gov (United States)

    Berghaus, Londa J; Giguère, Steeve; Guldbech, Kristen

    2013-10-25

    The objectives of this study were to determine the mutant prevention concentration (MPC), time above the MPC and mutant selection window for 10 antimicrobial agents against Rhodococcus equi and to determine if the combination of a macrolide with rifampin would decrease emergence of resistant mutants. Antimicrobial agents investigated (erythromycin, clarithromycin, azithromycin, rifampin, amikacin, gentamicin, enrofloxacin, vancomycin, imipenem, and doxycycline) were selected based on in vitro activity and frequency of use in foals or people infected with R. equi. Each antimicrobial agent or combination of agents was evaluated against four virulent strains of R. equi. MPC were determined using an agar plate assay. Pharmacodynamic parameters were calculated using published plasma and pulmonary pharmacokinetic variables. There was a significant (Pequi. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Characterisation of cuticular mutants in Arabidopsis thaliana

    OpenAIRE

    Faust, Andrea

    2006-01-01

    Plants are protected by the extracellular cuticle, which is made up of cutin, cutan and waxes. The cutin composition of a variety of plants has been known and models of the biosynthesis of cutin monomers exist but not many enzymes have been identified. It is generally accepted that a defect in the cuticle leads to an organ fusion phenotype. In the model plant A. thaliana many fusion mutants have been identified but the identification of genes involved have not lead to a complete picture of th...

  4. Using of AFLP to evaluate gamma-irradiated amaranth mutants

    Directory of Open Access Journals (Sweden)

    Labajová Mária

    2013-01-01

    Full Text Available To determine which of several gamma-irradiated mutants of amaranth Ficha cultivar and K-433 hybrid are most genetically similar to their non-irradiated control genotypes, we performed amplified fragment length polymorphism (AFLP based analysis. A total of 40 selective primer combinations were used in reported analyses. First analyses of gamma-irradiated amaranth mutant lines were done used the AFLP. In the study, primers with the differentiation ability for all analysed mutant lines are reported. The very specific changes in the mutant lines´ non-coding regions based on AFLP length polymorphism were analysed. Mutant lines of the Ficha cultivar (C15, C26, C27, C82, C236 shared a genetic dissimilarity of 0,11 and their ISSR profiles are more similar to the Ficha than those of K-433 hybrid mutant lines. The K-433 mutant lines (D54, D279, D282 shared genetic dissimilarity of 0,534 but are more distinct to their control plant as a whole, as those of the Ficha mutant lines. Different AFLP fingerprints patters of the mutant lines when compared to the Ficha cultivar and K-433 hybrid AFLP profiles may be a consequence of the complex response of the intergenic space of mutant lines to the gamma-radiance. Although a genetic polymorphism was detected within accessions, the AFLP markers successfully identified all the accessions. The AFLP results are discussed by a combination of biochemical characteristics of mutant lines and their control genotypes.

  5. Distribution of soluble amino acids in maize endosperm mutants

    Directory of Open Access Journals (Sweden)

    Toro Alejandro Alberto

    2003-01-01

    Full Text Available For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L. mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form.

  6. Auxin physiology of the tomato mutant diageotropica

    Science.gov (United States)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  7. Indy mutants: live long and prosper

    Directory of Open Access Journals (Sweden)

    Stewart eFrankel

    2012-02-01

    Full Text Available Indy encodes the fly homologue of a mammalian transporter of di and tricarboxylatecomponents of the Krebs cycle. Reduced expression of fly Indy or two of the C. elegansIndy homologs leads to an increase in life span. Fly and worm tissues that play key roles inintermediary metabolism are also the places where Indy genes are expressed. One of themouse homologs of Indy (mIndy is mainly expressed in the liver. It has been hypothesizedthat decreased INDY activity creates a state similar to caloric restriction (CR. Thishypothesis is supported by the physiological similarities between Indy mutant flies on highcalorie food and control flies on CR, such as increased physical activity and decreases inweight, egg production, triglyceride levels, starvation resistance, and insulin signaling. Inaddition, Indy mutant flies undergo changes in mitochondrial biogenesis also observed inCR animals. Recent findings with mIndy knockout mice support and extend the findingsfrom flies. mIndy-/- mice display an increase in hepatic mitochondrial biogenesis, lipidoxidation and decreased hepatic lipogenesis. When mIndy-/- mice are fed high calorie foodthey are protected from adiposity and insulin resistance. These findings point to INDY as apotential drug target for the treatment of metabolic syndrome, type 2 diabetes and obesity.

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

    Science.gov (United States)

    McCammon, M T

    1996-09-01

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

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

    Science.gov (United States)

    Neuffer, M G; Sheridan, W F

    1980-08-01

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

  10. Mutants of downy mildew resistance in Lactuca sativa (lettuce).

    Science.gov (United States)

    Okubara, P A; Anderson, P A; Ochoa, O E; Michelmore, R W

    1994-07-01

    As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

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

    Science.gov (United States)

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

    2015-07-06

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

  12. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  13. Plant genetics: increased outcrossing in hothead mutants.

    Science.gov (United States)

    Peng, Peng; Chan, Simon W-L; Shah, Govind A; Jacobsen, Steve E

    2006-09-28

    Arising from: S. J. Lolle, J. L. Victor, J. M. Young & R. E. Pruitt 434, 505-509 (2005); Lolle et al. reply. Lolle et al. report that loss-of-function alleles of the HOTHEAD (HTH) gene in Arabidopsis thaliana are genetically unstable, giving rise to wild-type revertants. On the basis of the reversion of many other genetic markers in hth plants, they suggested a model in which a cache of extragenomic information could cause genes to revert to the genotype of previous generations. In our attempts to reproduce this phenomenon, we discovered that hth mutants show a marked tendency to outcross (unlike wild-type A. thaliana, which is almost exclusively self-fertilizing). Moreover, when hth plants are grown in isolation, their genetic inheritance is completely stable. These results may provide an alternative explanation for the genome wide non-mendelian inheritance reported by Lolle et al.

  14. Google: a narrativa de uma marca mutante

    Directory of Open Access Journals (Sweden)

    Elizete de Azevedo Kreutz

    2010-01-01

    Full Text Available As marcas mutantes já fazem parte de nossa realidade, embora ainda não totalmente percebidas e/ou aceitas como tal. O presente artigo busca refletir sobre a relevância dessas novas estratégias de comunicação e branding, identificando suas principais características. Para isso, utilizamos o método de estudo de caso, o Google, ancorado nos métodos de pesquisa bibliográfica e de internet. A escolha foi intencional, posto que a organização é referência em sua categoria, mecanismo de busca, e reflete essa estratégia comunicacional contemporânea. Como resultado, as informações obtidas nos possibilitam compreender essa tendência de comportamento de marca que busca a interação com seus públicos.

  15. Mutants of Cercospora kikuchii altered in cercosporin synthesis and pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    Upchurch, R.G.; Walker, D.C.; Rollins, J.A.; Ehrenshaft, M.; Daub, M.E. (North Carolina State Univ., Raleigh (United States))

    1991-10-01

    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.

  16. Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

    Science.gov (United States)

    Upchurch, R G; Walker, D C; Rollins, J A; Ehrenshaft, M; Daub, M E

    1991-10-01

    We 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.

  17. Colony mutants of compatible nocardiae displaying variations in recombining capacity.

    Science.gov (United States)

    Brownell, G H; Walsh, R S

    1972-03-01

    Colonial morphology mutants of Nocardia erythropolis were isolated following ultraviolet (UV) irradiation. The alleles rou-1/smo-1 were located by recombinant analysis and found to be linked to previously mapped characters. On the basis of recombinant class type patterns obtained from various selective characters it was postulated that the rou-1 allele may span a region of unique nucleotides in the Mat-Ce genome. Recombination frequencies of rou-1 and smo-2 bearing mutants of the Mat-Ce mating type were found to differ by over 1000 fold. Attempts to demonstrate that low recombination frequencies produced by the Smo mutants were due to Rec(-) genes were unsuccessful. No increased sensitivity to either UV or X irradiation was observed by the Smo mutants. Acriflavine treatment of either Rou or Smo colony mutants failed to accelerate reversion or to alter the recombining potentials of the mutants.

  18. Screening and identification of mutants of Magnaporthe grisea by REMI

    Institute of Scientific and Technical Information of China (English)

    XIONG Ruyi; LIU Juan; ZHOU Yijun; FAN Yongjian; ZHENG Xiaobo

    2007-01-01

    The plasmid pUCATPH was used to establish a transformation system in wild-type isolate M131 of Magnaporthe grisea.Six hundred and thirty-nine transformants were obtained by restriction enzyme-mediated integration (REMI) with hygromycin B (hyg B) resistance as a tag.Morphological analysis of two of the REMI mutants confirmed that they produced little melanin under black light and continued for three generations.Pathogenicity identification of six mutants screened proved that they made pathogenicity changes on three sets of differential varieties with different resistance genes.Rep-PCR analyses showed that two morphological mutants and two pathogenicity mutants differed from wild-type isolate M131 at the molecular level.RFLP analyses were performed to study the four mutants at the molecular level and the integration sites of the plasmid DNA.The results showed that the plasmid was inserted into all four mutants and that the insertion sites were random.

  19. Mutant p53: multiple mechanisms define biologic activity in cancer

    Directory of Open Access Journals (Sweden)

    Michael Paul Kim

    2015-11-01

    Full Text Available The functional importance of p53 as a tumor suppressor gene is evident through its pervasiveness in cancer biology. The p53 gene is the most commonly altered gene in human cancer; however, not all genetic alterations are biologically equivalent. The majority of p53 alterations involve missense mutations that result in the production of mutant p53 proteins. Such mutant p53 proteins lack normal p53 function and may acquire novel functions, often with deleterious effects. Here, we review characterized mechanisms of mutant p53 gain of function in multiple model systems. In addition, we review mutant p53 addiction as emerging evidence suggests that tumors may depend on sustained mutant p53 activity for continued growth. We also discuss the role of p53 in stromal elements and their contribution to tumor initiation and progression. Lastly, current genetic mouse models of mutant p53 are reviewed and their limitations discussed.

  20. Preliminary study on a gravity-insensitive rice mutant

    Institute of Scientific and Technical Information of China (English)

    金静; 朱诚; 张红心; 孙宗修

    2004-01-01

    A gravity-insensitive mutant was isolated from rice (Oryza sativa L. Cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism.Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type

  1. Preliminary study on a gravity-insensitive rice mutant

    Institute of Scientific and Technical Information of China (English)

    金静; 朱诚; 张红心; 孙宗修

    2004-01-01

    A gravity-insensitive mutant was isolated from rice (Oryza sativa L. cv. Zhonghua 11) transformed by Agrobacterium tumefaciens. The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism. Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

  2. [Eremothecium ashbyii mutants resistant to 2,6-diaminopurine].

    Science.gov (United States)

    Stepanov, A I; Beburov, M Iu; Zhdanov, V G

    1975-01-01

    3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

  3. Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

    Science.gov (United States)

    Goodrich-Tanrikulu, M; Stafford, A E; Lin, J T; Makapugay, M I; Fuller, G; McKeon, T A

    1994-10-01

    New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

  4. Characterization of host-range mutants of cyanophage N-1.

    Science.gov (United States)

    Sarma, T A; Kaur, B

    1997-10-01

    Fifteen host-range (h) mutants of cyanophage N-1 were characterized with reference to their efficiency of plating, time of appearance, morphology and size of plaques on Nostoc muscorum and its three phage-resistant (Nm 1/N-1, Nm 2/N-1 and Nm 8/N-1) mutants. While phage N-1 did not adsorb to the three phage-resistant mutants, the h mutants differed one from the other in having lower or higher adsorption rate constants on N. muscorum or the phage-resistant mutants. The inability of majority of h mutants isolated on Nm 1/N-1 to grow in Nm 8/N-1 was shown to be due to a failure of adsorption. The h mutants also differed one from the other in their reversion (back mutation) frequencies. The lethal doses (LD37) required to kill 37% of free phage particles after UV-irradiation, heating and ethylenediamine tetraacetate (EDTA) treatment greatly varied. Most of the h mutants were found to be considerably more sensitive to UV and thermic inactivation than N-1 while they were resistant to EDTA. The h mutants except five of them were unable to multiply at 40 degrees C. The significance of these features is discussed.

  5. plenty, a novel hypernodulation mutant in Lotus japonicus.

    Science.gov (United States)

    Yoshida, Chie; Funayama-Noguchi, Sachiko; Kawaguchi, Masayoshi

    2010-09-01

    Nitrogen fixation in nodules that contain symbiotic rhizobial bacteria enables legumes to thrive in nitrogen-poor soils. However, this symbiosis is energy consuming. Therefore, legumes strictly control nodulation at both local and systemic levels. Mutants deficient in such controls exhibit a range of phenotypes from non-nodulation to hypernodulation. Here, we isolated a novel hypernodulation mutant from the M(2) progeny derived from Lotus japonicus MG-20 seeds mutagenized by irradiation with a carbon ion beam. We named the mutant 'plenty' because it formed more nodules than the wild-type MG-20. The nodulation zone in the plenty mutant was wider than that in the wild type, but not as enhanced as those in other previously reported hypernodulation mutants such as har1, klv or tml of L. japonicus. Unlike these hypernodulation mutants, the plenty mutant developed nodules of the same size as MG-20. Overall, the plenty mutant exhibited a unique phenotype of moderate hypernodulation. However, a biomass assay indicated that this unique pattern of hypernodulation was a hindrance to host plant growth. The plenty mutant displayed some tolerance to external nitrates and a normal triple response to ethylene. Grafting experiments demonstrated that the root of plenty was responsible for its hypernodulation phenotype. Genetic mapping indicated that the PLENTY gene was located on chromosome 2.

  6. Induction and selection of citrus mutant by gamma-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, In Jung; Oh, Seung Kyu; Lee, Hyo Yeon [Jeju National University, Jeju (Korea, Republic of)

    2010-09-15

    We have subjected to gamma-irradiation to citrus buds and then grafted onto mature citrus tree. Mutant citrus branch lines have been induced. As a result of first selection, we found the several mutant lines showing interesting phenotypes such as higher sugar content. We have selected several branches showing good qualities such as higher sweetness and/or lower acidity. Some branch lines showed over 13 .deg. Brix sugar content and below 0.9% acidity. Other mutant branch lines showed the changes of shape, size, peel thickness, and fiber contents or distribution of fruits. The results suggest that gamma-irradiation is an effective tool for induction of citrus mutant lines.

  7. Chemotyping of yeast mutants using robotics.

    Science.gov (United States)

    Rieger, K J; El-Alama, M; Stein, G; Bradshaw, C; Slonimski, P P; Maundrell, K

    1999-07-01

    By now, the EUROFAN programme for the functional analysis of genes from the yeast genome has attained its cruising speed. Indeed, several hundreds of yeast mutants with no phenotype as tested by growth on standard media and no significant sequence similarity to proteins of known function are available through the efforts of various laboratories. Based on the methodology initiated during the pilot project on yeast chromosome III (Yeast 13, 1547-1562, 1997) we adapted it to High Throughput Screening (HTS), using robotics. The first 100 different gene deletions from EUROSCARF, constructed in an FY1679 strain background, were run against a collection of about 300 inhibitors. Many of these inhibitors have not been reported until now to interfere in vivo with growth of Saccharomyces cerevisiae. In the present paper we provide a list of novel growth conditions and a compilation of 49 yeast deletants (from chromosomes II, IV, VII, X, XIV, XV) corresponding to 58% of the analysed genes, with at least one clear and stringent phenotype. The majority of these deletants are sensitive to one or two compounds (monotropic phenotype) while a distinct subclass of deletants displays a hyper-pleiotropic phenotype with sensitivities to a dozen or more compounds. Therefore, chemotyping of unknown genes with a large spectrum of drugs opens new vistas for a more in-depth functional analysis and a more precise definition of molecular targets.

  8. Pharmacological correctors of mutant CFTR mistrafficking

    Directory of Open Access Journals (Sweden)

    Nicoletta ePedemonte

    2012-10-01

    Full Text Available The lack of phenylalanine 508 (∆F508 mutation in the CFTR Cl- channel represents the most frequent cause of cystic fibrosis (CF, a genetic disease affecting multiple organs such lung, pancreas, and liver. ∆F508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g. histone deacetylase inhibitors or a single CFTR-interacting protein. The presence of multiple defects caused at the CFTR protein level by ∆F508 mutation and the redundancy of quality control mechanisms detecting ∆F508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.

  9. New types of Escherichia coli recombination-deficient mutants.

    Science.gov (United States)

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  10. Characterization of Gibberellin Receptor Mutants of Barley (Hordeum vulgare L.)

    Institute of Scientific and Technical Information of China (English)

    Peter M.Chandler; Carol A.Harding; Anthony R.Ashton; Mark D.Mulcair; Nicholas E.Dixon; Lewis N.Mander

    2008-01-01

    The sequence of Gidl (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthoIogue from barley.This was expressed in E.coil,and produced a protein that was able to bind GA in vitro with both structural specificity and saturability.Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gsel mutants).Sixteen different gsel mutants each carried a unique nucleotide substitution in this sequence.In all but one case,these changes resulted in single amino acid substitutions,and,for the remaining mutant,a substitution in the 5' untranslated region of the mRNA is proposed to interfere with translation initiation.There was perfect linkage in segregating populations between new mutant alleles and the gsel phenotype,leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gsel locus.Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA1,and a deficit of C20 GA precursors.All of the gsel mutants had reduced sensitivity to exogenous GA3,and to AC94377 (a GA analogue) at concentrations that are normally 'saturating',but,at much higher concentrations,there was often a considerable response.The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

  11. Mutants of Pseudomonas putida affected in poly-3-hydroxyalkanoate synthesis

    NARCIS (Netherlands)

    Ren, Q; Kessler, B; van der Leij, F; Witholt, B.

    1998-01-01

    The generation and characterization of Pseudomonas putida KT2442 mutants affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. The mutants from P. putida KT2442 carrying several copies of the PHA-polymerase-encoding gene (phaC) were isolated via N-methyl-N'-nitro-N-nitrosoguanidine chemi

  12. A Mutant Hunt Using the C-Fern (Ceratopteris Richardii)

    Science.gov (United States)

    Calie, Patrick J.

    2005-01-01

    A modification of the popular C-Fern system, the tropical fern Ceratopteris richardii is developed in which students plate out a genetically mixed set of fern spores and then select for specific mutants. This exercise can provide students with an experience in plant mutant selection and can be used as a platform to expose students to a diverse…

  13. Characterization of peroxisome-deficient mutants of Hansenula polymorpha

    NARCIS (Netherlands)

    Tan, Xuqiu; Titorenko, Vladimir I.; Klei, Ida J. van der; Sulter, Grietje J.; Haima, Peter; Waterham, Hans R.; Evers, Melchior; Harder, Willem; Veenhuis, Marten; Cregg, James M.

    1995-01-01

    In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut(-)) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of pel mutants, termed Pim(-), are characterized by the presence of

  14. Absence of Pneumocystis dihydropteroate synthase mutants in Brittany, France.

    Science.gov (United States)

    Le Gal, Solène; Robert-Gangneux, Florence; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Damiani, Céline; Totet, Anne; Gangneux, Jean-Pierre; Nevez, Gilles

    2013-05-01

    Archival Pneumocystis jirovecii specimens from 84 patients monitored at Rennes University Hospital (Rennes, France) were assayed at the dihydropteroate synthase (DHPS) locus. No patient was infected with mutants. The results provide additional data showing that P. jirovecii infections involving DHPS mutants do not represent a public health issue in Brittany, western France.

  15. 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.

  16. Phenotypic Characterization of a Female Sterile Mutant in Rice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A female sterile mutant, derived from a spontaneous mutation, wasfirst discovered in rice (Oryza sativa L. ssp.indica) restorer line 202R. With normal flowering, the mutant exhibits an extremely Iow seed-setting rate. When the mutant is crossed as a pollen donor, the seeds set normally; whereas when it is used as a pollen receiver,no seeds are obtained even with mixed pollen grains of different varieties sprinkled over the stigmas. The floret of the mutant, consisting of six stamens and one pistil, looks the same as that of the wild type in the malefemale organs, except that less than 10% of the mutant florets have three stigmas on the ovary. Although the mutant has a low seed-setting rate, Its pollen fertility is approximately 87.1%, which is equal to that of the wild type. In addition, more than 90% of the mature embryo sacs of the mutant have complete inner structures. At every stage after pollination, the sperm, embryo, and endosperm are not found in the mutant embryo sac,whereas the disintegration of the egg cell that does not accomplish fertilization is visible. Through observations with a fluorescence microscope, we have found that the pollen grains germinate normally, whereas the pollen tube abnormally elongates in the style-transmitting tissue. The mutant pollen tubes display various defects in the style, such as slower elongation, conversed elongation, distorted elongation, swollen tips, or branched tips. As a result, the growth of the pollen tubes ceases in the style, and, therefore, the pollen tubes cannot reach the embryo sac and the process of double fertilization is blocked. Based on these observations,we conclude that this mutant, designated as fs-202R, is a novel type of female sterile mutation in rice, which causes the arrest of the elongation of the pollen tube.

  17. Isolation and Characterization of mAMSA-hypersensitive Mutants

    Science.gov (United States)

    Rogojina, Anna T.; Nitiss, John L.

    2008-01-01

    Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro473 to Leu and Gly737 to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly737 → Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro473 → Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro473 → Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro473 → Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs. PMID:18723844

  18. Mapping pathological phenotypes in reelin mutant mice.

    Science.gov (United States)

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

    2014-01-01

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

  19. Ethanol production using nuclear petite yeast mutants

    Energy Technology Data Exchange (ETDEWEB)

    Hutter, A.; Oliver, S.G. [Department of Biomolecular Sciences, UMIST, Manchester (United Kingdom)

    1998-12-31

    Two respiratory-deficient nuclear petites, FY23{Delta}pet191 and FY23{Delta}cox5a, of the yeast Saccharomyces cerevisiae were generated using polymerase-chain-reaction-mediated gene disruption, and their respective ethanol tolerance and productivity assessed and compared to those of the parental grande, FY23WT, and a mitochondrial petite, FY23{rho}{sup 0}. Batch culture studies demonstrated that the parental strain was the most tolerant to exogenously added ethanol with an inhibition constant. K{sub i}, of 2.3% (w/v) and a specific rate of ethanol production, q{sub p}, of 0.90 g ethanol g dry cells{sup -1} h{sup -1}. FY23{rho}{sup 0} was the most sensitive to ethanol, exhibiting a K{sub i} of 1.71% (w/v) and q{sub p} of 0.87 g ethanol g dry cells{sup -1} h{sup -1}. Analyses of the ethanol tolerance of the nuclear petites demonstrate that functional mitochondria are essential for maintaining tolerance to the toxin with the 100% respiratory-deficient nuclear petite, FY23{Delta}pet191, having a K{sub i} of 2.14% (w/v) and the 85% respiratory-deficient FY23{Delta}cox5a, having a K{sub i} of 1.94% (w/v). The retention of ethanol tolerance in the nuclear petites as compared to that of FY23{rho}{sup 0} is mirrored by the ethanol productivities of these nuclear mutants, being respectively 43% and 30% higher than that of the respiratory-sufficient parent strain. This demonstrates that, because of their respiratory deficiency, the nuclear petites are not subject of the Pasteur effect and so exhibit higher rates of fermentation. (orig.)

  20. Mapping pathological phenotypes in Reelin mutant mice

    Directory of Open Access Journals (Sweden)

    Caterina eMichetti

    2014-09-01

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

  1. Mutant prevention concentrations of pradofloxacin for susceptible and mutant strains of Escherichia coli with reduced fluoroquinolone susceptibility.

    Science.gov (United States)

    Marcusson, Linda L; Komp Lindgren, Patricia; Olofsson, Sara K; Hughes, Diarmaid; Cars, Otto

    2014-10-01

    Pharmacodynamic and mutant prevention properties of the fluoroquinolone pradofloxacin (PRA) were measured against a set of 17 Escherichia coli strains carrying no, one or two known mutations conferring reduced fluoroquinolone susceptibility. The strains included susceptible wild-types, isogenic constructed mutants, isogenic selected mutants and clinical isolates. The effectiveness of PRA was determined with regard to preventing the selection of resistant mutants, using static and changing concentrations of drug. Ciprofloxacin was used as a reference drug. Minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of PRA for the susceptible wild-type strains were in the range 0.012-0.016mg/L and 0.2-0.3mg/L, respectively, giving a mean±standard deviation mutant prevention index (MPI=MPC/MIC) of 17.7±1.1. The mean MPI PRA of the 14 mutant strains was 19.2±12, and the mean MPI across all 17 strains was 18.9±10.8. In an in vitro kinetic model in which PRA was diluted with a half-life of 7h to mimic in vivo conditions, an initial concentration of PRA of 1.6-2.4mg/L (8-10× MPC), giving a PRA AUC/MPC ratio of 73-92, and a T>MPC of 21-23h was sufficient to prevent the selection of resistant mutants from the three susceptible wild-type strains. Dosing to reduce selection for antibiotic resistance in veterinary therapy has a role in reducing the reservoir of resistant mutants. We conclude that a level of dosing that prevents the selection of resistant mutants during therapy should be achievable in vivo. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  2. Effect of different immunosuppressive drugs on calcineurin and its mutants

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis.Their phosphatase activity and the corresponding solution conformation were examined.Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immunosuppressive drugs with CN.The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN.Furthermore,circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein.Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN,and had no effects on the phosphatase activity of mutants in Loop7 region,which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN.Examination of the activities of these mutants resulted in the presence of immunosuppressive component from traditional Chinese drugs.The component of Chinese drug,ZIP1,could directly inhibit both CN and CN mutants without drug binding protein.These results suggest that the Loop7 region is an important structural area involved in the inhibition by CyP-CsA.It is valuable to further study the inhibition by ZIP1.

  3. Methods of producing protoporphyrin IX and bacterial mutants therefor

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Epigenetic Suppression of T-DNA Insertion Mutants in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yangbin Gao; Yunde Zhao

    2013-01-01

    T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants.Here,we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis.When the two T-DNA insertion mutants,yucl-1 and ag-TD,were crossed together,the defects in all of the ag-TD plants in the F2 population were partially suppressed regardless of the presence of yucl-1.Conversion of ag-TD to the suppressed ag-TD (named as ag-TD*) did not follow the laws of Mendelian genetics.The ag-TD* could be stably transmitted for many generations without reverting to ag-TD,and ag-TD* had the capacity to convert ag-TD to ag-TD*.We show that epigenetic suppression of T-DNA mutants is not a rare event,but certain structural features in the T-DNA mutants are needed in order for the suppression to take place.The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences.We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two ToDNA insertions.This work shows that caution is needed when intronic T-DNA mutants are used.

  5. Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Lalonde, R; Strazielle, C

    2011-04-15

    Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage.

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis. To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC...... 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...

  7. Mutant γPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant's cytotoxicity.

    Science.gov (United States)

    Ogawa, Kota; Seki, Takahiro; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2013-10-11

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is misfolded, susceptible to aggregation and cytotoxic. Molecular chaperones assist the refolding and degradation of misfolded proteins and prevention of the proteins' aggregation. In the present study, we found that the expression of mutant γPKC-GFP increased the levels of heat-shock protein 70 (Hsp70) in SH-SY5Y cells. To elucidate the role of this elevation, we investigated the effect of siRNA-mediated knockdown of Hsp70 on the aggregation and cytotoxicity of mutant γPKC. Knockdown of Hsp70 exacerbated the aggregation and cytotoxicity of mutant γPKC-GFP by inhibiting this mutant's degradation. These findings suggest that mutant γPKC increases the level of Hsp70, which protects cells from the mutant's cytotoxicity by enhancing its degradation.

  8. Selection of mutants of capsicum annuum induced by gamma ray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Y. I.; Lee, Y. B. [Korea Atomic Energy Research Institute, Taejeon (Korea, Republic of); Lee, E. K. [Chungnam National Univ., Taejeon (Korea, Republic of)

    1998-06-01

    For induction and selection of mutations of Capsicum annuum L., dry seeds of pure lines No.1 and No.2 were irradiated with gamma ray of 150Gy, 200Gy and 250Gy. Various mutants were selected such as showing early maturity, short plant height, long fruit and chlorophyll mutations. Mutation frequency of No.1 line was 3.4% in the dose of 150Gy, while the frequency of No.2 line was 2.7% in the dose of 250Gy. For selection of resistant mutant to amino acid analog, the optimum concentration of 5-methyltryptophan (5-MT) and S-(2-aminoethyl)-L-cysteine were 25 ppm and 30 ppm, respectively. Four resistant mutant lines to 5-MT were selected among 400 mutant lines.

  9. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne; Hove-Jensen, Bjarne; Garber, Bruce B.;

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosine......-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5...... phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5...

  10. Generation of Peroxisome-Deficient Somatic Animal Cell Mutants.

    Science.gov (United States)

    Okumoto, Kanji; Fujiki, Yukio

    2017-01-01

    Cell mutants with a genetic defect affecting various cellular phenotypes are widely utilized as a powerful tool in genetic, biochemical, and cell biological research. More than a dozen complementation groups of animal somatic mutant cells defective in peroxisome biogenesis have been successfully isolated in Chinese hamster ovary (CHO) cells and used as a model system reflecting fatal human severe genetic disorders named peroxisome biogenesis disorders (PBD). Isolation and characterization of peroxisome-deficient CHO cell mutants has allowed the identification of PEX genes and the gene products peroxins, which directly leads to the accomplishment of isolation of pathogenic genes responsible for human PBDs, as well as elucidation of their functional roles in peroxisome biogenesis. Here, we describe the procedure to isolate peroxisome-deficient mammalian cell mutants from CHO cells, by making use of an effective, photo-sensitized selection method.

  11. Status and Perspectives on the Researches of Rice Glutelin Mutants

    Institute of Scientific and Technical Information of China (English)

    ZHENG Tian-qing; SHEN Wen-biao; ZHU Su-song; ZHAI Hu-qu; WAN Jian-min

    2003-01-01

    Rice (Oryza sativa L. ) is one of the model plants for genomics research. As the raising offunctional rice breeding for special usage, glutelin mutants play a more and more important role in the func-tional rice breeding as well as eukaryotic gene expression and regulation research materials. For example, therice cultivar special for the patients suffering from kidney disease and diabetes could be developed from the riceglutelin mutants. In this paper, current researches on characterization, mutation mechanism and breeding us-age of various rice glutelin mutants, especially the low glutelin content cultivars, were all discussed with per-spectives on the trends of the glutelin mutant researches in the era of post-genomics.

  12. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  13. Assessment of Genetic diversity in mutant cowpea lines using ...

    African Journals Online (AJOL)

    FKOLADE

    2016-11-09

    Nov 9, 2016 ... option of NTSYS, a rooted tree was also generated from the .... Dellarporta SF, Wood J, Hicks JB (1983). ... genetic diversity in Pigeon Pea (Cajanus sp). ... diversity in somatic mutants of grape (Vitis vinifera) cultivar Italia.

  14. Analysis of the aspartic acid metabolic pathway using mutant genes.

    Science.gov (United States)

    Azevedo, R A

    2002-01-01

    Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

  15. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    . Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate......, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  16. Characterization of Glutamine-Requiring Mutants of Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Joosten, Han M.L.J.; Herst, Patricia M.; Drift, Chris van der

    1982-01-01

    Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthetase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation conferri

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

    Science.gov (United States)

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

    2014-02-01

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

  18. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    Science.gov (United States)

    2014-08-01

    regulation of synaptic and neuronal functions.17 A point mutation in DHHC21 was identified in the depilated (dep) mouse mutant, resulting in hair follicle ...and hair follicle differentiation. PLoS Genet. 5, e1000748. (19) Mansilla, F., Birkenkamp-Demtroder, K., Kruhoffer, M., Sorensen, F. B., Andersen, C...AWARD NUMBER: W81XWH-13-1-0203 TITLE: Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma PRINCIPAL INVESTIGATOR: Xu Wu

  19. A relaxed mutant with an altered ribosomal protein L11.

    Science.gov (United States)

    Parker, J; Watson, R J; Friesen, J D

    1976-02-27

    Relaxed mutants of Escherichia coli have been isolated which have an altered electrophoretic mobility of ribosomal protein L11. It can be shown that reversion to stringency in one of these mutants occurs simultaneously with a reversion of L11 protein to tis normal mobility. The L11 structural gene, rplK, maping near rif, is carried by the bacteriophage lambdacI857S7drifd18, and is most likely identical with relC.

  20. Multiple defects in Escherichia coli mutants lacking HU protein.

    OpenAIRE

    Huisman, O; Faelen, M; Girard, D; Jaffé, A; Toussaint, A; Rouvière-Yaniv, J

    1989-01-01

    The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize,...

  1. 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.

  2. 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.

  3. Molecular analysis of mutants of the Neurospora adenylosuccinate synthetase locus

    Indian Academy of Sciences (India)

    A. Wiest; A. J. McCarthy; R. Schnittker; K. McCluskey

    2012-08-01

    The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5′ end of the gene and no mutants were identified in the 3′ untranslated region of this gene. We additionally analysed ad-8+ alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.

  4. Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities.

    Science.gov (United States)

    Koduru, P R; Rao, M K

    1980-05-01

    Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these - 'white', 'white tipped green', 'patchy white', 'white virescent', 'white striping 1', 'white striping 2', 'white striping 4', 'fine striping', 'chlorina' and 'yellow virescent' showed monogenic recessive inheritance and the remaining three - 'yellow striping', 'yellow green' and 'light green' seedling phenotypes showed digenic recessive inheritance. The genes for (i) 'white tipped green' (wr) and 'yellow virescent' (yv) and (ii) 'patchy white' (pw) and 'white striping 1' (wst 1) showed independent assortment. Further, the genes for 'white' (w), 'white tipped green' (wr) and 'yellow virescent' (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants - 'white tipped green', 'patchy white', 'white striping 1', 'white striping 2', 'fine striping', 'chlorina', 'yellow virescent', 'yellow striping', 'yellow green' and 'light green' phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in 'white virescent' there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F1's (mutant x control green). In F1's of six of the mutants - 'white tip', 'patchy white', 'chlorina', 'yellow virescent', 'fine striping' and 'yellow striping' the quantity of chlorophyll was almost equal to the wild type. In the F1's of three of the mutants - 'white striping 1', 'white striping 2' and 'light green' an intermediate value between the mutant and wild types was observed. In 'yellow virescent' retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants - 'white tipped green, 'white virescent', 'fine striping', 'chlorina', 'yellow striping', 'yellow green' and 'light green' defective

  5. Isolation and characterization of selenate resistant mutants of Acremonium chrysogenum

    Directory of Open Access Journals (Sweden)

    Airton Vialta

    1999-01-01

    Full Text Available Mutants unable to convert exogenous sulfate to sulfite were isolated using the toxic analogue selenate. Three of twenty-eight isolated mutants were chromate sensitive. They showed a possible lesion in the gene that codes the ATP sulfurylase. The others were chromate resistant, and probably had a lesion in one or both of the genes that code the sulfate permease. Methionine increased the resistance levels to selenate. In addition, the frequency of spontaneous mutants obtained in a medium containing methionine was higher (between 2.4 x 10-6 and 18.0 x 10-6 than that obtained using a medium without any intentional source of sulfur (between 0.7 x 10-6 and 5.0 x 10-6. The original strain, as well as the mutants, were able to grow in a sulfur-free liquid medium even after 4 consecutive inoculation procedures. These results indicated the existence of sulfur traces in the medium and/or an efficient intracellular storage system. There was no significant difference between cephalosporin C production in mutants and the original strain.Mutantes incapazes de converter o sulfato extracelular em sulfito foram isolados utilizando o análogo tóxico selenato. De 28 mutantes isolados, apenas 3 foram sensíveis ao cromato, provavelmente apresentando lesão no gene que codifica a ATP sulfurilase. Os demais foram resistentes ao cromato e devem conter lesão no gene sB ou também no gene sC. A metionina elevou os níveis de resistência ao selenato e a freqüência de mutantes espontâneos obtida em meio contendo este aminoácido foi maior (entre 2,42 x 10-6 e 18,04 x 10-6 do que a obtida no meio sem a adição de qualquer fonte intencional de enxofre (entre 0,71 x 10-6 e 5,0 x 10-6. A linhagem original e os mutantes foram capazes de crescer, mesmo depois de quatro etapas de inóculo, fato que pode ser explicado pela existência de traços do referido elemento no meio e/ou a presença de um sistema eficiente de estocagem intracelular. A produção de cefalosporina C

  6. Isolation of Escherichia coli mutants defective in uptake of molybdate.

    Science.gov (United States)

    Hemschemeier, S; Grund, M; Keuntje, B; Eichenlaub, R

    1991-10-01

    For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.

  7. 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.

  8. Auditory development in progressive motor neuronopathy mouse mutants.

    Science.gov (United States)

    Volkenstein, Stefan; Brors, Dominik; Hansen, Stefan; Berend, Achim; Mlynski, Robert; Aletsee, Christoph; Dazert, Stefan

    2009-11-06

    The present study was performed to elucidate the hearing development in the progressive motor neuronopathy (pmn) mouse mutant. This mouse has been used as a model for human motoneuron disease. A missense mutation in the tubulin-specific chaperon E (Tbce) gene on mouse chromosome 13 was localized as the underlying genetic defect. The protein encoded by the Tbce gene is essential for the formation of primary tubulin complexes. Studies on motoneurons show disorganization in microtubules and disturbed axonal transport, followed by retrograde degeneration of the motoneurons. A similar pathomechanism is also possible for hearing disorders where disrupted microtubules could cause functional deficits in spiral ganglion neurons or in cochlear hair cells. Click auditory brainstem response (ABR) audiometry in homozygous pmn mutants showed a normal onset of hearing, but an increasing hearing threshold from postnatal day 26 (P26) on to death, compared to heterozygous mutants and wild-type mice. Histological sections of the cochlea at different ages showed a regular morphology. Additionally, spiral ganglion explants from mutant and wild-type mice were cultured. The neurite length from pmn mutants was shorter than in wild-type mice, and the neurite number/explant was significantly decreased in pmn mutants. We show that the pmn mouse mutant is a model for a progressive rapid hearing loss from P26 on, after initially normal hearing development. Heterozygous mice are not affected by this defect. With the knowledge of the well-known pathomechanism of this defect in motoneurons, a dysfunction of cellular mechanisms regulating tubulin assembling suggests that tubulin assembling plays an essential role in hearing function and maintenance.

  9. EMMA—mouse mutant resources for the international scientific community

    Science.gov (United States)

    Wilkinson, Phil; Sengerova, Jitka; Matteoni, Raffaele; Chen, Chao-Kung; Soulat, Gaetan; Ureta-Vidal, Abel; Fessele, Sabine; Hagn, Michael; Massimi, Marzia; Pickford, Karen; Butler, Richard H.; Marschall, Susan; Mallon, Ann-Marie; Pickard, Amanda; Raspa, Marcello; Scavizzi, Ferdinando; Fray, Martin; Larrigaldie, Vanessa; Leyritz, Johan; Birney, Ewan; Tocchini-Valentini, Glauco P.; Brown, Steve; Herault, Yann; Montoliu, Lluis; de Angelis, Martin Hrabé; Smedley, Damian

    2010-01-01

    The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org. PMID:19783817

  10. Effect of different immunosuppressive drugs on calcineurin and its mutants

    Institute of Scientific and Technical Information of China (English)

    阎力君; 于翠娟; 张丽芳; 魏群

    2000-01-01

    Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these

  11. Normal aging modulates the neurotoxicity of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Elsa Diguet

    Full Text Available Aging likely plays a role in neurodegenerative disorders. In Huntington's disease (HD, a disorder caused by an abnormal expansion of a polyglutamine tract in the protein huntingtin (Htt, the role of aging is unclear. For a given tract length, the probability of disease onset increases with age. There are mainly two hypotheses that could explain adult onset in HD: Either mutant Htt progressively produces cumulative defects over time or "normal" aging renders neurons more vulnerable to mutant Htt toxicity. In the present study, we directly explored whether aging affected the toxicity of mutant Htt in vivo. We studied the impact of aging on the effects produced by overexpression of an N-terminal fragment of mutant Htt, of wild-type Htt or of a beta-Galactosidase (beta-Gal reporter gene in the rat striatum. Stereotaxic injections of lentiviral vectors were performed simultaneously in young (3 week and old (15 month rats. Histological evaluation at different time points after infection demonstrated that the expression of mutant Htt led to pathological changes that were more severe in old rats, including an increase in the number of small Htt-containing aggregates in the neuropil, a greater loss of DARPP-32 immunoreactivity and striatal neurons as assessed by unbiased stereological counts.The present results support the hypothesis that "normal" aging is involved in HD pathogenesis, and suggest that age-related cellular defects might constitute potential therapeutic targets for HD.

  12. Mutant alpha-synuclein and autophagy in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Kangyong Liu; Chunfeng Liu; Chuancheng Ren; Yaping Yang; Liwei Shen; Xuezhong Li; Fen Wang; Zhenghong Qin

    2011-01-01

    Several studies have demonstrated that overexpression of mutant α-synuclein in PC12 cells is related to occurrence of autophagy.The present study established mutant a-synuclein (A30P)-transfected PC12 cells and treated them with the autophagy inducer rapamycin and autophagy inhibitor wortmannin, respectively.Results demonstrated that mutant o-synuclein resulted in cell death via autophagy and involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity.Mutant α-synuclein (A30P) also mediated toxicity of1-methyl-4-phenylpyridinium ion.Moreover, rapamycin inhibited a-synuclein aggregation, while wortmannin promoted o-synuclein aggregation and cell death.To further determine the role of autophagy due to mutant a-synuclein, the present study measured expression of microtubule-associated protein light chain 3.Results revealed that wortmannin and 1-methyl-4-phenylpyridinium ion inhibited expression of microtubule-associated protein light chain 3,while rapamycin promoted its expression.These findings suggested that abnormal aggregation of a-synuclein induced autophagic programmed cell death in PC12 cells.

  13. Cytokinin production by plant growth promoting rhizobacteria and selected mutants.

    Science.gov (United States)

    García de Salamone, I E; Hynes, R K; Nelson, L M

    2001-05-01

    One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.

  14. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    Science.gov (United States)

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  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. Hepatitis B escape mutants in Scottish blood donors.

    Science.gov (United States)

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

    2013-06-01

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

  17. Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes.

    Science.gov (United States)

    Majtan, Tomas; Liu, Lu; Carpenter, John F; Kraus, Jan P

    2010-05-21

    Missense mutations represent the most common cause of many genetic diseases including cystathionine beta-synthase (CBS) deficiency. Many of these mutations result in misfolded proteins, which lack biological function. The presence of chemical chaperones can sometimes alleviate or even restore protein folding and activity of mutant proteins. We present the purification and characterization of eight CBS mutants expressed in the presence of chemical chaperones such as ethanol, dimethyl sulfoxide, or trimethylamine-N-oxide. Preliminary screening in Escherichia coli crude extracts showed that their presence during protein expression had a significant impact on the amount of recovered CBS protein, formation of tetramers, and catalytic activity. Subsequently, we purified eight CBS mutants to homogeneity (P49L, P78R, A114V, R125Q, E176K, P422L, I435T, and S466L). The tetrameric mutant enzymes fully saturated with heme had the same or higher specific activities than wild type CBS. Thermal stability measurements demonstrated that the purified mutants are equally or more thermostable than wild type CBS. The response to S-adenosyl-L-methionine stimulation or thermal activation varied. The lack of response of R125Q and E176K to both stimuli indicated that their specific conformations were unable to reach the activated state. Increased levels of molecular chaperones in crude extracts, particularly DnaJ, indicated a rather indirect effect of the chemical chaperones on folding of CBS mutants. In conclusion, the chemical chaperones present in the expression medium were able to fully restore the activity of eight CBS mutants by improving their protein folding. This finding could have direct implications for the development of a therapeutical approach to pyridoxine unresponsive homocystinuria.

  18. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M;

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...... sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could...

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

    Science.gov (United States)

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

    2016-07-01

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

  20. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.

    2003-01-01

    or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...... of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...

  1. Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

    Science.gov (United States)

    Liu, Wan-Chun; Kohn, Jessica; Szwed, Sarah K; Pariser, Eben; Sepe, Sharon; Haripal, Bhagwattie; Oshimori, Naoki; Marsala, Martin; Miyanohara, Atsushi; Lee, Ramee

    2015-11-01

    Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

  2. The use of mutants to probe models of gravitropism.

    Science.gov (United States)

    Firn, R D; Wagstaff, C; Digby, J

    2000-08-01

    It has been widely believed for more than 70 years that auxin plays a central role in the induction of differential growth which causes gravitropic curvature. However, this long-standing consensus about a role for auxin in gravitropism has only been achieved by allowing several mutually exclusive models to coexist. Furthermore, because there is no detailed model which is unchallenged by evidence, consensus is now centred on ill-defined models which have a low predictive value, hence are harder to challenge experimentally. An increasing number of mutants with abnormal gravitropic behaviour are becoming available. Such mutants should be very helpful in challenging existing models of gravitropism and in providing new evidence on which to build improved, more precise models. However, to date, most studies of mutants with abnormal gravitropism have been guided, experimentally and conceptually, by the old inadequate and vague models. Consequently, the full potential of modern molecular analysis in aiding our understanding of gravitropism has yet to be realized.

  3. Candida albicans mutant construction and characterization of selected virulence determinants.

    Science.gov (United States)

    Motaung, T E; Albertyn, J; Pohl, C H; Köhler, Gerwald

    2015-08-01

    Candida albicans is a diploid, polymorphic yeast, associated with humans, where it mostly causes no harm. However, under certain conditions it can cause infections ranging from superficial to life threatening. This ability to become pathogenic is often linked to the immune status of the host as well as the expression of certain virulence factors by the yeast. Due to the importance of C. albicans as a pathogen, determination of the molecular mechanisms that allow this yeast to cause disease is important. These studies rely on the ability of researchers to create deletion mutants of specific genes in order to study their function. This article provides a critical review of the important techniques used to create deletion mutants in C. albicans and highlights how these deletion mutants can be used to determine the role of genes in the expression of virulence factors in vitro.

  4. How does the NPM1 mutant induce leukemia?

    Directory of Open Access Journals (Sweden)

    Paolo Sportoletti

    2011-06-01

    Full Text Available NPM1 is the most frequently mutated gene in AML and the role of the NPM1 mutant in acute myeloid leukemia along with its leukemogenic potential are still under investigation. NPM1 genetic alterations can contribute to leukemogenesis through the direct oncogenic effect of the mutant protein and the concomitant loss of one functional allele. Npm1 loss determines tumor development in the mouse while in human NPM1 maps in a chromosomal region frequently loss in myelodysplastic syndrome (MDS. The NPM1 mutant cytoplasmic delocalization in leukemic blasts alters multiple cellular pathways through either loss or gain of function effects on different protein partners. Here we discuss the most relevant studies on the role of the NPM1 molecule in hematological malignancies and both in vitro and in vivo studies that are trying to elucidate the way by which the NPM1 mutation induces leukemia.

  5. Antisense downregulation of mutant huntingtin in a cell model

    DEFF Research Database (Denmark)

    Hasholt, L.; Abell, K.; Norremolle, A.;

    2003-01-01

    of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique. Methods NT2 precursor cells and differentiated postmitotic NT2-N neurons, respectively, were...... transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS-ASHD/20+) or a control oligonucleotide either by cotransfection...... or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...

  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. Isolation of a Defective Prion Mutant from Natural Scrapie

    Science.gov (United States)

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

    2016-01-01

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

  8. Callus cultures of tomato mutants: I. Nutritional requirements.

    Science.gov (United States)

    Ulrich, J M; Mackinney, G

    1969-01-01

    Callus from hypocotyl, stem, and fruit tissue of tomato mutants was grown on a complex pea extract medium. The genotypes responded differently to the levels of nutrients and stimulators or inhibitors in the medium. Hypocotyl callus of yellow (r) tomato required K(2) SO(4) for quick establishment and continued steady growth for several months; callus of this mutant could also grow with 0.5 % dimethyl sulfoxide in the medium, although growth was less than the control. The red ghost (r(+) gh) mutant is sensitive to a toxic component in the pea extract, and makes its best growth with the standard minerals and vitamins, but in 1/2 concentration pea extract plus 5 % coconut water. Tangerine (t), red lutescent stem (r(+) l(2) ), and r(+) gh are mutants which respond differently to thiourea: t grows about the same at all concentrations, r(+) gh grows best at low thiourea, and r(+) l(2) grows best at the specific level of 20 mg/l thiourea. The recent active t or r(+) l(1) and r(+) l(2) isolates require supplementary auxin to which the older, slow-growing isolates do not respond. However, there is variation in growth response of different isolates of the same mutant. The several red (r(+) ) cultures are similar in their slow growth, but somewhat different in responses to specific nutrients. The recent (+) isolate is one of the most active cultures, in comparison to the slow growth of t callus isolated in 1964. It is therefore concluded that growth is affected both by the specific requirements of the mutant and by the age and vigor of isolates.

  9. Modeling of gap gene expression in Drosophila Kruppel mutants.

    Directory of Open Access Journals (Sweden)

    Konstantin Kozlov

    Full Text Available The segmentation gene network in Drosophila embryo solves the fundamental problem of embryonic patterning: how to establish a periodic pattern of gene expression, which determines both the positions and the identities of body segments. The gap gene network constitutes the first zygotic regulatory tier in this process. Here we have applied the systems-level approach to investigate the regulatory effect of gap gene Kruppel (Kr on segmentation gene expression. We acquired a large dataset on the expression of gap genes in Kr null mutants and demonstrated that the expression levels of these genes are significantly reduced in the second half of cycle 14A. To explain this novel biological result we applied the gene circuit method which extracts regulatory information from spatial gene expression data. Previous attempts to use this formalism to correctly and quantitatively reproduce gap gene expression in mutants for a trunk gap gene failed, therefore here we constructed a revised model and showed that it correctly reproduces the expression patterns of gap genes in Kr null mutants. We found that the remarkable alteration of gap gene expression patterns in Kr mutants can be explained by the dynamic decrease of activating effect of Cad on a target gene and exclusion of Kr gene from the complex network of gap gene interactions, that makes it possible for other interactions, in particular, between hb and gt, to come into effect. The successful modeling of the quantitative aspects of gap gene expression in mutant for the trunk gap gene Kr is a significant achievement of this work. This result also clearly indicates that the oversimplified representation of transcriptional regulation in the previous models is one of the reasons for unsuccessful attempts of mutant simulations.

  10. HBV genotypes prevalence, precore and basal core mutants in Morocco.

    Science.gov (United States)

    Baha, Warda; Ennaji, My Mustapha; Lazar, Fatiha; Melloul, Marouane; El Fahime, Elmostafa; El Malki, Abdelouahad; Bennani, Abdelouaheb

    2012-08-01

    The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. The objective of the current study was to find out the pattern of HBV genotypes circulating in Morocco and to investigate the precore (PC) and basal core promoter (BCP) mutants' status in Moroccan chronic hepatitis B patients. Viral genotypes were determined in 221 chronic carriers using INNO-LiPA HBV assay and hemi-nested PCR. Phylogenetic analysis was performed in 70 samples, and multiplex PCR method was used to confirm some genotyping results. PC and CP mutants were determined using Inno-Lipa. All isolates were successfully genotyped. The genotype distribution was D in 90.45% of cases, A (5.9%), E (1 case), and mixed genotypes (5 A/D and 2 D/F) in 3.17% patients. HBV carried in the HBV/D samples could be assigned to D7 (63.3%), D1 (32.7%) and 2% of strains to each D4 and D5, all HBV/A belonged to A2 subgenotype and HBV/E strain could not be sub-genotyped. In 70 studied strains, HBV mutants were detected in 88.6% of cases; PC mutants were detected in (40%) of patients and 21.5% present a mixture of wild type and G1896A mutation. BCP mutants were observed in 65.7% of cases, 22.9% were found to have the T1762/1764A double mutation, 18.6% had A1762/1764T mutation and 22.9% of patients showed the A1762T/G1764A double mutation with either A1762T/G1764T mutation. Co-infection by PC and BCP mutants was detected in 52.9% of cases. Movement from place to place most likely shapes the observed genotype distribution and consequent prevalence of genotypes other than A2 or D7 in this population. High circulation of PC and BCP mutants is common in chronic hepatitis B infection in Morocco.

  11. Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

    DEFF Research Database (Denmark)

    Fukai, Eigo; Malolepszy, Anna; Sandal, Niels Nørgaard

    2014-01-01

    The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next......-generation sequencing approaches. The LORE1 mutant lines are freely available and can be ordered online. Endogenous retrotransposons are also active in many other plant species. Based on the methods developed for LORE1 mutagenesis, it should be simple to establish similar systems in other species, once an appropriate...

  12. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    Science.gov (United States)

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  13. Repair effects of laser on mutants of filamentous fungi

    Science.gov (United States)

    Zhao, Yansheng; Xiao, Canpeng; Qian, Hailun; Su, Baoliang; Hu, Yujun; Deng, Jianhui

    1999-09-01

    The paper reports that penicillin-producing strains and lovastatin-producing strains were irradiated by UV and subsequently by laser (632.8 nm), and the reparation rate reached 297% and 264%. High-yield mutant was selected with improved potency of 24.5% and 30%, respectively; Gibberellin producing strains were treated with chemical agent LiCl, and then irradiated with 632.8 nm laser. One mutant with 189.6% increased potency was obtained. The experimental results indicated that using laser irradiation after UV or chemical agent mutation was a new useful method in breeding high-yield strains.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, Susan I.

    2009-06-08

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

  15. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization.

    Science.gov (United States)

    Bellemann, P; Geider, K

    1992-05-01

    Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

  16. High-throughput identification of protein mutant stability computed from a double mutant fitness landscape.

    Science.gov (United States)

    Wu, Nicholas C; Olson, C Anders; Sun, Ren

    2016-02-01

    The effect of a mutation on protein stability is traditionally measured by genetic construction, expression, purification, and physical analysis using low-throughput methods. This process is tedious and limits the number of mutants able to be examined in a single study. In contrast, functional fitness effects can be measured in a high-throughput manner by various deep mutational scanning tools. Using protein GB 1, we have recently demonstrated the feasibility of estimating the mutational stability effect ( ΔΔG) of single-substitution based on the functional fitness profile of all double-substitutions. The principle is to identify genetic backgrounds that have an exhausted stability margin. The functional effect of an additional substitution on these genetic backgrounds can then be used to compute the mutational ΔΔG based on the biophysical relationship between functional fitness and thermodynamic stability. However, to identify such genetic backgrounds, the approach described in our previous study required a benchmark dataset, which is a set of known mutational ΔΔG. In this study, a benchmark-independent approach is developed. The genetic backgrounds of interest are identified using k-means clustering with the integration of structural information. We further demonstrated that a reasonable approximation of ΔΔG can also be obtained without taking structural information into account. In summary, this study describes a novel method for computing ΔΔG from double-substitution functional fitness profiles alone, without relying on any known mutational ΔΔG as a benchmark.

  17. Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents

    OpenAIRE

    Rogers, Palmer; Palosaari, Neil

    1987-01-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced n...

  18. Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group 2 mutants.

    NARCIS (Netherlands)

    M. van Duin (Mark); J.H. Janssen; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); L.H. Thompson; D. Bootsma (Dirk); A. Westerveld (Andries)

    1988-01-01

    textabstractThe human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In ord

  19. Modeling dynamics of mutants in heterogeneous stem cell niche

    Science.gov (United States)

    Shahriyari, L.; Mahdipour-Shirayeh, A.

    2017-02-01

    Studying the stem cell (SC) niche architecture is a crucial step for investigating the process of oncogenesis and obtaining an effective stem cell therapy for various cancers. Recently, it has been observed that there are two groups of SCs in the SC niche collaborating with each other to maintain tissue homeostasis: border stem cells (BSCs), which are responsible in controlling the number of non-stem cells as well as stem cells, and central stem cells (CeSCs), which regulate the SC niche. Here, we develop a bi-compartmental stochastic model for the SC niche to study the spread of mutants within the niche. The analytic calculations and numeric simulations, which are in perfect agreement, reveal that in order to delay the spread of mutants in the SC niche, a small but non-zero number of SC proliferations must occur in the CeSC compartment. Moreover, the migration of BSCs to CeSCs delays the spread of mutants. Furthermore, the fixation probability of mutants in the SC niche is independent of types of SC division as long as all SCs do not divide fully asymmetrically. Additionally, the progeny of CeSCs have a much higher chance than the progeny of BSCs to take over the entire niche.

  20. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... Our results hopefully shed light on the further study of molecular mechanism of white ... 2002). The mutant white egg 3 (w-3) has white eyes and eggs with ... As of other insects, the color of the eggs of silkworm mainly depends ...

  1. Early ripening mutants induced by colchicine in rice

    Institute of Scientific and Technical Information of China (English)

    CAIGuohai; YANWanchao; CAOXin

    1993-01-01

    In 1981-1983, the frequency and range of the mutants induced by colchicine were investigated in M2 and M3 of two indica-rice cultivars.Seedlings of M2 and M3 were treated with 0.05% colchicine solution at 4-5 leaf stage.

  2. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    Science.gov (United States)

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia.

  3. Identification of a Gravitropism-Deficient Mutant in Rice

    Directory of Open Access Journals (Sweden)

    He Yan

    2017-03-01

    Full Text Available A gravitropism-deficient mutant M96 was isolated from a mutant bank, generated by ethyl methane sulfonate (EMS mutagenesis of indica rice accession ZJ100. The mutant was characterized as prostrate growth at the beginning of germination, and the prostrate growth phenotype ran through the whole life duration. Tiller angle and tiller number of M96 increased significantly in comparison with the wild type. Tissue section observation analysis indicated that asymmetric stem growth around the second node occurred in M96. Genetic analysis and gene mapping showed that M96 was controlled by a single recessive nuclear gene, tentatively termed as gravitropism-deficient M96 (gdM96, which was mapped to a region of 506 kb flanked by markers RM5960 and InDel8 on the long arm of chromosome 11. Sequencing analysis of the open reading frames in this region revealed a nucleotide substitution from G to T in the third exon of LOC_Os11g29840. Additionally, real-time fluorescence quantitative PCR analysis showed that the expression level of LOC_Os11g29840 in the stems was much higher than in the roots and leaves in M96. Furthermore, the expression level was more than four times in M96 stem than in the wild type stem. Our results suggested that the mutant gene was likely a new allele to the reported gene LAZY1. Isolation of this new allele would facilitate the further characterization of LAZY1.

  4. Development of sparse-seeded mutant kinnow (Citrus reticulata ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... The mutant was put to the conventional propagation up to mV5, for confirmation of the continuity of ... techniques with fruits such as citrus, grape (Vitis vinifera ... irradiated bud scions were grafted onto Citrus jambhiri rootstock,.

  5. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    Science.gov (United States)

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  6. Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

    Science.gov (United States)

    Konen, Jessica; Koo, Junghui; Robinson, Brian S.; Wiles, Walter Guy; Huang, Chunzi; Martin, W. David; Behera, Madhusmita; Smith, Geoffrey H.; Hill, Charles E.; Rossi, Michael R.; Sica, Gabriel L.; Rupji, Manali; Chen, Zhengjia; Kowalski, Jeanne; Kasinski, Andrea L.; Ramalingam, Suresh S.; Khuri, Fadlo R.; Marcus, Adam I.

    2017-01-01

    Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre–induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.

  7. Siim Nestor soovitab : Mutant Disco. Azymuth. Klubis Hollywood / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2003-01-01

    Mutant Disco klubis Prive 4. juulil. Brasiilia jazz-trio Azmuth klubis BonBon 5. juulil. Pidustuste sarja Hip Hop Cafe sünnipäeva tähistamisest klubis Hollywood 4. juulil, üritusest Ibiza Night 5. juulil

  8. Susceptibility genes for schizophrenia: mutant models, endophenotypes and psychobiology.

    Science.gov (United States)

    O'Tuathaigh, Colm M P; Desbonnet, Lieve; Moran, Paula M; Waddington, John L

    2012-01-01

    Schizophrenia is characterised by a multifactorial aetiology that involves genetic liability interacting with epigenetic and environmental factors to increase risk for developing the disorder. A consensus view is that the genetic component involves several common risk alleles of small effect and/or rare but penetrant copy number variations. Furthermore, there is increasing evidence for broader, overlapping genetic-phenotypic relationships in psychosis; for example, the same susceptibility genes also confer risk for bipolar disorder. Phenotypic characterisation of genetic models of candidate risk genes and/or putative pathophysiological processes implicated in schizophrenia, as well as examination of epidemiologically relevant gene × environment interactions in these models, can illuminate molecular and pathobiological mechanisms involved in schizophrenia. The present chapter outlines both the evidence from phenotypic studies in mutant mouse models related to schizophrenia and recently described mutant models addressing such gene × environment interactions. Emphasis is placed on evaluating the extent to which mutant phenotypes recapitulate the totality of the disease phenotype or model selective endophenotypes. We also discuss new developments and trends in relation to the functional genomics of psychosis which might help to inform on the construct validity of mutant models of schizophrenia and highlight methodological challenges in phenotypic evaluation that relate to such models.

  9. Genetic characterization of glossy-leafed mutant broccoli lines

    Science.gov (United States)

    Glossy mutants of Brassica oleracea L. have reduced or altered epicuticular wax on the surface of their leaves as compared to wild-type plants, conveying a shiny green appearance. Mutations conferring glossiness are common and have been found in most B. oleracea crop varieties, including cauliflower...

  10. A wilty mutant of rice has impaired hydraulic conductance.

    Science.gov (United States)

    Koizumi, Koji; Ookawa, Taiichiro; Satoh, Hikaru; Hirasawa, Tadashi

    2007-08-01

    The rice CM2088 mutant is the wilty phenotype and wilts markedly under well-watered sunny conditions. The leaf water potential and epidermal (mainly stomatal) conductance of CM2088 plants decreased significantly under conditions that induced intense transpiration, as compared with those of wild-type plants, revealing that the wilty phenotype was not the result of abnormal stomatal behavior but was due to an increase in resistance to water transport. The resistance to water transport was dramatically elevated in the node and the sheath and blade of a leaf of the mutant, but not in the root or stem. The diameter of xylem vessels in the large vascular bundles of the leaf sheath and the internode tended to be small, and the numbers of vessel elements with narrowed or scalariform perforation plates in the leaf blade and sheath were greater in the mutant than in the wild type. Most xylem vessels were occluded, with air bubbles in the leaf sheath of the mutant during the midday hours under intense transpiration conditions, while no bubbles were observed in plants that were barely transpiring, revealing that the significant increase in resistance to water transport was a result of the cavitation. The additive effects of cavitation in xylem vessels and the decreased diameter and deformed plates of vessel elements might be responsible for the wilty phenotype of CM2088.

  11. Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus

    NARCIS (Netherlands)

    Oklejewicz, Malgorzata; Daan, Serge

    2002-01-01

    The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span

  12. Siim Nestor soovitab : Mutant Disco. Azymuth. Klubis Hollywood / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2003-01-01

    Mutant Disco klubis Prive 4. juulil. Brasiilia jazz-trio Azmuth klubis BonBon 5. juulil. Pidustuste sarja Hip Hop Cafe sünnipäeva tähistamisest klubis Hollywood 4. juulil, üritusest Ibiza Night 5. juulil

  13. Some Experiments with Respiratory Deficient Mutants of Yeast (Saccharomyces cerevisiae)

    Science.gov (United States)

    Freeland, P. W.

    1978-01-01

    Methods are described for the induction and identification of respiratory deficient mutants in yeast. Practical schemes are given to enable students to obtain dose-response information for physical and chemical mutagens such as heat, ultraviolet light, or acriflavine. A simple test for environmental mutagens is described. (Author/MA)

  14. Morphology and inheritance of a new rice leaf death mutant

    Institute of Scientific and Technical Information of China (English)

    CHENGZhakuan; GUMingkang

    1998-01-01

    A new mutant showing successive leaf death was selected in a mutmion populalion from an indica rice variety Zhongxian 3037 trealed with 94Co-γ rays. The leaves of this rnutanl began lodie on the emergence of tlle third leaf tip, and all leaves died sooner alter flowering. Consequently,

  15. Rest mutant zebrafish swim erratically and display atypical spatial preferences.

    Science.gov (United States)

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

    2015-05-01

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

  16. Metabolic reprogramming in mutant IDH1 glioma cells.

    Directory of Open Access Journals (Sweden)

    Jose L Izquierdo-Garcia

    Full Text Available Mutations in isocitrate dehydrogenase (IDH 1 have been reported in over 70% of low-grade gliomas and secondary glioblastomas. IDH1 is the enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate while mutant IDH1 catalyzes the conversion of α-ketoglutarate into 2-hydroxyglutarate. These mutations are associated with the accumulation of 2-hydroxyglutarate within the tumor and are believed to be one of the earliest events in the development of low-grade gliomas. The goal of this work was to determine whether the IDH1 mutation leads to additional magnetic resonance spectroscopy (MRS-detectable changes in the cellular metabolome.Two genetically engineered cell models were investigated, a U87-based model and an E6/E7/hTERT immortalized normal human astrocyte (NHA-based model. For both models, wild-type IDH1 cells were generated by transduction with a lentiviral vector coding for the wild-type IDH1 gene while mutant IDH1 cells were generated by transduction with a lentiviral vector coding for the R132H IDH1 mutant gene. Metabolites were extracted from the cells using the dual-phase extraction method and analyzed by 1H-MRS. Principal Component Analysis was used to analyze the MRS data.Principal Component Analysis clearly discriminated between wild-type and mutant IDH1 cells. Analysis of the loading plots revealed significant metabolic changes associated with the IDH1 mutation. Specifically, a significant drop in the concentration of glutamate, lactate and phosphocholine as well as the expected elevation in 2-hydroxyglutarate were observed in mutant IDH1 cells when compared to their wild-type counterparts.The IDH1 mutation leads to several, potentially translatable MRS-detectable metabolic changes beyond the production of 2-hydroxyglutarate.

  17. Arabidopsis mutant bik1 exhibits strong resistance to Plasmodiophora brassicae

    Directory of Open Access Journals (Sweden)

    Tao Chen

    2016-09-01

    Full Text Available Botrytis-induced kinase1 (BIK1, a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1 and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2 and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2 and npr1-1 (non-expresser of PR genes that regulate systemic acquired resistance (SAR mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms enhance the resistance to clubroot disease.

  18. Comparative stability of dihydrofolate reductase mutants in vitro and in vivo.

    Science.gov (United States)

    Leontiev, V V; Uversky, V N; Gudkov, A T

    1993-01-01

    Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

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

    Science.gov (United States)

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

    2007-11-01

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

  20. Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; McKinney, Lea V; Pike, Helen

    2008-01-01

    The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11 ...

  1. Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis.

    Science.gov (United States)

    Fukushima, Atsushi; Kusano, Miyako; Mejia, Ramon Francisco; Iwasa, Mami; Kobayashi, Makoto; Hayashi, Naomi; Watanabe-Takahashi, Akiko; Narisawa, Tomoko; Tohge, Takayuki; Hur, Manhoi; Wurtele, Eve Syrkin; Nikolau, Basil J; Saito, Kazuki

    2014-05-14

    Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/.

  2. Mutants of GABA transaminase (POP2 suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh mutants in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Frank Ludewig

    Full Text Available BACKGROUND: The gamma-aminubutyrate (GABA shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD, the mitochondrial enzymes GABA transaminase (GABA-T; POP2 and succinic semialdehyde dehydrogenase (SSADH. We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA, gamma-hydroxybutyrate (GHB and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.

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

    Science.gov (United States)

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

    2016-01-10

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

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

    Science.gov (United States)

    Rizal, Govinda; Karki, Shanta; Thakur, Vivek; Wanchana, Samart; Alonso-Cantabrana, Hugo; Dionora, Jacque; Sheehy, John E; Furbank, Robert; von Caemmerer, Susanne; Quick, William Paul

    2017-01-01

    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 in the

  5. Improved solubility of replication factor C (RFC) Walker A mutants.

    Science.gov (United States)

    Marzahn, Melissa R; Bloom, Linda B

    2012-06-01

    Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.

  6. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila.

    Science.gov (United States)

    Seki, Yuuichi; Tanimura, Teiichi

    2014-09-01

    A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf 01) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.

  7. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila

    Indian Academy of Sciences (India)

    Yuuichi Seki; Teiichi Tanimura

    2014-09-01

    A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf01) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.

  8. Induction and use of artificial mutants in sweet potato

    Energy Technology Data Exchange (ETDEWEB)

    Marumine, Shokichi

    1984-03-01

    X-ray, ethylene imine, TSP and WCo were used as mutagen for sweet potato mutation breeding and visible variations were observed for all mutagen. In the case of WCo irradiation, mutation rate of skin color is 0.5-1.3% based on cutting. Direction and variation of dry matter and tuber yield of mutants which were induced by TSP and/or WCo irradiation showed more deteriorative variation than progressive variation but some induced mutant lines show same or superior characters than original line. In the case of TSP irradiation to tuber, obstruction is not so much up to dese of 10,000 ci per tuber but treatment of 330 ci per cutting approximate to LD50. By tuber treatment with WCo gamma rays, suppression of sprouting occurred in dose of 30kR. Tendency to increase a variation was not observed at higher doses. 50-200 ci per cutting or 300-500 ci per tuber in TSP treatment and 15 kR in WCo gamma-irradiation for tuber seemed to be optimum dosages. Hybrid seed of mutant selected for dry matter content was compared with that of original line and it was concluded that the variation of selected line was genetic. Mutant induced by TSP and WCo treatment was used as a parental material and progeny of the cross was selected for practical characters. As a result, a line of higher starch yield with high resistance to pest and disease was selected and this line was used as parental material of further breeding. (author).

  9. Azotobacter vinelandii mutS: nucleotide sequence and mutant analysis.

    OpenAIRE

    Le, O; Shen, B.; Iismaa, S E; Burgess, B K

    1993-01-01

    An Azotobacter vinelandii homolog to the Salmonella typhimurium mutS gene was discovered upstream of the fdxA gene. The product of this gene is much more similar to S. typhimurium MutS than either is to the HexA protein of Streptococcus pneumoniae. An A. vinelandii delta mutS mutant strain was shown to have a spontaneous mutation frequency 65-fold greater than that of the wild type.

  10. Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

    OpenAIRE

    de Montaigu Amaury; Magneschi Leonardo; Catalanotti Claudia; Yang Wenqiang; Mus Florence; Pootakham Wirulda; Gonzalez-Ballester David; Higuera Jose J; Prior Matthew; Galván Aurora; Fernandez Emilio; Grossman Arthur R

    2011-01-01

    Abstract A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment...

  11. Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

    Science.gov (United States)

    2015-09-01

    transition occurs is unknown. In this proposal, we are studying the molecular and cellular mechanisms that cause this transition to occur in...weekly cancer center and pathology grand rounds amongst others. d. How were the results disseminated to communities of interest? Nothing to report...process and obtain high quality data recently. Therefore, we will be performing RNA sequencing on samples of EGFR mutant SCLC in the next couple of

  12. Functional analysis of Burkitt's lymphoma mutant c-Myc proteins

    OpenAIRE

    1996-01-01

    The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the retinoblastoma-related protein p107. In Burkitt’s lymphoma, missense mutations within the c-Myc transactivation domain have been found with high frequency. It has been reported that mutant c-Myc proteins derived ...

  13. Aconitase causes iron toxicity in Drosophila pink1 mutants.

    Directory of Open Access Journals (Sweden)

    Giovanni Esposito

    2013-04-01

    Full Text Available The PTEN-induced kinase 1 (PINK1 is a mitochondrial kinase, and pink1 mutations cause early onset Parkinson's disease (PD in humans. Loss of pink1 in Drosophila leads to defects in mitochondrial function, and genetic data suggest that another PD-related gene product, Parkin, acts with pink1 to regulate the clearance of dysfunctional mitochondria (mitophagy. Consequently, pink1 mutants show an accumulation of morphologically abnormal mitochondria, but it is unclear if other factors are involved in pink1 function in vivo and contribute to the mitochondrial morphological defects seen in specific cell types in pink1 mutants. To explore the molecular mechanisms of pink1 function, we performed a genetic modifier screen in Drosophila and identified aconitase (acon as a dominant suppressor of pink1. Acon localizes to mitochondria and harbors a labile iron-sulfur [4Fe-4S] cluster that can scavenge superoxide to release hydrogen peroxide and iron that combine to produce hydroxyl radicals. Using Acon enzymatic mutants, and expression of mitoferritin that scavenges free iron, we show that [4Fe-4S] cluster inactivation, as a result of increased superoxide in pink1 mutants, results in oxidative stress and mitochondrial swelling. We show that [4Fe-4S] inactivation acts downstream of pink1 in a pathway that affects mitochondrial morphology, but acts independently of parkin. Thus our data indicate that superoxide-dependent [4Fe-4S] inactivation defines a potential pathogenic cascade that acts independent of mitophagy and links iron toxicity to mitochondrial failure in a PD-relevant model.

  14. Dihydrodipicolinate synthase in opaque and floury maize mutants

    NARCIS (Netherlands)

    Varisi, V.A.; Medici, L.O.; Meer, van der I.M.; Lea, P.J.; Azevedo, J.L.

    2007-01-01

    Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) was isolated and studied in four high-lysine maize mutants (Oh43o1, Oh43o2, Oh43fl1 and Oh43fl2). The activity of DHDPS was analyzed at 16, 20, and 24 DAP and characterized in the presence of the amino acids, lysine, S-(2-aminoethyl)-l-cysteine (AEC)

  15. Metabolite profiling of two low phytic acid (lpa) rice mutants.

    Science.gov (United States)

    Frank, Thomas; Meuleye, Bertrand Seumo; Miller, Andreas; Shu, Qing-Yao; Engel, Karl-Heinz

    2007-12-26

    Two low phytic acid (lpa) rice mutant lines, Os-lpa-XS110-1 and Os-lpa-XS110-2, were grown together with their parent wild-type variety Xiushui 110 in four field trials. HPLC analysis of inositol phosphates in the seeds produced demonstrated that compared to the wild-type, the reduction in phytic acid content in Os-lpa-XS110-1 (-46%) was more pronounced than that in Os-lpa-XS110-2 (-23%). Lower inositol phosphates (InsP 3, InsP 4, InsP 5) were not detected in the mutants. The lpa mutants and the wild-type rice were subjected to comparative metabolite profiling by capillary gas chromatography. On average, 34% (Os-lpa-XS110-1) and 42% (Os-lpa-XS110-2) of the detected peaks were statistically significantly different between wild-type and mutants. However, only a few of these differences could be consistently observed for all field trials. Identification and quantification of the consistently different metabolites revealed that contents of myo-inositol and raffinose were increased in Os-lpa-XS110-1 but decreased in Os-lpa-XS110-2 compared to the wild-type. In addition, Os-lpa-XS110-1 exhibited increased levels of galactose and galactinol. Consideration of these metabolic changes in light of the routes involved in the biosynthesis of phytic acid indicated a disturbance in the early biosynthetic pathway of phytic acid in Os-lpa-XS110-2 (similar to the lpa-1 type mutation in maize) and a mutation event affecting phosphorylation of myo-inositol in Os-lpa-XS110-1 (similar to the lpa-3-type mutation).

  16. GREEN FLUORESCENT PIGMENT ACCUMULATED BY A MUTANT OF CELLVIBRIO GILVUS.

    Science.gov (United States)

    LOVE, S H; HULCHER, F H

    1964-01-01

    Love, Samuel H. (Bowman Gray School of Medicine, Winston-Salem, N.C.), and Frank H. Hulcher. Green fluorescent pigment accumulated by a mutant of Cellvibrio gilvus. J. Bacteriol. 87:39-45. 1964.-A mutant of Cellvibrio gilvus, designated strain 139A, liberated a green, fluorescent pigment into the surrounding culture medium. A study of the factors which affected the accumulation of this pigment led to the development of a chemically defined medium which supported maximal pigment accumulation in aerated, liquid cultures. d-Glucose, glycine or l-serine, l-phenylalanine, l-proline, and l-lysine comprised the organic components of this medium. The visible absorption spectrum of the pigment showed a maximal band at 400 mmu (pH 7.0). A difference spectrum between reduced and oxidized pigment showed loss of the band at 400 mmu upon oxidation. However, a methanol-extractable, flavinelike compound occurred in the wild strain but not in the mutant. Ferric ions added to the defined medium stimulated growth, with a concomitant reduction of pigment accumulation. Pigment was formed at a maximal rate during the stationary growth phase, and the highest yield was obtained by 18 hr. Organic solvents did not extract the pigment from water solutions. One and sometimes two, compounds absorbing at 400 mmu could be eluted by ion-exchange chromatography on Cellex-P (H(+)), which was used to separate the pigment from other components in the culture supernatants so that the radioactivity of the pigment could be measured. The mutant synthesized C(14)-labeled pigment from d-glucose-U-C(14) and from each of four amino acids (glycine-1-C(14), l-phenylalanine-U-C(14), l-proline-U-C(14), and l-lysine-U-C(14). Delta-Amino-levulenic acid-4-C(14) did not contribute C(14) to the pigment.

  17. GPNMB ameliorates mutant TDP-43-induced motor neuron cell death.

    Science.gov (United States)

    Nagahara, Yuki; Shimazawa, Masamitsu; Ohuchi, Kazuki; Ito, Junko; Takahashi, Hitoshi; Tsuruma, Kazuhiro; Kakita, Akiyoshi; Hara, Hideaki

    2017-08-01

    Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP-43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP-43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)-positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba1)-positive microglia. GPNMB aggregates were localized in the microtubule-associated protein 2 (MAP-2)-positive neuron and neurofilament H non-phosphorylated (SMI-32)-positive neuron, and these were co-localized with TDP-43 aggregates in the spinal cord of ALS patients. Mock or TDP-43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP-43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP-43 plasmids and serum-free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP-43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP-43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Histological Characterization of the Dicer1 Mutant Zebrafish Retina

    Directory of Open Access Journals (Sweden)

    Saeed Akhtar

    2015-01-01

    Full Text Available DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA and RNA-interference (RNAi functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE cells are normal with no sign of degeneration at the age of 20 months.

  19. Recombination Phenotypes of Escherichia coli greA Mutants

    Directory of Open Access Journals (Sweden)

    Poteete Anthony R

    2011-03-01

    Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

  20. 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.

  1. 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.

  2. [Minor beta thalassemia masked by a hemoglobin A2 mutant].

    Science.gov (United States)

    Omar, Souheil; Hammami, Mohamed Bassem; Taeib, Sameh Haj; Feki, Moncef; Abbes, Salem; Kaabachi, Naziha

    2010-09-01

    The elevation of hemoglobin A2 (HbA2) is an essential criterion in the diagnosis of minor ss thalassemia. To report a case of minor ss thalassemia HbA2 with normal HbA2 rate. We report the case of ten years old boy, with hypochromic microcytic anemia, refractory to iron treatment. The study of hemoglobin (Hb) has revealed the presence of a minor abnormal fraction of Hb, amounted to 2.8%, associated with the presence of HbF and normal levels of HbA2. Family study revealed the presence of two Hb abnormalities (ss thalassemia trait and HbA2 mutant) transmitted to offspring in isolation or associated. The genotypic study confirmed the presence of minor, 0 thalassemia and a ⁰/₀₀ gene mutation, causing a new mutant HbA2 named HbA2 Pasteur-Tunis [⁰/₀₀ 59(E3)LysgAsn(AAGgAAC)]. The presence of ⁰/₀₀ mutant reduces HbA2 level and could hide ss thalassemia trait. Rigorous and methodical interpretation of phenotypic data is crucial to not overlook the presence of ss thalassemia trait, whose diagnosis is crucial for genetic counseling and prenatal diagnosis.

  3. Characterization and Fine Mapping of the ibf Mutant in Rice

    Institute of Scientific and Technical Information of China (English)

    Jiajun Cui; Shengci Fan; Tian Shao; Zejun Huang; Dali Zheng; Ding Tang; Ming Li; Qian Qian; Zhukuan Cheng

    2007-01-01

    The pigment is an important character in plant development. In the present study, we characterized and fine mapped one inhibitor for brown furrows gene (ibf) in rice (Oryza sativa L.). in the Ibf mutant, brown pigments specifically accumulate in the furrows of hulls as seeds mature and reach a maximum level in dry seeds. Genetic analysis showed that the mutant phenotype is controlled by one recessive nuclear gene, which was finally mapped in a 90-kb reglon on the long arm of chromosome 9. Polymerase chain reaction and Southern blotting analysis revealed that there was a 26 kb deletion in the 90-kb region in the mutant. Since all the open reading frames outside the gap in the delimited reglon had no detectable difference in DNA sequence with the wild-type, we postulated that the Ibf locus should be located in the gap. Through gene annotation and reverse transcription-polymerase chain reaction (RT-PCR) analysis, we selected OsKF1 encoding a kelch repeat-containing F-box family protein as the candidate gene of ibf.

  4. Potent inhibition of HIV-1 replication by a Tat mutant.

    Science.gov (United States)

    Meredith, Luke W; Sivakumaran, Haran; Major, Lee; Suhrbier, Andreas; Harrich, David

    2009-11-10

    Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

  5. Potent inhibition of HIV-1 replication by a Tat mutant.

    Directory of Open Access Journals (Sweden)

    Luke W Meredith

    Full Text Available Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection.

  6. Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

    Science.gov (United States)

    Elmonem, Mohamed A.; Khalil, Ramzi; Khodaparast, Ladan; Khodaparast, Laleh; Arcolino, Fanny O.; Morgan, Joseph; Pastore, Anna; Tylzanowski, Przemko; Ny, Annelii; Lowe, Martin; de Witte, Peter A.; Baelde, Hans J.; van den Heuvel, Lambertus P.; Levtchenko, Elena

    2017-01-01

    The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis. PMID:28198397

  7. Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

    Science.gov (United States)

    Yamagami, Kanako; Yamamoto, Takaharu; Sakai, Shota; Mioka, Tetsuo; Sano, Takamitsu; Igarashi, Yasuyuki; Tanaka, Kazuma

    2015-01-01

    In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

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

    Science.gov (United States)

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

    2015-12-01

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

  9. Identification of Pfdhfr mutant variants in Plasmodium berghei model

    Directory of Open Access Journals (Sweden)

    Chairat Uthaipibull

    2011-12-01

    Full Text Available Parasite resistance to antimalarials is a major burden in controlling malaria disease. Genetic mutations within the parasites are found to be the factor in conferring resistance to drugs. In this study, the power of random mutant library and transgenic parasite systems were employed to identify mutations on the antimalarial drug target, viz. Plasmodium falciparum dihydrofolate reductase (DHFR, which could contribute to resistance, and to elucidate the functionality of resistant mutant parasites in P. berghei. Using the moderate drug-resistant PfdhfrS108N gene as template, we generated a library of Pfdhfr mutants by error-prone PCR followed by transfection and selection in P. berghei. Two clones of transgenic P. berghei expressing PfDHFR of interest due to the position of mutations, i.e. PbPfDHFR3m1 (M55I+S108N+S189C and PbPfDHFR3m2 (C50Y+S108N+F116S, were selected for drug sensitivity test. Although these transgenic parasite clones showed similar reproducibility with the parental transgenic P. berghei, expressing PfDHFR with mutation at S108N (PbPfS108N in response to antifolate pyrimethamine, this study reconfirms that this P. berghei model is effective in predicting the evolution of Pfdhfr mutations in vivo. This approach can be applied during the development of new antifolates with better effective properties against drug resistant parasites.

  10. Genes and Alcohol Consumption: Studies with Mutant Mice

    Science.gov (United States)

    Mayfield, Jody; Arends, Michael A.; Harris, R. Adron; Blednov, Yuri A.

    2017-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test. PMID:27055617

  11. Genes and Alcohol Consumption: Studies with Mutant Mice.

    Science.gov (United States)

    Mayfield, J; Arends, M A; Harris, R A; Blednov, Y A

    2016-01-01

    In this chapter, we review the effects of global null mutant and overexpressing transgenic mouse lines on voluntary self-administration of alcohol. We examine approximately 200 publications pertaining to the effects of 155 mouse genes on alcohol consumption in different drinking models. The targeted genes vary in function and include neurotransmitter, ion channel, neuroimmune, and neuropeptide signaling systems. The alcohol self-administration models include operant conditioning, two- and four-bottle choice continuous and intermittent access, drinking in the dark limited access, chronic intermittent ethanol, and scheduled high alcohol consumption tests. Comparisons of different drinking models using the same mutant mice are potentially the most informative, and we will highlight those examples. More mutants have been tested for continuous two-bottle choice consumption than any other test; of the 137 mouse genes examined using this model, 97 (72%) altered drinking in at least one sex. Overall, the effects of genetic manipulations on alcohol drinking often depend on the sex of the mice, alcohol concentration and time of access, genetic background, as well as the drinking test.

  12. Pattern formation mechanisms in motility mutants of Myxococcus xanthus

    CERN Document Server

    Starruss, Joern; Jakovljevic, Vladimir; Sogaard-Andersen, Lotte; Deutsch, Andreas; Baer, Markus

    2016-01-01

    Formation of spatial patterns of cells is a recurring theme in biology and often depends on regulated cell motility. Motility of M. xanthus depends on two motility machineries: the S-engine and A-engine. Moving M. xanthus cells can organize into spreading colonies or spore-filled fruiting bodies depending on their nutritional status. To understand these two pattern formation processes and the contributions by the two motility machineries, as well as cell reversal, we analyze spatial self-organization in 3 strains: i) a mutant that moves unidirectionally without reversing by the A-motility system only, ii) a unidirectional mutant that is also equipped with the S-motility system, and iii) the wild-type that, in addition to the two motility systems, reverses its direction of movement. The mutant moving by the A-engine illustrates that collective motion in the form of large moving clusters can arise in gliding bacteria due to steric interactions of the rod-shaped cells, without the need of invoking any biochemica...

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

    Directory of Open Access Journals (Sweden)

    Martin R. Hediger

    2013-08-01

    Full Text Available 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.

  14. dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants.

    Science.gov (United States)

    Torrey, T A; Atlung, T; Kogoma, T

    1984-01-01

    The possible allelic relationship between dasF (dnaA suppressor) and sdrA/rnh (stable DNA replication/RNase H) mutations was examined. dasF mutations could not only suppress various dnaA(ts) mutations, but also the insertional inactivation of the dnaA gene or deletion of the oriC sequence, as could sdrA mutations. dasF mutants were found to exhibit the stable DNA replication phenotype, and the sensitivity to rich media, of sdrA mutants. The dasF and sdrA mutations were mapped very closely between metD and proA on the E. coli genetic map. The mutations were recessive to the wild-type allele for all the above phenotypes. It was concluded that dasF is allelic to sdrA/mh.

  15. Properties of Streptomyces fradiae Mutants Blocked in Biosynthesis of the Macrolide Antibiotic Tylosin

    OpenAIRE

    Baltz, Richard H.; Seno, Eugene T.

    1981-01-01

    We isolated numerous mutants of Streptomyces fradiae blocked in tylosin biosynthesis after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. These mutants were classified into nine groups, based upon the tylosin-like compounds produced and upon cofermentation analyses. More than 80% of the mutants isolated produced no tylosin-like compounds, and the majority of these were blocked only in the formation of tylactone. Four classes of mutants blocked in the biosynthesis or addition of tylosin sug...

  16. Gibberellins and Carotenoids in the Wild Type and Mutants of Gibberella fujikuroi

    OpenAIRE

    1991-01-01

    A new screening procedure was used to isolate 14 gib mut ts of Gi!'berella_ fujikurof wit modi cations the production of gibberellins. The production of carotenmds and g bberellms was mvestlgated m the gib mutants and in representative car mutants with various modifications of carotenoid biosynthesis. The determinations of gibberellins were carried out with a simplified ftuorescence method. One of the mutants lacked both gibberellins and carotenoids. In many mutants the two path...

  17. Mutants as an aid to the study of higher plant photomorphogenesis.

    OpenAIRE

    Adamse, P.

    1988-01-01

    Study of photomorphogenesis is often complicated by the interaction of different photoreceptors regulating a given process or by the induction of multiple effects by a single photoreceptor. Mutants in which particular components of the morphogenetic pathways are eliminated provide the possibility of studying a more simplified form of photomorphogenesis. Three classes of photomorphogenetic mutants are proposed: photoreceptor, transduction chain and response mutants. In this study three mutants...

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

    OpenAIRE

    Murchison, H; Larrimore, S; Curtiss, R

    1981-01-01

    A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non...

  19. Production of Extracellular Pigment by a Mutant of Monascus kaoliang sp. nov

    OpenAIRE

    Lin, Ching-Fwu; Iizuka, Hiroshi

    1982-01-01

    A hyperpigment-producing mutant, R-10847, was derived from Monascus kaoliang F-2 (ATCC 26264) through a series of mutagenesis steps. The mutant produced a large quantity of Monascus pigment when grown in mantou (steamed bread) by solid culture. The mutant produced pigments extracellularly by extruding the pigments outside the cell in a lump together with some viscous substances. The productivity of pigment was about 100-fold greater than that of the wild type. The mutant lost the capability o...

  20. Properties of Streptomyces fradiae Mutants Blocked in Biosynthesis of the Macrolide Antibiotic Tylosin

    OpenAIRE

    Baltz, Richard H; Seno, Eugene T.

    1981-01-01

    We isolated numerous mutants of Streptomyces fradiae blocked in tylosin biosynthesis after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. These mutants were classified into nine groups, based upon the tylosin-like compounds produced and upon cofermentation analyses. More than 80% of the mutants isolated produced no tylosin-like compounds, and the majority of these were blocked only in the formation of tylactone. Four classes of mutants blocked in the biosynthesis or addition of tylosin sug...

  1. 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)

  2. New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...

  3. Photocontrol of seed germination of wild type and long-hypocotyl mutants of Arabidopsis thaliana

    NARCIS (Netherlands)

    Cone, J.W.

    1985-01-01

    This thesis reports research on the photocontrol of seed germination of wildtype and long-hypocotyl mutants of Arabidopsis thaliana. The mutants show reduced photoinhibition of hypocotyl growth in white light in comparison to that of wildtype. In monochromatic light some of the mutants also show no

  4. Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli

    DEFF Research Database (Denmark)

    Brown, S; Brickman, E R; Beckwith, J

    1981-01-01

    We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon inser...

  5. An improved method for rapid generation and screening of Bacillus thuringiensis phage-resistant mutants.

    Science.gov (United States)

    Gillis, Annika; Mahillon, Jacques

    2014-11-01

    A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.

  6. Inverse polymerase chain reaction for rapid gene isolation in Arabidopsis thaliana insertion mutants

    NARCIS (Netherlands)

    Vanderhaeghen, R.; Scheres, B.J.G.; Montagu, M. van; Lijsebetten, M. van

    1992-01-01

    Recently, many mutants have been isolated in the model plant Arabidopsis thaliana by the insertion of the Agrobacterium tumefaciens T-DNA into the plant genome. Instead of applying Southern analysis on these insertion mutants and to avoid the construction of mutant- derived genomic libraries, we pro

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

    Directory of Open Access Journals (Sweden)

    Hiroki Otani

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

  8. Agronomic Characterization of Wheat Mutants (Triticum aestivum of M3 Generation Planted in Sukabumi

    Directory of Open Access Journals (Sweden)

    Laela Sari

    2016-11-01

    Full Text Available The purpose of this study was to identify the selection criteria to obtain a superior mutant derived from the wheat plants of such varieties as Dewata, Selayar and Alibey, adaptive in medium land. The analysis of agronomic growth characters showed a significantly effect on a growth percentage of the initial growth (8 mutants, flowering time (1 mutant, panicle stem length (15 mutants, number of panicles (7 mutants, the number of grains per panicle (8 mutants, grain weight observed (8 mutants, grain weight per genotype (6 mutants, leaf area (2 mutants and leaf greenness (5 mutants. The effects on the characters of ripe time, harvest, panicle length and plant height were not significant. The mutants of Dewata, Selayar and Alibey could be selected based on the characters of panicle stem length, number of grains per panicle and grain weight per observation because these characters generated more mutants than the other characters. The correlation analysis between the characters of growth and yield components of wheat mutants showed that the number of grains per panicle was positively correlated with the grain weight observed, while the length of panicle stem was positively correlated with grain weight per genotype, number of panicles and leaf area. Hopefully some mutants produced could adapt to the tropical medium land, thus adding to the diversity of wheat germplasm in Indonesia, thereby reducing the import of wheat to Indonesia.How to CiteSari, L., Purwito, A., Sopandie, D., Purnamaningsih, R. & Sudarmonowati, E. (2016. Agronomic Characterization of Wheat Mutants (Triticum aestivum of M3 Generation Planted in Sukabumi. Biosaintifika: Journal of Biology & Biology Education, 8(3, 353-361. 

  9. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Rodermel, Steven

    2015-11-16

    The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

  10. Helicobacter pylori arginase mutant colonizes arginase Ⅱ knockout mice

    Institute of Scientific and Technical Information of China (English)

    Songhee H Kim; Melanie L Langford; Jean-Luc Boucher; Traci L Testerman; David J McGee

    2011-01-01

    AIM: To investigate the role of host and bacterial argi-nases in the colonization of mice by Helicobacter pylori (H. Pylori).METHODS: H. Pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. Pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. Pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase Ⅱ, since this enzyme is expressed in the stomach, and H. Pylori has previously been shown to induce the expres-sion of murine gastric arginase Ⅱ. To test this hypoth-esis, wild-type and arginase (rocF) mutant H. Pylori strain SS1 were inoculated into arginase Ⅱ knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase Ⅱ knock-out mice. Moreover, feeding arginase Ⅱ knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginase Ⅰactivity in several tissues, did not block the ability of the rocF mutant H. Pylori to colonize. In con-trast, BEC poorly inhibited H. Pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. Pylori urease is neither bacterial arginase nor host arginase Ⅱ; instead, either residual host arginase Ⅰor agmatinase is probably responsible.

  11. The antiandrogenic effect of finasteride against a mutant androgen receptor.

    Science.gov (United States)

    Wu, Yue; Chhipa, Rishi Raj; Zhang, Haitao; Ip, Clement

    2011-05-15

    Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild type AR. When PC-3 cells, which are AR-null, were transfected with either the wild type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy.

  12. 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...

  13. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced......, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib...

  14. Demarcation of mutant-carrying regions in barley plants after ethylmethane-sulfonate seed treatment

    DEFF Research Database (Denmark)

    Jacobsen, P.

    1966-01-01

    was obtained.The absence of cluster sharing allows the recognition in the barley plant of 8 mutually exclusive mutant sectors which never had a mutant cluster in common. The anatomical analysis proves that the barley embryo contains at least 6 separate shoot meristems or prospective shoot meristems, which...... will constitute mutually exclusive mutant sectors in the plant. The combined genetical and anatomical analysis reveals that in large seeds there are always 9 meristems leading to 9 mutually exclusive mutant sectors. Up to 7 additional meristems leading to mutually exclusive mutant sectors can be present...

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

    Science.gov (United States)

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

    2007-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-04-06

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

  17. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish

    OpenAIRE

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 sy...

  18. Executionary pathway for apoptosis: lessons from mutant mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis or programmed cell death (PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms. Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer. The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apop totic pathways that exist in mammals. In this review, we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.

  19. Isolation and mapping of phosphotransferase mutants in Escherichia coli.

    Science.gov (United States)

    Epstein, W; Jewett, S; Fox, C F

    1970-11-01

    Mutants of Escherichia coli K-12 defective in enzyme I or Hpr, the two common components of the phosphoenolpyruvate-dependent phosphotransferase system, were isolated by a simple, direct method. The ptsI locus, the structural gene for enzyme I, and the ptsH locus, the site of mutations leading to loss of Hpr activity, are adjacent genes and could be part of a single operon. These two genes lie between the purC and supN markers in the order: strA... guaB-purC-ptsI-ptsH-supN-dsdA... his.

  20. Executionary pathway for apoptosis:lessons from mutant mice

    Institute of Scientific and Technical Information of China (English)

    WOOMINNA; RAZQALLAHHAKEM; 等

    2000-01-01

    Apoptosis or programmed cell death(PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms.Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer.The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apoptotic pathways that exist in mammals.In this review,we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.

  1. Developmental genes during placentation: insights from mouse mutants

    Institute of Scientific and Technical Information of China (English)

    Jinhu a LU; Qiang WANG; Bingyan WANG; Fengchao WANG; Haibin WANG

    2011-01-01

    Placenta,a temporary organ first formed during the development of a new life is essential for the survival and growth of the fetus in eutherian mammals.It serves as an interface for the exchange of nutrients,gases and wastes between the maternal and fetal compartments.During the past decades,studies employing gene-engineered mouse mutants have revealed a wide range of signaling molecules governing the trophoblast development and function during placentation under various pathophysiological conditions.Here,we summarize the recent progress with particular respect to the involvement of developmental genes during placentation.

  2. Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

    Directory of Open Access Journals (Sweden)

    de Montaigu Amaury

    2011-07-01

    Full Text Available Abstract A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker and in the strategies used to maintain and store transformants.

  3. Ultra-violet-resistant mutants of Bacillus thuringiensis

    Energy Technology Data Exchange (ETDEWEB)

    Jones, D.R.; Karunakaran, V. (Polytechnic of Central London (UK). Faculty of Engineering and Science, School of Biological and Health Sciences); Burges, H.D. (Institute of Horticultural Research, Littlehampton (UK)); Hacking, A.J. (Reading Univ. (UK). Dextra Labs.Ltd.)

    1991-06-01

    One of the main disadvantages of using Bacillus thuringiensis as an insecticide is that the spore and crystal preparations applied to foliage are readily washed away by rain and are inactivated by sunlight. Spores from some strains of B. thuringiensis have been shown to be highly sensitive to u.v. light. This study has demonstrated how mutants with increased resistance to u.v., isolated by successive rounds of u.v. irradiation, and additionally with increased specific pathogenicity can be isolated. These techniques should be applied to strains that are frequently used in the industrial production of B.thuringiensis toxin. (author).

  4. Characterization of a Deswapped Triple Mutant Bovine Odorant Binding Protein

    Directory of Open Access Journals (Sweden)

    Roberto Favilla

    2011-04-01

    Full Text Available The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the β-barrel lipocalin scaffold.

  5. Generation of targeted mouse mutants by embryo microinjection of TALENs.

    Science.gov (United States)

    Wefers, Benedikt; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

    2014-08-15

    Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step, without the need for embryonic stem cells. Thereby, knockout and knockin alleles can be generated fast and efficiently by embryo microinjection of TALEN mRNAs and targeting vectors. In this article we present an introduction into the TALEN technology and provide protocols for the application of TALENs in mouse zygotes.

  6. Radiation induced useful mutants of Japanese mint (Mentha arvesis L. )

    Energy Technology Data Exchange (ETDEWEB)

    Kak, S.N.; Kaul, B.L.

    1980-01-01

    With a view to inducing mutations, dormant suckers of Mentha arvensis var. piperascens were exposed to X- and gamma rays. Individual clones raised from these treatments were screened for morphological and biochemical traits. Clones with apparent mutations in the first vegetative generation were propagated through second generation and those breeding true for the mutated character were isolated. In this way several mutants were isolated, some of which were found to be novel with respect to the quality of oil, besides those economically useful. The study shows that induced mutations could be very useful in the breeding of vegetatively propagated plants.

  7. Changes in Chlorophyll Fluorescence of Rice Mutants Induced by High Hydrostatic Pressure

    Institute of Scientific and Technical Information of China (English)

    BAI Cheng-ke; LI Gui-shuang; PENG Chang-lian; DUAN Jun

    2003-01-01

    Three mutants of rice (Oryza sativa L. ), Mutant 1, Mutant 2and Mutant 3, which were selected by high hydrostatic pressure (75 MPa), and their parent Yuexiangzhan were used to study the changes in chlorophyll fluorescence during different growth stages. In all the three mutants, the function of PSⅡ was improved, Fy/Fm ratio of mutants increased compared to their parent at tillering and heading stage, and φPS Ⅱ also improved except for Mutant 2 at heading stage. Similar to their parent, the mutants exhibited slight photoinhibition at noon and almost complete recovery to initial levels of 6:00 after 18:00 at heading stage. At milking stage, the photoinhibition in the mutants was obvious, and recovered rapidly compared to the parent. Yields of individual plant and grain/straw ratio were also higher in three mutants than the parent. Results indicated that characteristics of chlorophyll fluorescence in leaves of mutants and their photoinhibition in the field had changed. It is suggested that high hydrostatic pressure induction could be applied as a new effective approach in high-yield rice breeding in the future.

  8. Phenotypic characterization and virulence of a sae- agr- mutant of Staphylococcus aureus.

    Science.gov (United States)

    Giraudo, A T; Rampone, H; Calzolari, A; Nagel, R

    1996-02-01

    A sae::Tn551 agr::tetM double mutant was constructed and characterized. The production of several exoproteins (e.g., beta-hemolysin, DNase, and proteases) by this mutant was determined and found to be lower than the already diminished production of either isogenic single mutant sae- or agr-. The double mutant also showed, like the agr- mutant, null production of alpha- and delta-hemolysins and diminished levels of lipase. The reduced levels of many exoproteins in the double mutant as compared with their already diminished levels in either single mutant suggest that there is an additive or synergistic interaction between the two mutations involved, sae- and agr-. However, inactivation of both loci, sae and agr, had a different effect on the two exoproteins that are up regulated in the agr- mutant; thus, coagulase dropped to levels close to the null levels of the sae- parental strain, while extracellular protein A displayed the high levels characteristic of the agr- single mutant. The virulence of the sae- agr- double mutant, determined by intraperitoneal injection in mice, was found to be significantly diminished as compared with that of the sae+ agr+ parental strain or the sae- agr+ single mutant.

  9. Isolation and characterization of Rhizobium meliloti mutants affected in exopolysaccharide production.

    Science.gov (United States)

    Rodríguez-Navarro, D N; Palomares, A J; Casadesús, J

    1991-06-01

    Rhizobium meliloti mutants affected in the production of exopolysaccharide (EPS) were isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutants were classified into three phenotypic classes: (I) Exo-, rough mutants lacking exopolysaccharide; (II) Exos (for "small") which form tiny, compact colonies and synthesize reduced amounts of EPS; and (III) Exoc (for "constitutive"), hypermucoid mutants which overproduce EPS. Hypermucoid strains showed increased resistance to desiccation. All the mutants were able to nodulate, although a significant decrease in infectivity degree and/or competitiveness was found in rough and compact strains. Two mutants proved to be deficient in nitrogen fixation. Complementation analysis with cloned R. meliloti exo genes could not be applied to the study of these Fix- mutants because introduction of plasmids derived from cosmid vector pLAFR1 caused loss of nodulating ability. However, complementation of calcofluor staining and EPS production was observed. Complementation with certain exo genes also caused a marked increase in motility.

  10. Characterization and Genetic Analysis of a Novel Mutant mst of Rice Defective in Flower Development

    Institute of Scientific and Technical Information of China (English)

    LI Yun; XU Pei-zhou; ZHANG Hong-yu; FU Shao-hong; YANG Jin; ZHANG Ru-quan; WU Xian-jun

    2009-01-01

    A spontaneous mutant with multiple stigmas (mst) was found in an indica rice line 466. The mst mutant exhibits normal at the vegetative development stage and produces normal inflorescence structures. The difference between the mutant and the wild type was observed when the stamen primordium began to develop. In the mst florets, palea and lemma opened, lodicules were homeotically transformed into palea/lemma-like structures, and stamens were homeotically transformed into carpel-like structures. It looked like multiple stigmas being full of the whole floret. The phenotypic changes of mst were very similar to that of B-like mutant spw1. Compared with other mutants with pistillate morphologies, the severe mst florets showed that the inner three floral organs were completely changed into palea/lemma-like structures. Moreover, the mutant was female sterile. Occasionally, with the changing environment, one or two stamens were fertile. Genetic analysis indicated that the mutant traits were controlled by a single recessive gene.

  11. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms

    Directory of Open Access Journals (Sweden)

    Qiong-Yao Tang

    2016-01-01

    Full Text Available Twelve sodium-activated potassium channel (KCNT1, Slack genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel’s sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na+ ([Na+]i concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po. We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na+]i concentration at which these mutants exert their pathological effects.

  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

    DEFF Research Database (Denmark)

    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. Características fisiológicas de microtomateiros fitocromo-mutantes Physiological characteristics of micro-tomato (Lycopersicon esculentum P. Miller) phytochrome-mutants

    National Research Council Canada - National Science Library

    Hyrandir Cabral de Melo; Evaristo Mauro de Castro; Ângela Maria Soares; Cynthia de Oliveira; Sílvio Júnio Ramos

    2009-01-01

    ... (Lycopersicon esculentum P. Miller cv. Micro-Tom) fitocromo-mutantes. A cultivar Micro-Tom e os mutantes aurea (deficiente na biossíntese do cromóforo dos fitocromos), atroviolacea (atv) e high pigment1 (hp1...

  14. Ethanol fermentation on glucose/xylose mixture by co-cultivation of restricted glucose catabolite repressed mutants of Pichia stipitis with respiratory deficient mutants of Saccharomyces cerevisiae.

    Science.gov (United States)

    Kordowska-Wiater, Monika; Targoński, Zdzisław

    2002-01-01

    Restricted glucose catabolite repressed mutants of P. stipiti CCY 39501 were selected using UV irradiation. Four mutants were obtained which assimilated glucose slower than the native strain of P. stipitis and the degree of glucose repression was about 2-fold lower for P5-90-133 and P5-200-16 mutants and about 10-fold lower for P5-80-7 and P5-80-35 mutants. P5-80-7 and P5-80-35 produced very small amounts of ethanol from glucose and xylose, whereas P5-90-133 and P5-200-16 fermented sugars at the wild-type level. These two mutants were selected for co-fermentation process with native strain of S. cerevisiae V30 or Ja(a), as well as with their respiratory deficient mutants. During co-culture process of P. stipitis mutants with native strains of S. cerevisiae the ethanol yields obtained ranged from 0.38 to 0.45 g/g, and this alcohol was produced mainly from glucose. But, when also xylose, besides glucose was fermented to ethanol during co-fermentation of both mutant strains, lower yields of ethanol (0.28-0.40 g/g) were obtained.

  15. Chloroplast ultrastructure in leaves of Cucumis sativus chlorophyll mutant

    Directory of Open Access Journals (Sweden)

    Irena Palczewska

    2014-02-01

    Full Text Available The developing and young leaves of Cucumis sativus chlorophyll mutants are yellow, when mature they become green and do not differ in their colour from those of control plants. The mesophyll of yellow leaves contains a diversiform plastid population with a varying degree of defectiveness, which is mainly manifested in the reduction or disorganization of the typical thylakoid system. DNA areas, ribosome-like particles and aggregates of electron-dense material are preserved in the stroma of mutated plastids. Starch grains are deficient. Apart from mutated plastids, chloroplasts with a normal structure, as in control plants, were also observed.The leaf greening process is accompanied by a reconstruction and rearrangement of the inner chloroplast lamellar system and an ability to accumulate starch. However, in the mutant chloroplasts as compared with control-plant ones, an irregular arrangement of grana and reduced number of inter-grana thylakoids can be seen. An osmiophilic substance stored in the stroma of mutated plastids and the vesicles formed from an internal plastid membrane take part in restoration of the membrane system.

  16. Boolean network model predicts knockout mutant phenotypes of fission yeast.

    Directory of Open Access Journals (Sweden)

    Maria I Davidich

    Full Text Available BOOLEAN NETWORKS (OR: networks of switches are extremely simple mathematical models of biochemical signaling networks. Under certain circumstances, Boolean networks, despite their simplicity, are capable of predicting dynamical activation patterns of gene regulatory networks in living cells. For example, the temporal sequence of cell cycle activation patterns in yeasts S. pombe and S. cerevisiae are faithfully reproduced by Boolean network models. An interesting question is whether this simple model class could also predict a more complex cellular phenomenology as, for example, the cell cycle dynamics under various knockout mutants instead of the wild type dynamics, only. Here we show that a Boolean network model for the cell cycle control network of yeast S. pombe correctly predicts viability of a large number of known mutants. So far this had been left to the more detailed differential equation models of the biochemical kinetics of the yeast cell cycle network and was commonly thought to be out of reach for models as simplistic as Boolean networks. The new results support our vision that Boolean networks may complement other mathematical models in systems biology to a larger extent than expected so far, and may fill a gap where simplicity of the model and a preference for an overall dynamical blueprint of cellular regulation, instead of biochemical details, are in the focus.

  17. Boolean Network Model Predicts Knockout Mutant Phenotypes of Fission Yeast

    Science.gov (United States)

    Davidich, Maria I.; Bornholdt, Stefan

    2013-01-01

    Boolean networks (or: networks of switches) are extremely simple mathematical models of biochemical signaling networks. Under certain circumstances, Boolean networks, despite their simplicity, are capable of predicting dynamical activation patterns of gene regulatory networks in living cells. For example, the temporal sequence of cell cycle activation patterns in yeasts S. pombe and S. cerevisiae are faithfully reproduced by Boolean network models. An interesting question is whether this simple model class could also predict a more complex cellular phenomenology as, for example, the cell cycle dynamics under various knockout mutants instead of the wild type dynamics, only. Here we show that a Boolean network model for the cell cycle control network of yeast S. pombe correctly predicts viability of a large number of known mutants. So far this had been left to the more detailed differential equation models of the biochemical kinetics of the yeast cell cycle network and was commonly thought to be out of reach for models as simplistic as Boolean networks. The new results support our vision that Boolean networks may complement other mathematical models in systems biology to a larger extent than expected so far, and may fill a gap where simplicity of the model and a preference for an overall dynamical blueprint of cellular regulation, instead of biochemical details, are in the focus. PMID:24069138

  18. Pyrin gene and mutants thereof, which cause familial Mediterranean fever

    Energy Technology Data Exchange (ETDEWEB)

    Kastner, Daniel L [Bethesda, MD; Aksentijevichh, Ivona [Bethesda, MD; Centola, Michael [Tacoma Park, MD; Deng, Zuoming [Gaithersburg, MD; Sood, Ramen [Rockville, MD; Collins, Francis S [Rockville, MD; Blake, Trevor [Laytonsville, MD; Liu, P Paul [Ellicott City, MD; Fischel-Ghodsian, Nathan [Los Angeles, CA; Gumucio, Deborah L [Ann Arbor, MI; Richards, Robert I [North Adelaide, AU; Ricke, Darrell O [San Diego, CA; Doggett, Norman A [Santa Cruz, NM; Pras, Mordechai [Tel-Hashomer, IL

    2003-09-30

    The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

  19. Flavonoid accumulation patterns of transparent testa mutants of arabidopsis

    Science.gov (United States)

    Peer, W. A.; Brown, D. E.; Tague, B. W.; Muday, G. K.; Taiz, L.; Murphy, A. S.

    2001-01-01

    Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

  20. JC polyomavirus mutants escape antibody-mediated neutralization.

    Science.gov (United States)

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  1. Overexpression of SOS genes in ciprofloxacin resistant Escherichia coli mutants.

    Science.gov (United States)

    Pourahmad Jaktaji, Razieh; Pasand, Shirin

    2016-01-15

    Fluoroquinolones are important antibiotics for the treatment of urinary tract infections caused by Escherichia coli. Mutational studies have shown that ciprofloxacin, a member of fluoroquinolones induces SOS response and mutagenesis in pathogenic bacteria which in turn develop antibiotic resistance. However, inhibition of SOS response can increase recombination activity which in turn leads to genetic variation. The aim of this study was to measure 5 SOS genes expressions in nine E. coli mutants with different MICs for ciprofloxacin following exposure to ciprofloxacin. Gene expression was assessed by quantitative real time PCR. Gene alteration assessment was conducted by PCR amplification and DNA sequencing. Results showed that the expression of recA was increased in 5 mutants. This overexpression is not related to gene alteration, and enhances the expression of polB and umuCD genes encoding nonmutagenic and mutagenic polymerases, respectively. The direct relationship between the level of SOS expression and the level of resistance to ciprofloxacin was also indicated. It was concluded that novel therapeutic strategy that inhibits RecA activity would enhance the efficiency of common antibiotics against pathogenic bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Optokinetic behavior is reversed in achiasmatic mutant zebrafish larvae.

    Science.gov (United States)

    Rick, J M; Horschke, I; Neuhauss, S C

    2000-05-18

    The vertebrate optokinetic nystagmus (OKN) is a compensatory oculomotor behavior that is evoked by movement of the visual environment. It functions to stabilize visual images on the retina. The OKN can be experimentally evoked by rotating a drum fitted with stripes around the animal and has been studied extensively in many vertebrate species, including teleosts. This simple behavior has earlier been used to screen for mutations affecting visual system development in the vertebrate model organism zebrafish. In such a screen, we have found a significant number of homozygous belladonna (bel) mutant larvae to be defective in the correct execution of the OKN [1]. We now show that about 40% of homozygous bel larvae display a curious reversal of the OKN upon visual stimulation. Monocular stimulation leads to primary activation of ipsilateral eye movements in larvae that behave like the wild type. In contrast, affected larvae display contralateral activation of eye movements upon monocular stimulation. Anatomical analysis of retinal ganglion cell axon projections reveal a morphological basis for the observed behavioral defect. All animals with OKN reversal are achiasmatic. Further behavioral examination of affected larvae show that OKN-reversed animals execute this behavior in a stimulus-velocity-independent manner. Our data support a parsimonious model of optokinetic reversal by the opening of a controlling feedback loop at the level of the optic chiasm that is solely responsible for the observed behavioral abnormality in mutant belladonna larvae.

  3. Analysis of Stomatal Patterning in Selected Mutants of MAPK Pathways

    KAUST Repository

    Felemban, Abrar

    2016-05-01

    Stomata are cellular valves in plants that play an essential role in the regulation of gas exchange and are distributed in the epidermis of aerial organs. In Arabidopsis thaliana, stomatal production and development are coordinated by the mitogen-activated protein kinase (MAPK) signalling pathway, which modulates a variety of other processes, including cell proliferation, regulation of cytokinesis, programed cell death, and response to abiotic and biotic stress. The environment also plays a role in stomatal development, by influencing the frequency at which stomata develop in leaves. This thesis presents an analysis of stomatal development in Arabidopsis mutants in two MAPK pathways: MEKK1-MKK1/MKK2-MPK4, and MAP3K17/18-MKK3. Obtained results demonstrate the effect of stress conditions on stomatal development and specify the involvement of analysed MAPK in stomatal patterning. First, both analysed pathways modulate stomatal patterning in Arabidopsis cotyledons. Second, plant growth-promoting bacteria tested enhance stomatal density and affect guard cell morphology. Third, the sucrose or mannitol treatment increases defects in stomatal patterning. Finally, salt stress or high temperature can suppress stomatal defects in mutants of the MEKK1-MKK1/MKK2-MPK4 pathway.

  4. Computational identification of adaptive mutants using the VERT system

    Directory of Open Access Journals (Sweden)

    Winkler James

    2012-04-01

    Full Text Available Background Evolutionary dynamics of microbial organisms can now be visualized using the Visualizing Evolution in Real Time (VERT system, in which several isogenic strains expressing different fluorescent proteins compete during adaptive evolution and are tracked using fluorescent cell sorting to construct a population history over time. Mutations conferring enhanced growth rates can be detected by observing changes in the fluorescent population proportions. Results Using data obtained from several VERT experiments, we construct a hidden Markov-derived model to detect these adaptive events in VERT experiments without external intervention beyond initial training. Analysis of annotated data revealed that the model achieves consensus with human annotation for 85-93% of the data points when detecting adaptive events. A method to determine the optimal time point to isolate adaptive mutants is also introduced. Conclusions The developed model offers a new way to monitor adaptive evolution experiments without the need for external intervention, thereby simplifying adaptive evolution efforts relying on population tracking. Future efforts to construct a fully automated system to isolate adaptive mutants may find the algorithm a useful tool.

  5. Structural dataset for the PPARγ V290M mutant

    Directory of Open Access Journals (Sweden)

    Ana C. Puhl

    2016-06-01

    Full Text Available Loss-of-function mutation V290M in the ligand-binding domain of the peroxisome proliferator activated receptor γ (PPARγ is associated with a ligand resistance syndrome (PLRS, characterized by partial lipodystrophy and severe insulin resistance. In this data article we discuss an X-ray diffraction dataset that yielded the structure of PPARγ LBD V290M mutant refined at 2.3 Å resolution, that allowed building of 3D model of the receptor mutant with high confidence and revealed continuous well-defined electron density for the partial agonist diclofenac bound to hydrophobic pocket of the PPARγ. These structural data provide significant insights into molecular basis of PLRS caused by V290M mutation and are correlated with the receptor disability of rosiglitazone binding and increased affinity for corepressors. Furthermore, our structural evidence helps to explain clinical observations which point out to a failure to restore receptor function by the treatment with a full agonist of PPARγ, rosiglitazone.

  6. The Herbicidal Activity of Mutant Isolates from Botrytis cinerea

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jin-lin; ZHANG Li-hui; LIU Ying-chao; MA Juan; LI Chuan; DONG Jin-gao

    2006-01-01

    Fifteen mutant isolates were obtained by ultraviolet mutation from parent isolate Botrytis cinerea BC-4. Among them three mutant isolates, BC4-1, BC4-2, and BC4-15, showed strong herbicidal activity. BC4-1 showed maximum herbicidal activity for inhibition of germination and growth of Digitaria sanguinalis L. and Amaranthus retroflexus L. The results also showed that herbicidal activity was influenced by differing pH of PD media, with pH value of 4.0 being the optimum.The crude toxin was extracted using chloroform, petroleum ether, and ethyl acetate, respectively, and the ethyl acetate extracts showed the strongest inhibitory activity on the germination and growth of D. sanguinalis L. and A. retroflexus L.Using HPLC, one fraction with an absorption peak at 271 nm was separated from the crude toxin. This fraction could strongly inhibit the growth of D. sanguinalis L. at a concentration of 100 mg L-1 and could completely inhibit the seed germination of D. sanguinalis L. and A. retroflexus L. at a concentration of 50 mg L-1.

  7. Improved production of spiramycin by mutant Streptomyces ambofaciens

    Institute of Scientific and Technical Information of China (English)

    金志华; 岑沛霖

    2004-01-01

    Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S. ambofaciens XC 2-37 were studied. The potency of S. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation. The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

  8. Improved production of spiramycin by mutant Streptomyces ambofaciens

    Institute of Scientific and Technical Information of China (English)

    金志华; 岑沛霖

    2004-01-01

    Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S.ambofaciens XC 2-37 were studied. The potency orS. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.

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

    Science.gov (United States)

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

    2014-02-01

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

  10. HDAC6 regulates mutant SOD1 aggregation through two SMIR motifs and tubulin acetylation.

    Science.gov (United States)

    Gal, Jozsef; Chen, Jing; Barnett, Kelly R; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-05-24

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.

  11. HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation*

    Science.gov (United States)

    Gal, Jozsef; Chen, Jing; Barnett, Kelly R.; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-01-01

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS. PMID:23580651

  12. Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression.

    Science.gov (United States)

    Lima, Juliana Oliveira; Pereira, Jorge Fernando; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de

    2017-02-09

    Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

  13. Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Anne Grünewald

    Full Text Available BACKGROUND: Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD. The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7, as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and

  14. 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

  15. Connexin mutant embryonic stem cells and human diseases

    Institute of Scientific and Technical Information of China (English)

    Kiyomasa; Nishii; Yosaburo; Shibata; Yasushi; Kobayashi

    2014-01-01

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin(Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells(ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  16. Extra-large Tribolium confusum: a new autosomal recessive mutant.

    Science.gov (United States)

    Vardell, H H; Brower, J H

    1975-12-01

    A new mutant of Tribolium confusum Jacquelin duVal (Coleoptera: Tenebrionidae), extra-large (designated xl), was isolated in mating competition tests with red-eye (re) and wild-type (+). Crosses showed that it was autosomal recessive gene with subvital effects. The pupal weights averaged 6.1 and 7.3 mg for males and females, respectively, about twice the weights of the ancestral wild-type. The generation time (egg to adult) was approximately 8 to 9 weeks compared with about 4 weeks for the wild-type. This increase resulted from a lengthening of the larval stage since the durations of the egg and pupal stages were within the ranges of the wild-type. Mean longivity of xl males and females was reduced to 8.5 and 6.0 weeks, respectively at 26.7 +/- 1 degree C and 60% RH.

  17. Origins and spread of pfdhfr mutant alleles in Plasmodium falciparum.

    Science.gov (United States)

    Mita, Toshihiro

    2010-06-01

    The emergence and spread of Plasmodium falciparum parasite resistant to sulfadoxine and pyrimethamine (SP) poses a serious public health problem. Resistance is caused by point mutations in dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps), the two key enzymes in the folate biosynthetic pathway. The use of microsatellite markers flanking pfdhfr has recently shown that the invasion of limited resistant lineages may explain the widespread SP resistance in many endemic regions. In Africa, however, multiple indigenous origins of pfdhfr triple mutants have been demonstrated. More new independent lineages and routes of geographical spread of resistance may be found by further molecular evolutionary analyses using samples from various endemic regions. Here, I review recent studies about the history of SP usage and the evolution and spread of resistant lineages while addressing the technical issue of microsatellite analysis.

  18. Management of a patient with advanced BRAF-mutant melanoma.

    Science.gov (United States)

    Ashworth, Michelle T; Daud, Adil

    2014-03-01

    A 49-year-old man initially diagnosed in 1995 with cutaneous melanoma presented to the authors' institution in 2009 with metastatic, BRAF V600E-mutant melanoma. His treatment course to date has included surgery, adjuvant radiotherapy, and interferon, metastasectomies, granulocyte-macrophage colony-stimulating factors, a clinical trial with the BRAF inhibitor vemurafenib (PLX-4032), clinical trial with combination BRAF plus MEK inhibition with vemurafenib plus GDC-0973, and combination targeted and immune therapy with vemurafenib plus the anti-CTLA4 antibody ipilimumab. This case report illustrates the long-term management of a patient with metastatic melanoma using targeted and immune therapy, evolution in treatment guidelines, next directions in research, and the critical role of clinical trials in advancement of patient care.

  19. Specific mistranslation in hisT mutants of Escherichia coli.

    Science.gov (United States)

    Parker, J

    1982-01-01

    Certain strains of Escherichia coli mistranslate at very high frequencies when starved for asparagine or histidine. This mistranslation is the result of misreading events on the ribosome. The introduction of a hisT mutation into such a strain decreases the frequency of mistranslation during histidine starvation but not during asparagine starvation. The most likely explanation is that the replacement of the pseudouridine residue in the anticodon loop of glutamine specific transfer ribonucleic acid by uridine in hisT mutants leads to an increase in fidelity of transfer ribonucleic acid function. The hisT gene in Escherichia coli has also been more accurately mapped, giving the gene order purF-hisT-aroC-fadL-dsdA.

  20. Kinetic stability of designed glycosylation mutants of Coprinus cinereus peroxidase.

    Science.gov (United States)

    Tams, J W; Welinder, K G

    2001-08-31

    The effect of glycans and surface mutations on protein unfolding induced by heat or urea has been studied. Removal of the only native high mannose type glycan in the N142P, N142T, and N142D CIP mutants reduced the lifetime to half of that of wtCIP at irreversible conditions of unfolding. The effect was moderate at reversible conditions. Five glycomutants designed to have 0, 1, 2, 4 and 6N glycans showed a correlation between increased carbohydrate mass and increased stability toward irreversible unfolding. The results are in agreement with a dampening effect of glycans on backbone fluctuation in both the native and the unfolded states. However, experiments in reversible conditions were less clear because of additional effects of an increasing number of amino acid substitutions and aggregation. Examples of strong effects from minor surface changes were also observed.

  1. EGFR-Mutant Lung Adenocarcinoma Mimicking a Pneumonia

    Directory of Open Access Journals (Sweden)

    Álvaro Taus

    2012-01-01

    Full Text Available PET-CT scan has demonstrated to be very effective in lung cancer diagnosis and staging, but lung cancer has multiple ways of presentation, which can lead to an error in diagnosis imaging and a delay on the beginning of specific treatment. We present a case of a 77-year-old man with an initial PET-CT scan showing high 18F-FDG intake, suggesting a bilateral pneumonia, who was finally diagnosed of an EGFR-mutant lung adenocarcinoma. EGFR-activating mutation allowed us to start treatment with the oral tyrosin kinase inhibitor Gefitinib, obtaining a rapid and sustained response. Histological confirmation of imaging findings is always necessary to avoid diagnostic errors.

  2. Connexin mutant embryonic stem cells and human diseases.

    Science.gov (United States)

    Nishii, Kiyomasa; Shibata, Yosaburo; Kobayashi, Yasushi

    2014-11-26

    Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

  3. Catalytic properties of ADAM12 and its domain deletion mutants

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Visse, Robert; Sørensen, Hans Peter

    2008-01-01

    Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting...... of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits...... restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most...

  4. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  5. A pedigree analysis of two homozygous mutant Gitelman syndrome cases.

    Science.gov (United States)

    Luo, Jiewei; Yang, Xiao; Liang, Jixing; Li, Weihua

    2015-01-01

    Gitelman syndrome (GS) is a salt-wasting tubulointerstitial disease of autosomal recessive inheritance (OMIM613395) caused by genic mutation of SLC12A3, which codes thiazide-sensitive Na-Cl cotransporter (NCCT) gene. The gene mutation of the majority of GS patients is compound heterozygous. This study analyzes two cases of GS gene mutation and the clinical phenotype. Twenty patients of two GS pedigrees underwent direct sequence alignment of 26 exons of SLC12A3 to spot and locate mutant site. Proband A of Pedigree I had three mutant sites: Arg928Cys, a homozygote, missense mutation, and two homozygous silent mutations, Ala122Ala and Thr465Thr, and 8 members of Pedigree I carried Arg928Cy heterozygous mutation. Proband B of Pedigree II had a homozygote, Ser710X, and a termination codon was spotted, which would inevitably be translated into abridged and defective protein, and 7 members had Ser710X heterozygous mutation. The heterozygous mutation carriers of the two pedigrees often have stimulus-controlled hypokalemia after strenuous exercise. The parents of Proband A are cousins, a case of intermarriage. Both probands show hypokalemia, hypochloraemia, hypocalcinuria, hyperreninemia, and hyperaldosteronemia; Proband A has normal serum magnesium and increased urinary sodium excretion, while Proband B has hypomagnesemia and increased urinary magnesium ion excretion. Both probands have normal or lower blood pressure, weakness and numbness of lower extremities, muscular soreness, and occasional palpitations and chest discomfort. Proband A wearies easily and Proband B has occasional joint numbness and pain. These two homozygous mutations are responsible for the morbidity of two GS families and they show heterogenicity of clinical phenotype.

  6. Boc modifies the holoprosencephaly spectrum of Cdo mutant mice

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    2011-05-01

    Holoprosencephaly (HPE is caused by a failure to form the midline of the forebrain and/or midface. It is one of the most common human birth defects, but clinical expression is extremely variable. HPE is associated with mutations in the sonic hedgehog (SHH pathway. Mice lacking the Shh pathway regulator Cdo (also called Cdon display HPE with strain-dependent penetrance and expressivity, implicating silent modifier genes as one cause of the variability. However, the identities of potential HPE modifiers of this type are unknown. We report here that whereas mice lacking the Cdo paralog Boc do not have HPE, Cdo;Boc double mutants on a largely Cdo-resistant genetic background have lobar HPE with strong craniofacial anomalies and defects in Shh target gene expression in the developing forebrain. Boc is therefore a silent HPE modifier gene in mice. Furthermore, Cdo and Boc have specific, selective roles in Shh signaling in mammals, because Cdo;Boc double-mutant mice do not display the most severe HPE phenotype seen in Shh-null mice, nor do they have major defects in digit patterning or development of vertebrae, which are also Shh-dependent processes. This is in contrast to reported observations in Drosophila, where genetic removal of the Cdo and Boc orthologs Ihog and Boi results in a complete loss of response to the hedgehog ligand. Therefore, there is evolutionary divergence between mammals and insects in the requirement of the hedgehog pathway for Cdo/Ihog family members, with mammalian development involving additional factors and/or distinct mechanisms at this level of pathway regulation.

  7. A human vitamin D receptor mutant activated by cholecalciferol.

    Science.gov (United States)

    Ousley, Amanda M; Castillo, Hilda S; Duraj-Thatte, Anna; Doyle, Donald F; Azizi, Bahareh

    2011-07-01

    The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.

  8. Selection and characterization of L-ethionine resistant mutants of Trichosporon cutaneum.

    Science.gov (United States)

    Georgieva, Nelly; Alexieva, Zlatka

    2005-01-01

    Trichosporon cutaneum R57 and its L-ethionine resistant mutant NZ94 strain were investigated. The amino acid analyses of cell content of both strains were carried out. The pool of free methionine in the mutant strain is enhanced 16.5 times. The total amount of sulphur-containing amino acids in the mutant cells was significantly increased from 36.8 in the wild strain to 113.4 mg/g protein in the mutant strain. In the process of mutant strain cultivation there was found a high excretion of free methionine (259 microg/ml) in the medium. It was shown that the amino acid content of both wild and mutant strains would be helpful for formulating of new improved animal nutritional diets.

  9. Comparison of the Immunogenicities of HIV-1 Mutants Based on Structural Modification of env

    Institute of Scientific and Technical Information of China (English)

    Jian-hui NIE; Chun-tao ZHANG; Hui-hui CHONG; Xue-ling WU; Chu-yu LIU; Yu WU; Chen-yan ZHAO; Lin-qi ZHANG; You-Chun WANG

    2008-01-01

    Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. In vivo fitness and virulence of a drug-resistant herpes simplex virus 1 mutant.

    Science.gov (United States)

    Pesola, Jean M; Coen, Donald M

    2007-05-01

    Two important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.

  12. Crescimento e desenvolvimento de frutos do tomateiro 'Santa Clara' e do seu mutante natural 'Firme' Growth and development of 'Santa Clara' tomato fruit and its mutant 'Firme'

    Directory of Open Access Journals (Sweden)

    Marcia Lima Moura

    2004-12-01

    Full Text Available Na região produtora de hortaliças de Viçosa, MG, identificaram-se plantas de tomate da cv. Santa Clara (Lycopersicon esculentum Mill., que apresentam senescência foliar precoce e estigmas amarelados, com frutos de coloração "amarelo-creme" quando imaturos e vermelho quando maduros, de maturação lenta, e mais firmes que o fenótipo normal. Objetivou-se com este trabalho avaliar o crescimento e desenvolvimento dos frutos normais e mutantes. O delineamento experimental foi de blocos casualizados com quatro repetições. Os frutos mutantes apresentaram matéria fresca total e diâmetro transversal e longitudinal menores do que o normal durante todo o seu desenvolvimento. A espessura do pericarpo foi significativamente menor nos frutos mutantes do que nos frutos normais, a partir dos 21 dias após a antese. Folhas medianas e basais de plantas mutantes apresentaram menores teores de clorofila do que o observado em plantas normais. O período de amadurecimento do fruto mutante foi de 14 dias, enquanto dos frutos normais foi de 7 dias, quando ligados à planta-mãe, demonstrando a maior longevidade dos frutos mutantes. Além disso, os frutos mutantes apresentaram atraso na elevação da produção de etileno durante o amadurecimento.'Santa Clara' tomato (Lycopersicon esculentum Mill. plants showing earlier leaf senescence and yellowish stigma, fruits with pale yellow when immature and red when reach full ripe stage, associated to a lower rate of ripening and firmer than the wild type, were found in Viçosa, MG. The objective of this study was to evaluate the fruit growth and development of the mutant and wild type tomatoes. Mutant fruits showed smaller total fresh weight than wild type throughout development and thinner pericarp after 21 days after flowering. Basal and intermediate mutant plant leaves showed lower chlorophyll levels. 'Santa Clara' fruit took 7 days to reach full red ripe stage, while mutant fruits took 14 days. Furthermore

  13. Características fisiológicas de microtomateiros fitocromo-mutantes Physiological characteristics of micro-tomato (Lycopersicon esculentum P. Miller phytochrome-mutants

    Directory of Open Access Journals (Sweden)

    Hyrandir Cabral de Melo

    2009-10-01

    Full Text Available Objetivou-se, neste trabalho, caracterizar aspectos fisiológicos de microtomateiros (Lycopersicon esculentum P. Miller cv. Micro-Tom fitocromo-mutantes. A cultivar Micro-Tom e os mutantes aurea (deficiente na biossíntese do cromóforo dos fitocromos, atroviolacea (atv e high pigment1 (hp1;ambos superexpressam eventos mediados por fitocromos foram cultivados em condições controladas de luz e temperatura e caracterizados no estágio de floração. O mutante hp1 obteve as maiores taxas de fotossíntese potencial e de conteúdo de carotenóides. O mutante aurea manteve taxas de fotossíntese potencial similares à cultivar Micro-Tom, mesmo expressando o mais baixo conteúdo de clorofilas, e também expressou o maior conteúdo de nitrogênio entre os demais microtomateiros. Os mutantes aurea e hp1 obtiveram os menores conteúdos de açúcares solúveis totais. O mutante atv expressou o maior conteúdo de clorofilas e também a menor razão clorofila a/b.The objective of this work was to characterize physiological aspects of micro-tomato (Lycopersicon esculentum P. Miller cv. Micro-Tom phytochrome-mutants. Plants of Micro-Tom cultivar and aurea (deficient in phytochrome chromophore biosynthesis, high pigment1 (hp1 and atroviolacea (atv (both super express phytochrome events-mediated mutants were cultivated under controlled light and temperature and evaluated in flowering stage. The hp1 mutant expressed the highest rates of potential photosynthesis and also the content of total carotenoids. Aurea mutant maintained similar potential photosynthesis rates as the Micro-Tom cultivar, even containing low chlorophyll content, and expressed the highest content of nitrogen among all micro-tomatoes studied. Total soluble sugars were lower in aurea and hp1 mutants. The atv mutant expressed the highest content of chlorophylls and also the lowest rate of chlorophyll a/b.

  14. Isolation of a Rhodobacter capsulatus mutant that lacks c-type cytochromes and excretes porphyrins.

    OpenAIRE

    Biel, S W; Biel, A J

    1990-01-01

    A Rhodobacter capsulatus mutant lacking cytochrome oxidase activity was isolated by Tn5 mutagenesis. Difference spectroscopy of crude extracts and extracted c-type cytochromes demonstrated that this mutant completely lacked all c-type cytochromes. The strain did, however, synthesize normal amounts of b-type cytochromes and nonheme iron. This mutant also excreted large amounts of coproporphyrin and protoporphyrin and synthesized reduced amounts of bacteriochlorophyll, suggesting a link between...

  15. The Transcriptional Targets of Mutant FOXL2 in Granulosa Cell Tumours

    OpenAIRE

    2012-01-01

    BACKGROUND: Despite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. METHODS: The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 wer...

  16. Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium.

    OpenAIRE

    Boominathan, K; Dass, S B; Randall, T A; Kelley, R.L.; Reddy, C A

    1990-01-01

    Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical...

  17. Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri.

    OpenAIRE

    McDonough, M. A.; Klei, H. E.; Kelly, J. A.

    1999-01-01

    Penicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild-type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6-bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6-bromohexana...

  18. lac Up-promoter mutants with increased homology to the consensus promoter sequence.

    OpenAIRE

    Mandecki, W; Goldman, R A; Powell, B S; Caruthers, M H

    1985-01-01

    Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.

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

    Science.gov (United States)

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

    1997-01-01

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

  20. Indole-3-Acetic Acid Biosynthesis in the Mutant Maize orange pericarp, a Tryptophan Auxotroph.

    Science.gov (United States)

    Wright, A D; Sampson, M B; Neuffer, M G; Michalczuk, L; Slovin, J P; Cohen, J D

    1991-11-15

    The maize mutant orange pericarp is a tryptophan auxotroph, which results from mutation of two unlinked loci of tryptophan synthase B. This mutant was used to test the hypothesis that tryptophan is the precursor to the plant hormone indole-3-acetic acid (IAA). Total IAA in aseptically grown mutant seedlings was 50 times greater than in normal seedlings. In mutant seedlings grown on media containing stable isotopelabeled precursors, IAA was more enriched than was tryptophan. No incorporation of label into IAA from tryptophan could be detected. These results establish that IAA can be produced de novo without tryptophan as an intermediate.

  1. Isolation and characterization of rice lesion mimic mutants from a T-DNA tagged population

    Institute of Scientific and Technical Information of China (English)

    LI Shutian; PEI Zhongyou; LUO Lijuan; TIAN Yingchuan; HE Chaozu

    2005-01-01

    A rice ( Oryza sativa L. ssp. japonica cv. Nipponbare) T-DNA tagged population consisting of about 7000 individual lines was generated and screened for rice lesion mimic mutants in the T1 generation. Ten lines were found to develop spontaneous lesions in the absence of pathogen infection and displayed distinct lesion phenotypes. These mutants were tentatively designated as lm1 -lm10 (for lesion mimic), respectively. Lesion formation of lm mutants was developmentally regulated, and all the mutants showed stunted growth and reduced fertility. Genetic analysis demonstrated that all the mutations were recessive, and five partially fertile mutants (lm4-lm8) were derived from different loci. Mimic lesions occurring on the leaves of lm mutants resulted from cell death as revealed by trypan blue staining. Six of them ( lm3 -lm8 ) exhibited enhanced resistance to five bacterial blight isolates, indicating their wide-spectrum resistance to this pathogen. These results imply that some lesion mimic mutations of rice might be involved in disease resistance signaling pathways,and that isolation of these mutated genes may be useful for elucidating molecular mechanisms of plant disease resistance. Among the mutants, only one mutant, lm6, was preliminarily shown to cosegregate with the inserted T-DNA in its T1 generation, making it feasible to isolate the gene responsible for the phenotype of this mutant.

  2. Generation of mutants with developmental defects in zebrafish by ENU mutagenesis

    Institute of Scientific and Technical Information of China (English)

    JIN Peng; TIAN Tian; SUN Zhihui; MENG Anming

    2004-01-01

    As a good model for studying early development of vertebrates, zebrafish (Danio rerio) is attracting more and more attention. Following ENU mutagenesis, 320 F2 families were established. Mutants, which showed defects in epiboly, axis, somite, head, and cardiac and blood systems, were identified by observing morphological changes in F3 embryos. So far, 35 mutant lines have been established, the majority of which showed anomalies in axis and somite formation. These mutant lines provide useful genetic resources for cloning of the mutant genes and for studying mechanisms of early development of vertebrate embryos.

  3. Mutant connexin 50 (S276F) inhibits channel and hemichannel functions inducing cataract

    Indian Academy of Sciences (India)

    Yuanyuan Liu; Chen Qiao; Tanwei Wei; Fang Zheng; Shuren Guo; Qiang Chen; Ming Yan; Xin Zhou

    2015-06-01

    This study was designed to detect the expression, detergent resistance, subcellular localization, and channel and hemichannel functions of mutant Cx50 to understand the forming mechanism for inducing congenital cataract by a novel mutation p.S276F in connexin 50 (Cx50) reported previously by us. HeLa and human lens epithelial (HLE) cells were transfected with wild-type Cx50 and mutant Cx50 (S276F). We examined the functional characteristics of mutant Cx50 (S276F) in comparison with those of wild-type Cx50 using immunoblot, confocal fluorescence microscopy, dye transfer analysis and dye uptake assay. The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly problems. Scrape loading dye transfer was significantly evident in cells transfected with wild-type Cx50 compared to those in cells transfected with mutant Cx50 and cotransfected with wild-type and mutant Cx50. The dye uptake was found to be significantly lower in cells transfected with mutant Cx50 than in cells transfected with wild-type Cx50 and cells cotransfected with wild-type and mutant Cx50. The transfected HeLa and HLE cell lines showed similar performance in all the experiments. These results indicated that the mutant Cx50 (S276F) might inhibit the function of gap junction channel in a dominant negative manner, but inhibit the hemichannel function in a recessive negative manner.

  4. Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents.

    Science.gov (United States)

    Rogers, P; Palosaari, N

    1987-12-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid, and the Spo mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.

  5. Clostridium acetobutylicum mutants that produce butyraldehyde and altered quantities of solvents

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, P.; Palosaari, N.

    1987-12-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid/sup -/) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo/sup -/) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo/sup -/ mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid/sup -/, and the Spo/sup -/ mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.

  6. Isolation and characterization of stable mutants of Streptomyces peucetius defective in daunorubicin biosynthesis

    Indian Academy of Sciences (India)

    K. S. Vetrivel; K. Dharmalingam

    2001-04-01

    Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, -rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed by dnrE (mutants SPAK and SPMAG), dnrS (SPFS and SPRHO) and doxA (SPDHC) gene products.

  7. Mutant frequency of radiotherapy technicians appears to be associated with recent dose of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Messing, K.; Ferraris, J.; Bradley, W.E.; Swartz, J.; Seifert, A.M. (Universite du Quebec a Montreal (Canada))

    1989-10-01

    The frequency of hypoxanthine phosphoribosyl transferase (HPRT) mutants among peripheral T-lymphocytes of radiotherapy technicians primarily exposed to 60Co was measured by the T-cell cloning method. Mutant frequencies of these technicians in 1984 and 1986 were significantly higher than those of physiotherapy technicians who worked in a neighboring service, and correlated significantly with thermoluminescence dosimeter readings recorded during the 6 mo preceding mutant frequency determination. Correlations decreased when related to dose recorded over longer time intervals. HPRT mutant frequency determination in peripheral lymphocytes is a good measure of recently received biologically effective radiation dose in an occupationally exposed population.

  8. Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

    DEFF Research Database (Denmark)

    Knecht, Wolfgang; Rozpedowska, E.; Le Breton, C.

    2007-01-01

    ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell...... to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced...

  9. Sequential evaluation of CALR mutant burden in patients with myeloproliferative neoplasms.

    Science.gov (United States)

    Cavalloni, Chiara; Rumi, Elisa; Ferretti, Virginia V; Pietra, Daniela; Roncoroni, Elisa; Bellini, Marta; Ciboddo, Michele; Casetti, Ilaria C; Landini, Benedetta; Fugazza, Elena; Troletti, Daniela; Astori, Cesare; Cazzola, Mario

    2017-05-16

    We investigated the variation of CALR-mutant burden during follow-up in 105 CALR-mutant MPN and compared it to the variation of JAK2-mutant burden in 226 JAK2-mutant MPN.The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR-mutant ET there was a difference between natural and therapy-related slope (P 0.006).In the JAK2-mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation (CALR vs JAK2) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (β = 0.19, P = 0.061) with no difference between diagnosis (P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2-mutant MPN.

  10. Fisiologia do amadurecimento na planta do tomate 'Santa Clara' e do mutante 'Firme' Physiology of vine-ripened tomato 'Santa Clara' and its mutant 'Firme'

    Directory of Open Access Journals (Sweden)

    Márcia Lima Moura

    2005-03-01

    Full Text Available O mutante natural de tomate 'Firme' da cv. Santa Clara tem frutos com coloração "amarelo-creme" quando imaturos, firmes e com amadurecimento lento. Estudou-se as alterações fisiológicas que ocorrem durante o processo de amadurecimento na planta de frutos de tomate da cv. Santa Clara e do mutante 'Firme'. Os frutos normais e mutantes foram colhidos em 6 diferentes estádios de maturidade, e em cada um deles foram avaliados a produção de etileno e CO2, os teores de açúcares solúveis totais do pericarpo e do tecido locular, e as atividades das enzimas oxidase do ACC e poligalacturonase. Os frutos mutantes apresentaram menores taxas respiratórias e de produção de etileno em todos os estádios de maturidade. A atividade da oxidase do ACC apresentou padrão de comportamento distinto durante o amadurecimento na planta dos frutos mutantes e normais, porém com semelhante atividade final. Os frutos mutantes apresentaram atraso no aumento da atividade da enzima poligalacturonase em relação aos frutos normais nas fases iniciais do amadurecimento. Frutos normais acumularam açúcares solúveis totais durante seu amadurecimento na planta, enquanto que nos frutos mutantes os teores foram inferiores nos estádios mais avançados do amadurecimento quando comparados com aqueles no início do climatério. O pericarpo dos frutos mutantes nos estádios mais avançados do amadurecimento teve teores de açúcares total inferiores.The natural tomato mutant 'Firme' from cv. Santa Clara presents a yellow-pale appearance when immature, firm texture and slow ripening. Some of the physiological changes throughout ripening of cv. Santa Clara and the natural mutant 'Firme' were evaluated on the vine. Fruit ethylene and CO2 production, locular and outer pericarp total soluble sugars content, ACC oxidase and poligalacturonase activities were evaluated in both genotypes at six maturity stages. Mutant fruits presented lower ethylene and CO2 production at all

  11. The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids.

    Science.gov (United States)

    Afitlhile, Meshack; Fry, Morgan; Workman, Samantha

    2015-02-01

    We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis.

  12. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    Energy Technology Data Exchange (ETDEWEB)

    Gindt, Y.M.

    1993-04-01

    The role of the L[sub cm] (I), [beta][sup 18] (II), and [alpha][sup AP-B] (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A[sub max] = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  13. Spectroscopic studies of Synechococcus sp PCC 7002 phycobilisome core mutants

    Energy Technology Data Exchange (ETDEWEB)

    Gindt, Y.M.

    1993-04-01

    The role of the L{sub cm} (I), {beta}{sup 18} (II), and {alpha}{sup AP-B} (III) chromoproteins in the phycobilisome (PBS) core was investigated using genetically engineered strains of Synechococcus missing different polypeptides. Intact cells, isolated PBS, and subcore preparations for each mutant were studied to determine the effect of that mutation on energy transfer within the PBS core and to the reaction centers. Three mutants lacked the II and/or III polypeptides, while the I chromophore was altered in others. A lower energy absorbing chromophore, A{sub max} = 695 nm, was substituted for the I chromophore. The deletion of the II and III subunits had no discernible effect on energy transfer from the PBS to PSII. In cells and isolated PBS, the altered I chromophore acts to quench the PBS complex and to redirect the energy which would be transferred to PSII. In the PBS and subcore preparations, deletion of the III subunit did not alter energy transfer within the core. The deletion of the II subunit from the PBS caused a small decrease in the excited state lifetimes of the final emitters indicating more disorder within the core. The I chromophore was found to absorb at 670nm and to emit at 683nm within the intact PBS. The II chromophore emits at 679nm while the III chromophore emits at 682nm. A strong interaction exists between the I chromophore and the II subunit. Upon deletion of the II subunit from the PBS core, the I chromophore emits at a higher energy. The II subunit could act to stabilize the I chromophore-binding pocket, or exciton coupling could be occurring between the two. The role of the III chromophore is still unclear at this time. The III chromophore does contribute to the RT emission of the isolated PBS, but it transfers energy to I at 77 K. One can conclude that the III subunit is adjacent to the trimer containing the I polypeptide.

  14. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish.

    Science.gov (United States)

    Leong, Ivone U S; Skinner, Jonathan R; Shelling, Andrew N; Love, Donald R

    2013-01-01

    Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients.

  15. Clinical significance of hepatitis B surface antigen mutants.

    Science.gov (United States)

    Coppola, Nicola; Onorato, Lorenzo; Minichini, Carmine; Di Caprio, Giovanni; Starace, Mario; Sagnelli, Caterina; Sagnelli, Evangelista

    2015-11-28

    Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the "a" determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications.

  16. Zebrafish atoh8 mutants do not recapitulate morpholino phenotypes

    Science.gov (United States)

    Place, Elsie S.; Smith, James C.

    2017-01-01

    Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it has been implicated in the regulation of pancreatic and endothelial cell differentiation, the phenotypic consequences of Atoh8 loss are uncertain. Conclusions from knockout studies in the mouse differ widely depending on the targeting strategy used, while atoh8 knockdown by interfering morpholino oligonucleotides (morpholinos) in zebrafish has led to a range of developmental defects. This study characterised zebrafish embryos homozygous for atoh8sa1465, a loss-of-function allele of atoh8, in order to provide genetic evidence for the developmental role of Atoh8 in this species. Embryos homozygous for atoh8sa1465 present normal body morphology, swimbladder inflation, and heart looping, and survive to adulthood. These embryos do not develop pericardial oedema by 72 hpf and are not sensitised to the loss of Fog1 protein, suggesting that this previously described abnormality is not a specific phenotype. Vascular patterning and primitive haematopoiesis are unaffected in atoh8sa1465/sa1465 mutant embryos. Together, the data suggest that Atoh8 is dispensible for zebrafish development under standard laboratory conditions. PMID:28182631

  17. Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

    Directory of Open Access Journals (Sweden)

    Jinglan Liu

    2009-05-01

    Full Text Available Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS. Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

  18. Natural variation of model mutant phenotypes in Ciona intestinalis.

    Directory of Open Access Journals (Sweden)

    Paolo Sordino

    Full Text Available BACKGROUND: The study of ascidians (Chordata, Tunicata has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. CONCLUSIONS/SIGNIFICANCE: Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.

  19. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    Science.gov (United States)

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-07

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  20. Clinical significance of hepatitis B surface antigen mutants

    Institute of Scientific and Technical Information of China (English)

    Nicola; Coppola; Lorenzo; Onorato; Carmine; Minichini; Giovanni; Di; Caprio; Mario; Starace; Caterina; Sagnelli; Evangelista; Sagnelli

    2015-01-01

    Hepatitis B virus(HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen(HBs Ag) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBs Ag, called the "a" determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBs Ag detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBs Ag mutants and their biological and clinical implications.

  1. Plasmodium yoelii: induction of attenuated mutants by irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Waki, S.; Yonome, I.; Suzuki, M.

    1986-12-01

    When erythrocytic forms of Plasmodium yoelii nigeriensis, which is invariably fatal in mice, were exposed to X rays, the dose to reduce surviving parasites to one millionth was 100 gray (10 Krad). A suspension of 5 X 10(6) per ml of parasitized erythrocyte was irradiated at 100 gray, and 0.2 ml aliquots were inoculated into 22 mice. Eleven mice showed patent parasitemia, and in these the growth curves were less steep than that found in nonirradiated parasites. The infections of 8 mice of the 11 were self-resolving, and the attenuated feature of the parasites maintained following a limited number of blood passages. The parasites were slowly growing even in nude mice and cause self-resolving infections in intact mice. BALB/c mice immunized with the attenuated parasites were protected against subsequent challenge infections with the original virulent erythrocytic and sporogonic forms. These findings indicate that attenuated mutants of malaria parasites can be readily induced by this method.

  2. Phenotypic analysis of Arabidopsis mutants: quantitative analysis of root growth.

    Science.gov (United States)

    Doerner, Peter

    2008-03-01

    INTRODUCTIONThe growth of plant roots is very easy to measure and is particularly straightforward in Arabidopsis thaliana, because the increase in organ size is essentially restricted to one dimension. The precise measurement of root apical growth can be used to accurately determine growth activity (the rate of growth at a given time) during development in mutants, transgenic backgrounds, or in response to experimental treatments. Root growth is measured in a number of ways, the simplest of which is to grow the seedlings in a Petri dish and record the position of the advancing root tip at appropriate time points. The increase in root length is measured with a ruler and the data are entered into Microsoft Excel for analysis. When dealing with large numbers of seedlings, however, this procedure can be tedious, as well as inaccurate. An alternative approach, described in this protocol, uses "snapshots" of the growing plants, which are taken using gel-documentation equipment (i.e., a video camera with a frame-grabber unit, now commonly used to capture images from ethidium-bromide-stained electrophoresis gels). The images are analyzed using publicly available software (NIH-Image), which allows the user simply to cut and paste data into Microsoft Excel.

  3. Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

    Science.gov (United States)

    Asad, Sedigheh; Dabirmanesh, Bahareh; Khajeh, Khosro

    2014-01-01

    Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

  4. Characteristics of fruit ripening in tomato mutant epi

    Institute of Scientific and Technical Information of China (English)

    WANG Zhong-feng; YING Tie-jin; BAO Bi-li; HUANG Xiao-dan

    2005-01-01

    The characteristics of fruit ripening and expression of ripening-related genes were investigated in epi, an ethylene overproduction mutant of tomato (Lycopersicon esculentum Mill.). The epi produces apparently more ethylene than its wild type VFN8 at every stage of vegetative and fruit growth and ripening; compared to VFN8, the epi fruit showed higher CO2 evolution,faster descending of chlorophyll, slightly quicker increase of carotenoid and lycopene, and faster reduction in pericarp firmness during maturation and ripening; and the mRNAs of three ripening-related genes including E8, pTOM5 andpTOM6 were at higher levels in epi. The ripening-related characteristics changing of the fruit are consistent with the increase of ethylene production and ripening-related genes expression. These results suggest that epi mutation possibly did not affect the ethylene perception and signaling during fruit ripening, and that the modified characteristics of fruit ripening possibly resulted from the ethylene overproduction and increased expression of ripening-related genes.

  5. 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...

  6. “Start” Mutants of Saccharomyces cerevisiae Are Suppressed in Carbon Catabolite-Derepressing Medium

    OpenAIRE

    Shuster, Jeffrey R.

    1982-01-01

    Temperature-sensitive cell division “start” mutants cdc28, cdc36, cdc37, and cdc39 of the yeast Saccharomyces cerevisiae arrested cell division in the G1 phase of the cell cycle in glucose medium. I report here that cdc28, cdc36, and cdc39 mutants were suppressed when grown in carbon catabolite-derepressing medium.

  7. Isolation and characterization of Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis.

    OpenAIRE

    Ankenbauer, R G; Cox, C D

    1988-01-01

    Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis were isolated after chemical mutagenesis by plating on a siderophore detection medium. Like the wild type, these mutants incorporated 7-[14C]salicylic acid into pyochelin, demonstrating that salicylic acid is an intermediate in the biosynthesis pathway of pyochelin.

  8. Isolation and study of two mutants of Streptomyces cattleya affected in DNA repair and genetic instability.

    Science.gov (United States)

    Hromic, A; Kirby, R

    1989-01-15

    Two mutants of Streptomyces cattleya affecting DNA repair were isolated. These mutants were analysed using spore survival curves and phage reactivation curves in the presence and absence of caffeine and arsenite. Two DNA repair systems (uvr1 and uvr2) were identified, the latter of which seems to influence genetic instability.

  9. Differential gene expression in salt-tolerant rice mutant and its parental variety

    Institute of Scientific and Technical Information of China (English)

    张劲松; 周骏马; 张驰; 陈受宜

    1996-01-01

    The differential expressions of three genes rbcL, salT and rab!6 in response to ABA, NaCl, PEG and heat shock were investigated in seedlings of a salt-tolerant rice mutant 20 (mutant 20) and its parental variety Oryza sativa var. japonica 77-170(170). By Northern blot analysis it was found that ABA induced the expression of all three genes of rbcL, salT and rab16 in shoots and roots of both 170 and mutant 20 with the exceptions of rab16 in shoots of mutant 20 and rbcL in roots of 170. Lower concentrations of NaCl induced rbcL expression in shoots of mutant 20 but not 170. Higher concentrations of NaCl decreased rbcL expression but induced expressions of salT and rab16 in shoots of both 170 and mutant 20. PEG(15%) and 37℃ heat shock showed almost no effects on the expression of the three genes in mutant 20. However, they caused a decrease in rbcL expression and slight induction of the rab16 gene in 170, with salT expression unaffected. These results indicated that mutant 20 was relatively less responsiv

  10. ISOLATION AND CHARACTERIZATION OF PEROXISOMAL PROTEIN IMPORT (PIM-) MUTANTS OF HANSENULA-POLYMORPHA

    NARCIS (Netherlands)

    WATERHAM, HR; TITORENKO, [No Value; VANDERKLEI, IJ; HARDER, W; VEENHUIS, M

    1992-01-01

    In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroxisomal matrix proteins. These mutants are characterized by the presence of few small intact per

  11. VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication

    Institute of Scientific and Technical Information of China (English)

    Jun Yi; Wei-Dong Gong; Ling Wang; Rui Ling; Jiang-Hao Chen; Jun Yun

    2005-01-01

    AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication.METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR). Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry.RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutantbearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups.CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.

  12. Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

    NARCIS (Netherlands)

    Johnson, Monique A.; Waterham, Hans R.; Ksheminska, Galyna P.; Fayura, Liubov R.; Cereghino, Joan Lin; Stasyk, Oleh V.; Veenhuis, Marten; Kulachkovsky, Aleksander R.; Sibirny, Andrei A.; Cregg, James M.

    1999-01-01

    We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a pe

  13. Impaired exercise tolerance and skeletal muscle myopathy in sulfonylurea receptor-2 mutant mice

    Science.gov (United States)

    Stoller, Douglas; Pytel, Peter; Katz, Sophie; Earley, Judy U.; Collins, Keith; Metcalfe, Jamie; Lang, Roberto M.

    2009-01-01

    By sensing intracellular energy levels, ATP-sensitive potassium (KATP) channels help regulate vascular tone, glucose metabolism, and cardioprotection. SUR2 mutant mice lack full-length KATP channels in striated and smooth muscle and display a complex phenotype of hypertension and coronary vasospasm. SUR2 mutant mice also display baseline cardioprotection and can withstand acute sympathetic stress better than normal mice. We now studied response to a form of chronic stress, namely that induced by 4 wk of daily exercise on SUR2 mutant mice. Control mice increased exercise capacity by 400% over the training period, while SUR2 mutant mice showed little increase in exercise capacity. Unexercised SUR2 mutant showed necrotic and regenerating fibers in multiple muscle skeletal muscles, including quadriceps, tibialis anterior, and diaphragm muscles. Unlike exercised control animals, SUR2 mutant mice did not lose weight, presumably due to less overall exertion. Unexercised SUR2 mutant mice showed a trend of mildly reduced cardiac function, measured by fractional shortening, (46 ± 4% vs. 57 ± 7% for SUR2 mutant and control, respectively), and this decrease was not exacerbated by chronic exercise exposure. Despite an improved response to acute sympathetic stress and baseline cardioprotection, exercise intolerance results from lack of SUR2 KATP channels in mice. PMID:19675276

  14. Photosynthetic Characteristics of Flag Leaves in Rice White Stripe Mutant 6001 During Senescence Process

    Directory of Open Access Journals (Sweden)

    Xiao-hui ZHEN

    2014-11-01

    Full Text Available Physiological, biochemical and electron microscopy analyses were used to investigate the photosynthetic performance of flag leaves in rice white stripe mutant 6001 during the senescence process. Results showed that the chlorophyll content at the heading and milk-ripe stages in rice mutant 6001 were about 34.78% and 3.00% less than those in wild type 6028, respectively. However, the chlorophyll content at the fully-ripe stage in rice mutant 6001 was higher than that in wild type 6028. At the heading stage, the net photosynthetic rate (Pn in rice mutant 6001 was lower than that in wild type 6028. Rice mutant 6001 also exhibited a significantly slower decrease rate of Pn than wild type 6028 during the senescence progress, especially at the later stage. Furthermore, Ca2+-ATPase, Mg2+-ATPase and photophosphorylation activities exhibited the similar trends as the Pn. During the senescence process, the 68 kDa polypeptide concentrations in the thylakoid membrane proteins exhibited a significant change, which was one of the critical factors that contributed to the observed change in photosynthesis. We also observed that the chloroplasts of rice mutant 6001 exhibited higher integrity than those of wild type 6028, and the chloroplast membrane of rice mutant 6001 disintegrated more slow during the senescence process. In general, rice mutant 6001 had a relatively slower senescence rate than wild type 6028, and exhibited anti-senescence properties.

  15. Induction, isolation, and characterization of aspergillus niger mutant strains producing elevated levels of beta-galactosidase.

    OpenAIRE

    1981-01-01

    An Aspergillus niger mutant strain, VTT-D-80144, with an improvement of three- to fourfold in the production of extracellular beta-galactosidase was isolated after mutagenesis. The production of beta-galactosidase by this mutant was unaffected by fermentor size, and the enzyme was also suitable for immobilization.

  16. Pseudomonas fluorescens Tn5-B20 mutant RA92 responds to carbon limitation in soil

    NARCIS (Netherlands)

    Overbeek, van L.S.; Elsas, van J.D.; Veen, van J.A.

    1997-01-01

    Tn5-B20 (lacZ as reporter gene) transcriptional fusion mutants of Pseudomonas fluorescens R2f Rpr were screened for their response to carbon limitation. Reporter gene expression was specifically induced by this stress factor in one mutant, designated RA92, and to a lower extent by phosphorus and nit

  17. Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis

    DEFF Research Database (Denmark)

    Tran, Phuoc T; Shroff, Emelyn H; Burns, Timothy F

    2012-01-01

    KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer...

  18. 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.

  19. Ensemble-based computational approach discriminates functional activity of p53 cancer and rescue mutants.

    Directory of Open Access Journals (Sweden)

    Özlem Demir

    2011-10-01

    Full Text Available The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain ("cancer mutants". Activity can be restored by second-site suppressor mutations ("rescue mutants". This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD, without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC metric was strongly correlated (r(2 = 0.77 with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i p53 cancer mutants were more flexible than wild-type protein, (ii second-site rescue mutations decreased the flexibility of cancer mutants, and (iii negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.

  20. Agronomic traits and RAPD analysis of two mutants derived from rice somatic cell culturing

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Genetic variation, including agronomic trait variation, often occurs in somatic cell culturing. In this study, we compared the main agronomic traits of two rice mutants, M3 and M14, which were derived from Shenxiangjing 5 somatic cell culturing. Significant differences were found between the two mutants and the wild rice Shenxiangjing 5 (Table 1). Results were as follows:

  1. Random Genetic Drift Determines the Level of Mutant mtDNA in Human Primary Oocytes

    OpenAIRE

    Brown, D T; Samuels, D C; Michael, E. M.; Turnbull, D.M.; Chinnery, P F

    2000-01-01

    We measured the proportion of mutant mtDNA (mutation load) in 82 primary oocytes from a woman who harbored the A3243G mtDNA mutation. The frequency distribution of mutation load indicates that random drift is the principal mechanism that determines the level of mutant mtDNA within individual oocytes.

  2. Locomotor and oculomotor impairment associated with cerebellar dysgenesis in Zic3-deficient (Bent tail) mutant mice.

    NARCIS (Netherlands)

    Aruga, J.; Ogura, H.; Shutoh, F.; Ogawa, M.; Franke, B.; Nagao, S.; Mikoshiba, K.

    2004-01-01

    We examined the adult neural phenotypes of the Bent tail mutant mouse. The Bent tail mutant mouse was recently shown to lack a submicroscopic part of the X chromosome containing the Zic3 gene, which encodes a zinc-finger protein controlling vertebrate neural development. While nearly one-fourth of

  3. Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis.

    Science.gov (United States)

    Valdés-Santiago, Laura; Cervantes-Chávez, José Antonio; Winkler, Robert; León-Ramírez, Claudia G; Ruiz-Herrera, José

    2012-03-01

    Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell.

  4. Direct Selection of Clostridium acetobutylicum Fermentation Mutants by a Proton Suicide Method

    OpenAIRE

    Cueto, Pablo H.; Méndez, Beatriz S.

    1990-01-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

  5. Comparison of arabidopsis stomatal density mutants indicates variation in water stress responses and potential epistatic effects

    Science.gov (United States)

    Shaneka S. Lawson; Paula M. Pijut; Charles H. Michler

    2014-01-01

    Recent physiological analysis of Arabidopsis stomatal density (SD) mutants indicated that SD was not the major factor controlling aboveground biomass accumulation. Despite the general theory that plants with fewer stomata have limited biomass acquisition capabilities, epf1 and several other Arabidopsis mutants varied significantly in leaf fresh...

  6. Sexual behavior of mutant strains of the medfly Ceratitis capitata (Diptera: Tephritidae

    Directory of Open Access Journals (Sweden)

    R.D Briceño

    2003-09-01

    Full Text Available Males of the mutant strains (blind, vestigal-winged of the Mediterranean fruit fly (medfly, Ceratits capitata (Wiedmann showed differences in behavior compared with control (mass-reared males. Mutant males made fewer mating attempts and achieved fewer matings than control males. Vestigal-winged females copulated less frequently with both mutants. Blind males climbed rather than jumped onto females and copulated in very low numbers compared with control and vestigal males. Blind females copulated normally with control, males and in very low numbers with both types of mutant malesMachos mutantes (ciegos, alas vestigiales de la mosca del mediterráneo Ceratitis capitata (Wiedmann mostraron diferencias en conducta comparados con los machos testigo (cría masiva. Los machos mutantes, realizaron menos intentos por aparearse y lograron menos apareamientos que los machos testigo. Las hembras con alas vestigiales, copularon menos con ambas clases de mutantes. Los machos ciegos, subieron en lugar de saltar sobre las hembras y copularon en números muy bajos comparados con los machos testigo y con los de alas vestigiales. Las hembras ciegas, copularon de forma normal con los machos testigo y en números muy bajos con ambos tipos de machos mutantes

  7. Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha : A physiological study

    NARCIS (Netherlands)

    Klei, Ida J. van der; Harder, Willem; Veenhuis, Marten

    1991-01-01

    We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wildtype (WT) levels, this mutant is unable to grow

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

    Science.gov (United States)

    Murchison, H; Larrimore, S; Curtiss, R

    1981-12-01

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

  9. A Nearly Non-Functional Mutant Allele of the Storage Protein Locus Hor2 in Barley

    DEFF Research Database (Denmark)

    Doll, Hans

    1980-01-01

    The low content of the storage protein fraction hordein-2 in the high-lysine mutant Risø 56 is due to a mutation at or near the locus Hor2 coding for hordein-2 polypeptides. The mutant gene is recessive in its qualitative effect on the electrophoretic banding pattern of hordein-2, but co...

  10. Muscle cells and motoneurons differentially remove mutant SOD1 causing familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Onesto, Elisa; Rusmini, Paola; Crippa, Valeria; Ferri, Nicola; Zito, Arianna; Galbiati, Mariarita; Poletti, Angelo

    2011-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  11. Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum

    Science.gov (United States)

    To evaluate the effects that genetic background has on two sorghum brown midrib (bmr) mutants, plant phenolics, lignin biosynthetic enzymes and stem anatomy were evaluated in wild-type (WT), bmr-6, bmr-12 and double-mutants (bmr-6 and bmr-12) in near isogenic , RTx430 and Wheatland backgrounds. The...

  12. Photosynthetic Characteristics of Flag Leaves in Rice White Stripe Mutant 6001 During Senescence Process

    Institute of Scientific and Technical Information of China (English)

    ZHEN Xiao-hui; XU Jin-gang; SHEN Wei-jun; ZHANG Xiao-juan; ZHANG Qi-jun; LU Chuan-gen; CHEN Guo-xiang; GAO Zhi-ping

    2014-01-01

    Physiological, biochemical and electron microscopy analyses were used to investigate the photosynthetic performance of flag leaves in rice white stripe mutant 6001 during the senescence process. Results showed that the chlorophyll content at the heading and milk-ripe stages in rice mutant 6001 were about 34.78% and 3.00% less than those in wild type 6028, respectively. However, the chlorophyll content at the fully-ripe stage in rice mutant 6001 was higher than that in wild type 6028. At the heading stage, the net photosynthetic rate (Pn) in rice mutant 6001 was lower than that in wild type 6028. Rice mutant 6001 also exhibited a significantly slower decrease rate ofPn than wild type 6028 during the senescence progress, especially at the later stage. Furthermore, Ca2+-ATPase, Mg2+-ATPase and photophosphorylation activities exhibited the similar trends as thePn. During the senescence process, the 68 kDa polypeptide concentrations in the thylakoid membrane proteins exhibited a significant change, which was one of the critical factors that contributed to the observed change in photosynthesis. We also observed that the chloroplasts of rice mutant 6001 exhibited higher integrity than those of wild type 6028, and the chloroplast membrane of rice mutant 6001 disintegrated more slow during the senescence process. In general, rice mutant 6001 had a relatively slower senescence rate than wild type 6028, and exhibited anti-senescence properties.

  13. Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence

    NARCIS (Netherlands)

    Jing, Hai-Chun; Sturre, Marcel J.G.; Hille, Jacques; Dijkwel, Paul P.

    2002-01-01

    The onset of leaf senescence is controlled by leaf age and ethylene can promote leaf senescence within a specific age window. We exploited the interaction between leaf age and ethylene and isolated mutants with altered leaf senescence that are named as onset of leaf death (old) mutants. Early leaf

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

    Science.gov (United States)

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

    2014-06-01

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

  15. Ethanol metabolism in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Sulter, G.J.; Klei, I.J. van der; Schanstra, J.P.; Harder, W.; Veenhuis, M.

    1991-01-01

    This paper describes ethanol metabolism in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. The PER mutant was able to use ethanol as sole-carbon source but showed reduced growth rates compared to wild-type cells together with a reduced rate of ethanol utilization under µmax conditions.

  16. Molecular mapping of three nuclear male sterility mutant genes in cultivated sunflower (Helianthus annuus L.)

    Science.gov (United States)

    The nuclear male sterility (NMS) trait is a useful tool for sunflower (Helianthus annuus L.) breeding and genetic programs. Previously, we induced NMS mutants in cultivated line HA 89. The mutants possessed single recessive genes, ms6, ms7, and ms8, respectively, in NMS HA 89-872, NMS HA 89-552, and...

  17. 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...

  18. Construction of a doramectin producer mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis.

    Science.gov (United States)

    Zhao, Xuejin; Wang, Yuanxin; Wang, Shiwei; Chen, Zhi; Wen, Ying; Song, Yuan

    2009-12-01

    The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain alpha-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant. In a fermentation medium supplemented with cyclohexanecarboxylic acid, this double mutant produced doramectin and its analogues but no oligomycin. The mutant proved to be genetically stable, without any antibiotic resistance markers inserted into its chromosome, and could potentially become an industrial doramectin-producing strain after further improvement.

  19. Rifampicin resistant initiation of chromosome replication from oriC in ihf mutants

    DEFF Research Database (Denmark)

    von Freiesleben, Ulrik; Rasmussen, Knud V.; Atlung, Tove

    2000-01-01

    IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows...... that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from ori......C in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration...

  20. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

    Science.gov (United States)

    Kok, Fatma O; Shin, Masahiro; Ni, Chih-Wen; Gupta, Ankit; Grosse, Ann S; van Impel, Andreas; Kirchmaier, Bettina C; Peterson-Maduro, Josi; Kourkoulis, George; Male, Ira; DeSantis, Dana F; Sheppard-Tindell, Sarah; Ebarasi, Lwaki; Betsholtz, Christer; Schulte-Merker, Stefan; Wolfe, Scot A; Lawson, Nathan D

    2015-01-12

    The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

  1. Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity.

    Science.gov (United States)

    Folch, J L; Antaramián, A; Rodríguez, L; Bravo, A; Brunner, A; González, A

    1989-12-01

    A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.

  2. Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc.

    Science.gov (United States)

    Intorne, Aline C; de Oliveira, Marcos Vinicius V; Lima, Mariana L; da Silva, Juliana F; Olivares, Fábio L; de Souza Filho, Gonçalo Apolinário

    2009-05-01

    Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca(5)(PO(4))(3)OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus.

  3. Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.

    Science.gov (United States)

    McFall, E

    1975-03-01

    A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.

  4. Purine transport by malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster.

    Science.gov (United States)

    Sullivan, D T; Bell, L A; Paton, D R; Sullivan, M C

    1979-06-01

    Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (pp) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.

  5. The transcriptional targets of mutant FOXL2 in granulosa cell tumours.

    Directory of Open Access Journals (Sweden)

    Roseanne Rosario

    Full Text Available BACKGROUND: Despite their distinct biology, granulosa cell tumours (GCTs are treated the same as other ovarian tumours. Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2 402C>G has been found in nearly all GCTs examined. This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. METHODS: The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Total RNA was hybridised to Affymetrix U133 Plus 2 microarrays. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2. RESULTS: The overexpression of wildtype and mutant FOXL2 in COV434, and the silencing of mutant FOXL2 expression in KGN, has shown that mutant FOXL2 is able to differentially regulate the expression of many genes, including two well known FOXL2 targets, StAR and CYP19A. We have shown that many of the genes regulated by mutant FOXL2 are clustered into functional annotations of cell death, proliferation, and tumourigenesis. Furthermore, TGF-β signalling was found to be enriched when using the gene annotation tools GATHER and GeneSetDB. This enrichment was still significant after performing a robust permutation analysis. CONCLUSION: Given that many of the transcriptional targets of mutant FOXL2 are known TGF-β signalling genes, we suggest that deregulation of this key antiproliferative pathway is one way mutant FOXL2 contributes to the pathogenesis of adult-type GCTs. We believe this pathway should be a target for future therapeutic interventions, if outcomes for women with GCTs are to improve.

  6. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    Science.gov (United States)

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

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

    Science.gov (United States)

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

    2014-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Beattie, G.A.; Lindow, S.E. (Univ. of California, Berkeley, CA (United States))

    1994-10-01

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

  9. A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain.

    Science.gov (United States)

    Middelhoven, W J; van Eijk, J; van Renesse, R; Blijham, J M

    1978-01-01

    NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.

  10. Catalytic properties of thimet oligopeptidase H600A mutant

    Energy Technology Data Exchange (ETDEWEB)

    Machado, Mauricio F.M.; Marcondes, Marcelo F. [Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil); Rioli, Vanessa [Laboratorio Especial de Toxinologia Aplicada, Instituto Butantan, 05467-010 Sao Paulo, SP (Brazil); Departamento de Biologia Celular e Desenvolvimento, Universidade de Sao Paulo, 05508-900 Sao Paulo, SP (Brazil); Ferro, Emer S. [Departamento de Biologia Celular e Desenvolvimento, Universidade de Sao Paulo, 05508-900 Sao Paulo, SP (Brazil); Juliano, Maria A.; Juliano, Luiz [Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil); Oliveira, Vitor, E-mail: vitor.oliveira@unifesp.br [Departamento de Biofisica, Universidade Federal de Sao Paulo, 04044-020 Sao Paulo, SP (Brazil)

    2010-04-02

    Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His{sup 600}. In the present work, the role of His{sup 600} of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S{sub 1} and S{sub 1}' specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His{sup 600} residue makes important interactions with the substrate, supporting the prediction that His{sup 600} moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K{sub m} and k{sub cat}, showing the importance of His{sup 600} for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His{sup 600} in TOP catalysis, transferring a proton to the newly generated NH{sub 2}-terminus or helping Tyr{sup 605} and/or Tyr{sup 612} in the intermediate oxyanion stabilization.

  11. Distinctive Klf4 mutants determine preference for DNA methylation status

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Hideharu; Wang, Dongxue; Steves, Alyse N.; Jin, Peng; Blumenthal, Robert M.; Zhang, Xing; Cheng, Xiaodong

    2016-09-04

    Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.

  12. Thermal radiosensitization in radiation-sensitive mutant mouse leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Toshikazu (Hiroshima Univ. (Japan). School of Dentistry)

    1994-06-01

    This study investigated thermal, radiation, and combined thermal radiation sensitization of mouse leukemic cells, L5178Y, and radiation-sensitive mutant cells, LX830. Radiation sensitivity (D[sub 0]) values were 0.41 Gy for LX830 and 1.39 Gy for L5178Y, with the ratio of D[sub 0] values in LX830 to in L5178Y being 3.4. Thus, LX830 was more radiosensitive than L5178Y. LX830 showed no shouldered survival curves. Although sublethal damage (SLD) repair was seen to the almost same degree in both LX830 and L5178Y, potential lethal damage (PLD) repair was scarcely observed in LX830. Both cell lines were similar in thermal sensitivity (T[sub 0]). Eosine staining suggested that cell killing due to hyperthermia had occurred in the interphase in both LX830 and L5178Y. L5178Y showed thermal sensitivity low in the G1 phase and high in the S phase; on the contrary, LX830 showed it high in the G1 phase and low in the S phase. Thermal radiosensitization was similar in both cell lines, although there was a great difference in radiation sensitivity between the cell lines. The difference in radiation sensitivity (D[sub 0]) between L5178Y and LX830 became small when radiation was given at the time of the maximum thermal resistance. This seemed to contribute to a decrease in radiation sensitivity in LX830. It can be concluded that thermal radiosensitization depends on thermal sensitivity and that radiation sensitivity decreases in radiation-sensitive cells when exposed to irradiation at the time of thermal resistance. (N.K.).

  13. Proton Sensing of CLC-0 Mutant E166D

    Science.gov (United States)

    Traverso, Sonia; Zifarelli, Giovanni; Aiello, Rita; Pusch, Michael

    2006-01-01

    CLC Cl− channels are homodimers in which each subunit has a proper pore and a (fast) gate. An additional slow gate acts on both pores. A conserved glutamate (E166 in CLC-0) is a major determinant of gating in CLC-0 and is crucially involved in Cl−/H+ antiport of CLC-ec1, a CLC of known structure. We constructed tandem dimers with one wild-type (WT) and one mutant subunit (E166A or E166D) to show that these mutations of E166 specifically alter the fast gate of the pore to which they belong without effect on the fast gate of the neighboring pore. In addition both mutations activate the common slow gate. E166A pores have a large, voltage-independent open probability of the fast gate (popen), whereas popen of E166D pores is dramatically reduced. Similar to WT, popen of E166D was increased by lowering pHint. At negative voltages, E166D presents a persistent inward current that is blocked by p-chlorophenoxy-acetic acid (CPA) and increased at low pHext. The pHext dependence of the persistent current is analogous to a similar steady inward current in WT CLC-0. Surprisingly, however, the underlying unitary conductance of the persistent current in E166D is about an order of magnitude smaller than that of the transient deactivating inward Cl− current. Collectively, our data support the possibility that the mutated CLC-0 channel E166D can assume two distinct open states. Voltage-independent protonation of D166 from the outside favors a low conductance state, whereas protonation from the inside favors the high conductance state. PMID:16380443

  14. Factors contributing to the biofilm-deficient phenotype of Staphylococcus aureus sarA mutants.

    Directory of Open Access Journals (Sweden)

    Laura H Tsang

    Full Text Available Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases. Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results

  15. Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo.

    Science.gov (United States)

    Kawamata, Hibiki; Peixoto, Pablo; Konrad, Csaba; Palomo, Gloria; Bredvik, Kirsten; Gerges, Meri; Valsecchi, Federica; Petrucelli, Leonard; Ravits, John M; Starkov, Anatoly; Manfredi, Giovanni

    2017-05-08

    Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43(A315T) mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. In the brain of TDP-43(A315T) mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43(A315T) peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43(A315T) mouse

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

    Science.gov (United States)

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

    2016-03-22

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

  17. An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Li, Xiaobo; Zhang, Ru; Patena, Weronika; Gang, Spencer S; Blum, Sean R; Ivanova, Nina; Yue, Rebecca; Robertson, Jacob M; Lefebvre, Paul A; Fitz-Gibbon, Sorel T; Grossman, Arthur R; Jonikas, Martin C

    2016-02-01

    The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

  18. Mutations of nuclear localization signals in mNANOG generate dominant negative mutants

    Institute of Scientific and Technical Information of China (English)

    ZHANG Juan; ZHANG XiaoFei; PEI DuanQing

    2009-01-01

    Mouse NANOG plays a critical role in maintaining self-renewal and pluripotency of embryonic stem cells.Yet,the precise mechanism of how mNANOG functions is still less known.Here,we report that mouse NANOG has two nuclear localization signals (NLS,RKQKMR and RMKCKR) which are respon-sible for the nuclear localization and transcriptional activity in the conserved homeobox domain.NLS mutants of mouse NANOG generate:3 mutants that are localized throughout the cells and lose the transectivation function.We further prove that all three NLS mutants may interact with the wild-type mouse NANOG like NANOG dimerization itself and inhibit the wild-type mouse NANOG activity,acting as dominant negative mutants.The NLS mutants of mouse NANOG may also inhibit activity of oct4 promoter in pluripotent cells,indicating that the NLS mutants can affect the endogenous mouse NANOG function in vivo.These data suggest that the NLS mutants of mouse NANOG may be used as a tool to regulate NANOG activity in pluripotent cells.

  19. A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans

    Science.gov (United States)

    Li, Shiwei; Xu, Shibin; Ma, Yanli; Wu, Shuang; Feng, Yu; Cui, Qingpo; Chen, Lifeng; Zhou, Shuang; Kong, Yuanyuan; Zhang, Xiaoyu; Yu, Jialei; Wu, Mengdi; Zhang, Shaobing O.

    2016-01-01

    To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 × 105 mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion. PMID:27261001

  20. An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

    Institute of Scientific and Technical Information of China (English)

    SUNJIANGE; XIAOLIYAO; 等

    1998-01-01

    In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

  1. Alterations in DNA methylation and genome structure in two rice mutant lines induced by high pressure

    Institute of Scientific and Technical Information of China (English)

    SHEN; Sile; WANG; Zhenwei; SHAN; Xiaohui; WANG; Hua; LI; Ling; LIN; Xuyun; LONG; Likun; WENG; Kenan; LIU; Bao; ZOU; Guangtian

    2006-01-01

    By using high-pressure treatment, two mutant lines were obtained from a genetically stable japonica rice cultivar Bijing38. Genomic DNA of the mutant lines, together with the original line (Bijing38), was either undigested or digested by Hpa II/Msp I, and then subjected to molecular analysis using two markers, ISSR and RAPD. Results indicated that changes in the PCR amplification profiles of both markers are apparent in the two mutant lines compared with the original rice cultivar, suggesting that there had been both sequence changes and DNA methylation modifications in the mutant lines. Southern blot analysis using diverse sequences, including two cellular genes (S2 and S3), a set of retrotransposons (Osr7, Osr36, Tos19 and more), and a MITE transposon family (mPing and Pong), confirmed the results, and indicated that changes in DNA methylation pattern, genomic structure, and possible activation of some transposons indeed occurred in the mutant lines. Moreover, these changes are stably maintained through selfed generations and in different organs. Thus, our results indicate that it is possible to obtain stable mutants in rice by high pressure treatments, and the molecular basis of the mutants may include both genetic and epigenetic changes. Therefore, high hydrostatic pressure seems a promising approach for plant mutagenesis.

  2. Diversity and Stability Study on Rice Mutants Induced in Space Environment

    Institute of Scientific and Technical Information of China (English)

    Wei-Hong Lu; Xin-Zhu Wang; Qi Zheng; Shuang-Hong Guan; Ping Xin; Ye-Qing Sun

    2008-01-01

    To further study the characteristics of changes on the molecular level of rice mutants induced in space environment, we analyzed proteins in leaves and seeds of four rice mutants (two high-tillering and two low-tillering) in the 8th and 9th generations after a 15-day spaceflight, and compared with their ground controls by two-dimentional polyacrylamide gel electrophoresis and reverse phase liquid chromatography (RPLC). In addition, the albumin, globulin, prolamine, glutelin, and amylose of the mutant seeds were analyzed by RPLC and ultra-violet spectrometry. The results showed that the low-abundance proteins of leaves in the peak tillering stage are more likely to be induced compared with their corresponding controls. The albumin, globulin, and prolamine of the mutant seeds revealed changes when compared with their controls, and the characteristics of changes in different mutants were stably inherited in the 8th and 9th generations, suggesting that they can be used as biomarkers to identity the mutants induced by spaceflight. Moreover, two proteins (SSP9111 and SSP6302) were found to be expressed with high intensity (two-fold change) in different mutants, which were both correlated with photosystem according to mass spectrometry and database searching.

  3. Mutant p53 and mTOR/PKM2 regulation in cancer cells.

    Science.gov (United States)

    Dando, Ilaria; Cordani, Marco; Donadelli, Massimo

    2016-09-01

    Mutations of TP53 gene are the most common feature in aggressive malignant cells. In addition to the loss of the tumor suppressive role of wild-type p53, hotspot mutant p53 isoforms display oncogenic proprieties notoriously referred as gain of functions (GOFs) which result in chemoresistance to therapies, genomic instability, aberrant deregulation of cell cycle progression, invasiveness and enhanced metastatic potential, and finally, in patient poor survival rate. The identification of novel functional oncogenic pathways regulated by mutant p53 represent and intriguing topic for emerging therapies against a broad spectrum of cancer types bearing mutant TP53 gene. Mammalian target of rapamycin (mTOR), as well as pyruvate kinase isoform M2 (PKM2) are master regulators of cancer growth, metabolism, and cell proliferation. Herein, we report that GOF mutant R175H and R273H p53 proteins trigger PKM2 phosphorylation on Tyr 105 through the involvement of mTOR signaling. Our data, together with the newly discovered connection between mutant p53 and mTOR stimulation, raise important implications for the potential therapeutic use of synthetic drugs inhibiting mTOR/PKM2 axis in cancer cells bearing mutant TP53 gene. We further hypothesize that mTOR/PKM2 pathway stimulation serves to sustain the oncogenic activity of mutant p53 through both the enhancement of chemoresistance and of aerobic glycolysis of cancer cells. © 2016 IUBMB Life, 68(9):722-726, 2016.

  4. Accumulation of Flavonoids in an ntra ntrb Mutant Leads to Tolerance to UV-C

    Institute of Scientific and Technical Information of China (English)

    Talaat Bashandy; Ludivine Taconnat; Jean-Pierre Renou; Yves Meyer; Jean-Philippe Reichheld

    2009-01-01

    NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of thioredoxins. There are two genes encoding NTRs (NTRA and NTRB) in the Arabidopsis genome, each encoding a cytosolic and a mitochondrial isoform. A double ntra ntrb mutant has recently been characterized and shows slower plant growth, slightly wrinkled seeds and a remarkable hypersensitivity to buthionine sulfoximine (BSO), a specific inhibitor of gluta-thione biosynthesis. In this paper, we demonstrate that this mutant also accumulates higher level of flavonoids. Analysis of transcriptome data showed that several genes of the flavonoid pathway are overexpressed in the ntra ntrb mutant. Accumulation of flavonoids is generally considered a hallmark of plant stress. Nevertheless, no elevation of the expression of genes encoding ROS-detoxification enzymes was observed, suggesting that the ntra ntrb plants do not suffer from oxidative disease. Another hypothesis suggests that flavonoids are specifically synthesized in the ntra ntrb mutant in order to rescue the inactivation of NTR. To test this, the ntra ntrb mutant was crossed with transparent testa 4 (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis. As ntra ntrb plants are more resistant to UV-C treatment than wild-type plants, this higher resistance was abolished in the ntra ntrb tt4 mutant, suggesting that accumulation of flavonoids in the ntra ntrb mutant protects plants against UV-light.

  5. Induction of two mutants in birdsfoot trefoil (Lotus corniculatus) by x-rays and chemical mutagens

    Energy Technology Data Exchange (ETDEWEB)

    Therrien, M.C.; Grant, W.F. (McGill Univ., Ste. Anne de Bellevue, Quebec (Canada). Macdonald Coll.)

    1982-10-01

    The mutagenic effects of X-rays, ethyl methanesulfonate (EMS), 8-ethoxycaffeine (EOC), N-hydroxyurea (HU) and 2-aminopurine (2AP) on seed treatment of birdsfoot trefoil (Lotus corniculatus L. 'Mirabel') were assessed over four generations. Mutants were recovered in the M/sub 2/, M/sub 3/ and M/sub 4/ generations from selfed lines, from crosses derived form selfed lines and from open pollination lines. Mutant plants exhibiting vestigial floret character were recovered from X-rays, EMS, EOC and HU treatments. Mutant chlorotica plants were obtained from EMS treatment only. No mutants were recovered from 2AP treatment, EMS, the most effective mutagen, produced nine vestigial floret and 12 chlorotica mutants. Mutants were obtained from only one exposure of X-rays (12 krad). There was evidence for preferential elimination of gametes. The chlorotica and vestigial floret mutants were inherited as tetrasomic recessives. Mutation frequencies of 0.4 - 3.1% in a tetrasomic background are indicative of the effectiveness of EMS in birdsfoot trefoil.

  6. Atrial fibrillation-linked germline GJA5/connexin40 mutants showed an increased hemichannel function.

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    Yiguo Sun

    Full Text Available Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40 have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca2+]o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.

  7. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

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    Ouyang Xiaosen

    2011-06-01

    Full Text Available Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity. Results In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy. Conclusions These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

  8. Morphological Characterization of a New and Easily Recognizable Nuclear Male Sterile Mutant of Sorghum (Sorghum bicolor)

    Science.gov (United States)

    Xin, Zhanguo; Huang, Jian; Smith, Ashley R.; Chen, Junping; Burke, John; Sattler, Scott E.

    2017-01-01

    Sorghum (Sorghum bicolor L. Moench) is one of the most important grain crops in the world. The nuclear male sterility (NMS) trait, which is caused by mutations on the nuclear gene, is valuable for hybrid breeding and genetic studies. Several NMS mutants have been reported previously, but none of them were well characterized. Here, we present our detailed morphological characterization of a new and easily recognizable NMS sorghum mutant male sterile 8 (ms8) isolated from an elite inbred BTx623 mutagenized by ethyl methane sulfonate (EMS). Our results show that the ms8 mutant phenotype was caused by a mutation on a single recessive nuclear gene that is different from all available NMS loci reported in sorghum. In fertile sorghum plants, yellow anthers appeared first during anthesis, while in the ms8 mutant, white hairy stigma emerged first and only small white anthers were observed, making ms8 plants easily recognizable when flowering. The ovary development and seed production after manual pollination are normal in the ms8 mutant, indicating it is female fertile and male sterile only. We found that ms8 anthers did not produce pollen grains. Further analysis revealed that ms8 anthers were defective in tapetum development, which led to the arrest of pollen formation. As a stable male sterile mutant across different environments, greenhouses, and fields in different locations, the ms8 mutant could be a useful breeding tool. Moreover, ms8 might be an important for elucidating male gametophyte development in sorghum and other plants. PMID:28052078

  9. The isolation of Staphylococcus aureus tea tree oil-reduced susceptibility mutants.

    Science.gov (United States)

    Cuaron, Jesus A; Dulal, Santosh; Cooke, Peter H; Torres, Nathanial J; Gustafson, John E

    2014-08-01

    Tea tree oil (TTO)-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. Revertants were also isolated from the TTORS mutants following growth in drug-free media, and all revertant strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that an SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared with parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in an SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

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

    Science.gov (United States)

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

    2009-08-01

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

  11. Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells.

    Science.gov (United States)

    Menendez, Daniel; Lowe, Julie M; Snipe, Joyce; Resnick, Michael A

    2016-09-20

    Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy.

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

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    Xue Xu

    2014-06-01

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

  13. Glycoprotein processing in mutants of HSV-1 that induce cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.; Kousoulas, K.G.; Knowles, R.W.; Read, G.S.; Holland, T.C.; Keller, P.M.; Warner, S.C.

    1982-01-01

    The synthesis of viral-specified glycoproteins, and their appearance on cell surfaces, were compared for cells infected either with syncytial mutants of HSV-1 or with the parental strains from which the mutants were derived. The mutants MP and tsB5, representatives of two different viral genes that affect fusion, were employed in these studies. Cells infected with either mutant gave rise to reduce surface labeling of major viral-specified glycoproteins throughout infection relative to the parental strains. Putative glycoprotein precursors were synthesized in similar amounts throughout infection in mutant and wild-type infected cells. However, during a chase period after a pulse of labeling, fully glycosylated species of glycoproteins accumulated more slowly in mutant than in parent infected cells. The effect was clearly visible early in MP-infected cells and by 9 hr after infection for tsB5-infected cells, and increased thereafter. Apparently the decrease in appearance of glycoproteins on the cell surface of mutant-infected cells is due to a decrease in their rate of processing. 26 references, 7 figures.

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

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    Chao Liu

    2015-08-01

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

  15. WHITE OYSTER MUSHROOM (p leurotus F lorida MUTANT WITH ALTERED ANTIOXIDANT CONTENTS

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    IRA DJAJANEGARA

    2008-01-01

    Full Text Available Radiation using gamma ray (60Co at 0.75 KGray with dose velocity of 1.149 KGray/hour on white oyster mushroom ( Pleurotus florida mycelia yielded several mutants. Based on isozyme analysis using two enzyme markers such as esterase (EST and acid phosphatase (ACP showed that 3 putative mutants (PO-3, PO-4 and PO-5 among 5 mutants are positive. Even though the isozyme patterns indicated that those 3 putative mutants are positively mutated, only PO-5 showed higher productivity compared to control (PO-K which is reflected by significantly higher number of fruit bodies, higher fresh weight and dry weight yield of three successive flush periods. It was assumed that the mutation which occurred in PO-3 and PO-4 may affect other trait(s of the white oyster mushroom. Antioxidant analysis of those mutants indicate that mutant PO-4 has significantly higher antioxidant content compared to control (PO-K and the two other mutants (PO-3 & PO-5. This finding leads to the possible applica -tion of white oyster mushroom as a natural antioxidant source.

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

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    Carmen Y S Chu

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

  17. Properties of mutants of haemophilus influenzae deficient in ATP-dependent deoxyribonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.

    1976-01-01

    Eight isogenic Haemophilus influenzae strains whose extracts lack ATP-dependent deoxyribonuclease activity (Add/sup -/ mutants) form three complementation and genetic linkage groups. Since there are known to be three subunits of the enzyme, these data suggest that each of the three genes specifies a different subunit. Gel electrophoresis of partially purified mutant extracts indicates that the smallest subunit is missing in one of the groups but is present in all the other mutants. The mutants are more sensitive to a variety of chemical agents than the wild type. The most sensitive mutants lack the ATPase activity associated with the enzyme. These strains exhibit aberrant incorporation of tritiated thymidine, which starts up more rapidly and shuts off sooner than in the wild type. An extracellular compound is responsible for most of this effect, in that wild type cells put into medium in which Add/sup -/ cells have been growing show a similar aberrant incorporation. The effect of these media can be mimicked by cyclic AMP and cyclic GMP, although millimolar concentrations are required. It is postulated that the Add/sup -/ mutants are more permeable to many substances than the wild type, partly because of the extracellular compound usually surrounding them, and the increased permeability might be responsible for the mutants' nonviability.

  18. A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans

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    Shiwei Li

    2016-08-01

    Full Text Available To identify genes that regulate the dynamics of lipid droplet (LD size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 × 105 mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10. The nine groups are named drop (lipid droplet abnormal and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.

  19. Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium

    Energy Technology Data Exchange (ETDEWEB)

    Boominathan, K.; Dass, S.B.; Randall, T.A.; Kelley, R.L.; Reddy, C.A. (Michigan State Univ., East Lansing (USA))

    1990-01-01

    Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study the authors isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity ({sup 14}C-labeled synthetic lignin {yields}{sup 14}CO{sub 2}) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidases, a key H{sub 2}O{sub 2}-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.

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

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    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.

  1. Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast.

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    Yuzy Matsuo

    Full Text Available Polypeptone is widely excluded from Schizosaccharomyces pombe growth medium. However, the reasons why polypeptone should be avoided have not been documented. Polypeptone dramatically induced cell lysis in the ura4 deletion mutant when cells approached the stationary growth phase, and this phenotype was suppressed by supplementation of uracil. To determine the specificity of this cell lysis phenotype, we created deletion mutants of other genes involved in de novo biosynthesis of uridine monophosphate (ura1, ura2, ura3, and ura5. Cell lysis was not observed in these gene deletion mutants. In addition, concomitant disruption of ura1, ura2, ura3, or ura5 in the ura4 deletion mutant suppressed cell lysis, indicating that cell lysis induced by polypeptone is specific to the ura4 deletion mutant. Furthermore, cell lysis was also suppressed when the gene involved in coenzyme Q biosynthesis was deleted. This is likely because Ura3 requires coenzyme Q for its activity. The ura4 deletion mutant was sensitive to zymolyase, which mainly degrades (1,3-beta-D glucan, when grown in the presence of polypeptone, and cell lysis was suppressed by the osmotic stabiliser, sorbitol. Finally, the induction of cell lysis in the ura4 deletion mutant was due to the accumulation of orotidine-5-monophosphate. Cell wall integrity was dramatically impaired in the ura4 deletion mutant when grown in the presence of polypeptone. Because ura4 is widely used as a selection marker in S. pombe, caution needs to be taken when evaluating phenotypes of ura4 mutants.

  2. Morphological Structure and Genetic Mapping of New Leaf-Color Mutant Gene in Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    LI Yu-hong; WANG Bao-he; DAI Zheng-yuan; LI Ai-hong; LIU Guang-qing; ZUO Shi-min; ZHANG Hong-xi; PAN Xue-biao

    2012-01-01

    Leaf-color mutations are a widely-observed class of mutations,playing an important role in the study of chlorophyll biosynthesis and plant chloroplast structure,function,genetics and development.A naturally-occurring leaf-color rice mutant,Baihuaidao 7,was analyzed.Mutant plants typically exhibited a green-white-green leaf-color progression,but this phenotype was only expressed in the presence of a stress signal induced by mechanical scarification such as transplantation.Prior to the appearance of white leaves,mutant plant growth,leaf color,chlorophyll content,and chloroplast ultrastructure appeared to be identical to those of the wild type.After the changeover to white leaf color,an examination of the mutated leaves revealed a decrease in total chlorophyll,chlorophyll a,chlorophyll b,and carotenoid content,a reduction in the number of chloroplast grana lamella and grana,and a gradual degradation of the thylakoid lamellas.At maturity,the mutant plant was etiolated and dwarfed compared with wild-type plants.Genetic analysis indicated that the leaf mutant character is controlled by a recessive nuclear gene.Genetic mapping of the mutant gene was performed using an F2 population derived from a Baihuaidao 7 ×Jiangxi 1587 cross.The mutant gene was mapped to rice chromosome 11,positioned between InDel markers L59.2-7 and L64.8-11,which are separated by approximately 740.5 kb.The mutant gene is believed to be a new leaf-color mutant gene in rice,and is tentatively designated as gwgl.

  3. Identification of a mutant PfCRT-mediated chloroquine tolerance phenotype in Plasmodium falciparum.

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    Stephanie G Valderramos

    2010-05-01

    Full Text Available Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ, the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1 suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2 implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3 uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt.

  4. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  6. Far-red light-insensitive, phytochrome A-deficient mutants of tomato.

    Science.gov (United States)

    van Tuinen, A; Kerckhoffs, L H; Nagatani, A; Kendrick, R E; Koornneef, M

    1995-01-20

    We have selected two recessive mutants of tomato with slightly longer hypocotyls than the wild type, one under low fluence rate (3 mumol/m2/s) red light (R) and the other under low fluence rate blue light. These two mutants were shown to be allelic and further analysis revealed that hypocotyl growth was totally insensitive to far-red light (FR). We propose the gene symbol fri (far-red light insensitive) for this locus and have mapped it on chromosome 10. Immunochemically detectable phytochrome A polypeptide is essentially absent in the fri mutants as is the bulk spectrophotometrically detectable labile phytochrome pool in etiolated seedlings. A phytochrome B-like polypeptide is present in normal amounts and a small stable phytochrome pool can be readily detected by spectrophotometry in the fri mutants. Inhibition of hypocotyl growth by a R pulse given every 4 h is quantitatively similar in the fri mutants and wild type and the effect is to a large extent reversible if R pulses are followed immediately by a FR pulse. After 7 days in darkness, both fri mutants and the wild type become green on transfer to white light, but after 7 days in FR, the wild-type seedlings that have expanded their cotyledons lose their capacity to green in white light, while the fri mutants de-etiolate. Adult plants of the fri mutants show retarded growth and are prone to wilting, but exhibit a normal elongation response to FR given at the end of the daily photoperiod. The inhibition of seed germination by continuous FR exhibited by the wild type is normal in the fri mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Photoinhibition of photosystem I in a pea mutant with altered LHCII organization.

    Science.gov (United States)

    Ivanov, A G; Morgan-Kiss, R M; Krol, M; Allakhverdiev, S I; Zanev, Yu; Sane, P V; Huner, N P A

    2015-11-01

    Comparative analysis of in vivo chlorophyll fluorescence imaging revealed that photosystem II (PSII) photochemical efficiency (Fv/Fm) of leaves of the Costata 2/133 pea mutant with altered pigment composition and decreased level of oligomerization of the light harvesting chlorophyll a/b-protein complexes (LHCII) of PSII (Dobrikova et al., 2000; Ivanov et al., 2005) did not differ from that of WT. In contrast, photosystem I (PSI) activity of the Costata 2/133 mutant measured by the far-red (FR) light inducible P700 (P700(+)) signal exhibited 39% lower steady state level of P700(+), a 2.2-fold higher intersystem electron pool size (e(-)/P700) and higher rate of P700(+) re-reduction, which indicate an increased capacity for PSI cyclic electron transfer (CET) in the Costata 2/133 mutant than WT. The mutant also exhibited a limited capacity for state transitions. The lower level of oxidizable P700 (P700(+)) is consistent with a lower amount of PSI related chlorophyll protein complexes and lower abundance of the PsaA/PsaB heterodimer, PsaD and Lhca1 polypeptides in Costata 2/133 mutant. Exposure of WT and the Costata 2/133 mutant to high light stress resulted in a comparable photoinhibition of PSII measured in vivo, although the decrease of Fv/Fm was modestly higher in the mutant plants. However, under the same photoinhibitory conditions PSI photochemistry (P700(+)) measured as ΔA820-860 was inhibited to a greater extent (50%) in the Costata 2/133 mutant than in the WT (22%). This was accompanied by a 50% faster re-reduction rate of P700(+) in the dark indicating a higher capacity for CET around PSI in high light treated mutant leaves. The role of chloroplast thylakoid organization on the stability of the PSI complex and its susceptibility to high light stress is discussed.

  8. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

    Science.gov (United States)

    Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

    2016-03-10

    Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. © 2016 by The American Society of Hematology.

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

    Science.gov (United States)

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

    2013-02-01

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

  10. Phenotypes of Myopathy-related Actin Mutants in differentiated C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    Machesky Laura M

    2007-01-01

    Full Text Available Abstract Background About 20 % of nemaline myopathies are thus far related to skeletal muscle alpha-actin. Seven actin mutants located in different parts of the actin molecule and linked to different forms of the disease were selected and expressed as EGFP-tagged constructs in differentiated C2C12 mytoubes. Results were compared with phenotypes in patient skeletal muscle fibres and with previous expression studies in fibroblasts and C2C12 myoblasts/myotubes. Results Whereas EGFP wt-actin nicely incorporated into endogenous stress fibres and sarcomeric structures, the mutants showed a range of phenotypes, which generally changed upon differentiation. Many mutants appeared delocalized in myoblasts but integrated into endogenous actin structures after 4–6 days of differentiation, demonstrating a poor correlation between the appearance in myotubes and the severity of the disease. However, for some mutants, integration into stress fibres induced aberrant structures in differentiated cells, like thickening or fragmentation of stress fibres. Other mutants almost failed to integrate but formed huge aggregates in the cytoplasm of myotubes. Those did not co-stain with alpha-actinin, a main component of nemaline bodies found in patient muscle. Interestingly, nuclear aggregates as formed by two of the mutants in myoblasts were found less frequently or not at all in differentiated cells. Conclusion Myotubes are a suitable system to study the capacity of a mutant to incorporate into actin structures or to form or induce pathological changes. Some of the phenotypes observed in undifferentiated myoblasts may only be in vitro effects. Other phenotypes, like aberrant stress fibres or rod formation may be more directly correlated with disease phenotypes. Some mutants did not induce any changes in the cellular actin system, indicating the importance of additional studies like functional assays to fully characterize the pathological impact of a mutant.

  11. Selective knockdown of mutant SOD1 in Schwann cells ameliorates disease in G85R mutant SOD1 transgenic mice.

    Science.gov (United States)

    Wang, Lijun; Pytel, Peter; Feltri, M Laura; Wrabetz, Lawrence; Roos, Raymond P

    2012-10-01

    Mutants of superoxide dismutase type 1 (mtSOD1) that have full dismutase activity (e.g., G37R) as well as none (e.g., G85R) cause familial amyotrophic lateral sclerosis (FALS), indicating that mtSOD1-induced FALS results from a toxicity rather than loss in SOD1 enzymatic activity. Still, it has remained unclear whether mtSOD1 dismutase activity can influence disease. A previous study demonstrated that Cre-mediated knockdown of G37R expression in Schwann cells (SCs) of G37R transgenic mice shortened the late phase of disease and survival. These results suggested that the neuroprotective effect of G37R expressed in SCs was greater than its toxicity, presumably because its dismutase activity counteracted reactive oxygen species (ROS). In order to further investigate this, we knocked down G85R in SCs by crossing G85R(flox) mice with myelin-protein-zero (P(0)):Cre mice, which express Cre recombinase in SCs. Knockdown of G85R in SCs of G85R mice delayed disease onset and extended survival indicating that G85R expression in SCs is neurotoxic. These results demonstrate differences in the effect on disease of dismutase active vs. inactive mtSOD1 suggesting that both a loss as well as gain in function of mtSOD1 influence FALS pathogenesis. The results suggest that mtSOD1-induced FALS treatment may have to be adjusted depending on the cell type targeted and particular mtSOD1 involved.

  12. Gain-of-function of mutant p53: mutant p53 enhances cancer progression by inhibiting KLF17 expression in invasive breast carcinoma cells.

    Science.gov (United States)

    Ali, Amjad; Shah, Abdus Saboor; Ahmad, Ayaz

    2014-11-01

    Kruppel-like-factor 17 (KLF17) is a negative regulator of metastasis and epithelial-mesenchymal-transition (EMT). However, its expression is downregulated in metastatic breast cancer that contains p53 mutations. Here, we show that mutant-p53 plays a key role to suppress KLF17 and thereby enhances cancer progression, which defines novel gain-of-function (GOF) of mutant-p53. Mutant-p53 interacts with KLF17 and antagonizes KLF17 mediated EMT genes transcription. Depletion of KLF17 promotes cell viability, decreases apoptosis and induces drug resistance in metastatic breast cancer cells. KLF17 suppresses cell migration and invasion by decreasing CD44, PAI-1 and Cyclin-D1 expressions. Taken together, our results show that KLF17 is important for the suppression of metastasis and could be a potential therapeutic target during chemotherapy.

  13. Reduced arbuscular mycorrhizal colonization in tomato ethylene mutants Reduzida formação de micorrízas arbusculares em tomateiros mutantes em etileno

    Directory of Open Access Journals (Sweden)

    Agustin Zsögön

    2008-01-01

    Full Text Available Plant hormones are likely key regulators of arbuscular mycorrhizae (AM development. However, their roles in AM are not well known. Here mutants in five hormone classes introgressed in a single tomato (Lycopersicon esculentum Mill. Syn Solanum lycopersicum L. background (cv. Micro-Tom were used to determine their effects on AM development and the expression of defense-related genes (chitinases and b-1,3-glucanases in roots. Under low P conditions, mutant epinastic (epi and Never ripe (Nr, ethylene overproducer and low sensitivity, respectively, had the intraradical colonization by Glomus clarum highly inhibited, as compared to the control Micro-Tom (MT. No significant alterations in fungal colonization were observed in mutants affecting other hormone classes. Under low P conditions, the steady state levels of transcripts encoding a class I basic chitinase (chi9 were higher in mycorrhizal epi and Nr mutant roots as compared to MT controls. In contrast the steady state levels of a class III acidic b-1,3-glucanase (TomPR-Q'a transcripts in mycorrhizal epi mutant roots were significantly lower than in mycorrhizal MT roots. Root colonization in epi mutants was accompanied by several alterations in fungal morphology, as compared to root colonization in MT controls. The data suggest that ethylene may play an important role in controlling intraradical arbuscular mycorrhizal fungal growth.Os hormônios vegetais são possíveis reguladores chave do desenvolvimento de micorrizas arbusculares (MAS. Contudo, seus papéis em MA são pouco conhecidos. No presente estudo, foram utilizados mutantes em cinco classes hormonais introgredidos em uma única cultivar (cv. Micro-Tom de tomateiro (Lycopersicon esculentum Mill. Syn Solanum lycopersicum L. para determinar seus efeitos no desenvolvimento de MA e expressão de genes relacionados à defesa (quitinases e b-1,3-glucanases em raízes. Sob condição de baixo P, os mutantes epinastic (epi e Never ripe (Nr, os

  14. Determining mutant spectra of three RNA viral samples using ultra-deep sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H

    2012-06-06

    RNA viruses have extremely high mutation rates that enable the virus to adapt to new host environments and even jump from one species to another. As part of a viral transmission study, three viral samples collected from naturally infected animals were sequenced using Illumina paired-end technology at ultra-deep coverage. In order to determine the mutant spectra within the viral quasispecies, it is critical to understand the sequencing error rates and control for false positive calls of viral variants (point mutantations). I will estimate the sequencing error rate from two control sequences and characterize the mutant spectra in the natural samples with this error rate.

  15. Hepatitis B surface gene 145 mutant as a minor population in hepatitis B virus carriers

    Directory of Open Access Journals (Sweden)

    Komatsu Haruki

    2012-01-01

    Full Text Available Abstract Background Hepatitis B virus (HBV can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145 is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted. Methods The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years; group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years. To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products. Results By mutant-specific real-time PCR, one subject (5.6% was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6% of the group 1 subjects and 10 (9.3% of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects of 12 subjects who had overlapped peaks at nt 587 in the electropherogram. Conclusions The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in

  16. Características fisiológicas de microtomateiros fitocromo-mutantes

    OpenAIRE

    Melo,Hyrandir Cabral de; Castro,Evaristo Mauro de; Soares,Ângela Maria; Oliveira,Cynthia de; Ramos,Sílvio Júnio

    2009-01-01

    Objetivou-se, neste trabalho, caracterizar aspectos fisiológicos de microtomateiros (Lycopersicon esculentum P. Miller cv. Micro-Tom) fitocromo-mutantes. A cultivar Micro-Tom e os mutantes aurea (deficiente na biossíntese do cromóforo dos fitocromos), atroviolacea (atv) e high pigment1 (hp1;ambos superexpressam eventos mediados por fitocromos) foram cultivados em condições controladas de luz e temperatura e caracterizados no estágio de floração. O mutante hp1 obteve as maiores taxas de fotoss...

  17. Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Doonan, C.J.; Wilson, H.L.; Rajagopalan, K.V.; Garrett, R.M.; Bennett, B.; Prince, R.C.; George, G.N.

    2009-06-02

    The molybdenum site of the Arginine 160 {yields} Glutamine clinical mutant of the physiologically vital enzyme sulfite oxidase has been investigated by a combination of X-ray absorption spectroscopy and density functional theory calculations. We conclude that the mutant enzyme has a six-coordinate pseudo-octahedral active site with coordination of Glutamine O{sup {epsilon}} to molybdenum. This contrasts with the wild-type enzyme which is five-coordinate with approximately square-based pyramidal geometry. This difference in the structure of the molybdenum site explains many of the properties of the mutant enzyme which have previously been reported.

  18. Comparative study of the mutant prevention concentrations of moxifloxacin, levofloxacin, and gemifloxacin against pneumococci.

    Science.gov (United States)

    Credito, Kim; Kosowska-Shick, Klaudia; McGhee, Pamela; Pankuch, Glenn A; Appelbaum, Peter C

    2010-02-01

    We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P<0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 microg/ml) within the susceptible range.

  19. Comparative Study of the Mutant Prevention Concentrations of Moxifloxacin, Levofloxacin, and Gemifloxacin against Pneumococci▿ †

    Science.gov (United States)

    Credito, Kim; Kosowska-Shick, Klaudia; McGhee, Pamela; Pankuch, Glenn A.; Appelbaum, Peter C.

    2010-01-01

    We tested the propensity of three quinolones to select for resistant Streptococcus pneumoniae mutants by determining the mutant prevention concentration (MPC) against 100 clinical strains, some of which harbored mutations in type II topoisomerases. Compared with levofloxacin and gemifloxacin, moxifloxacin had the lowest number of strains with MPCs above the susceptibility breakpoint (P < 0.001), thus representing a lower selective pressure for proliferation of resistant mutants. Only moxifloxacin gave a 50% MPC (MPC50) value (1 μg/ml) within the susceptible range. PMID:20008781

  20. Gamma-rays and EMS induced pentaphyllous mutant in black gram (Vigna mungo).

    Science.gov (United States)

    Singh, R K; Raghuvanshi, S S; Prakash, D

    1988-01-01

    Pentaphyllous mutants in black gram were isolated in M2 generation of a segregating family, irradiated at 20 kR. The genetic nature of mutants was tested by hybridizing with controls, and chi-square tests applied to the F2 population, proved it to be a monogenic recessive. The pentaphyllous mutant had a greater number of pods and leaves per plant and larger and more root nodules. It also showed improved nutritional value with increased seed protein percentage and no increase in TIA (trypsin inhibitor activity).

  1. Mildew-resistant mutants induced in North American two- and six-rowed malting barley cultivars

    DEFF Research Database (Denmark)

    Molina-Cano, J.L.; Simiand, J.P.; Sopena, A.;

    2003-01-01

    and were shown to have two new alleles at the mlo locus; these were designated, respectively, mlo31 and mlo32. Mutant URS2, showing partial resistance, was inherited as a dominant gene, but was not an allele at the Mla locus. The mean yield of each mutant was higher than that of its parental line......, but both URS1 and URS2 showed lower malt extract than Ursula. This lower extract might be due to the smaller grain size of the mutants that could, in turn, result from necrotic lesions in the leaves, as implied by the effects on grain yield....

  2. PHENOTYPIC ANALYSIS OF OsTPKb LOSS OF FUNCTION MUTANT RICE LINES

    Directory of Open Access Journals (Sweden)

    Isayenkov S. V.

    2015-08-01

    Full Text Available The results of screen and analysis of two OsTPKb rice mutant lines were described. The phenotypes and growth rate level of homozygous mutant plants of both rice lines were estimated. The electron microscopy of aleurone layer from forming seeds was performed. The OsTPKb mutant plants demonstrate lower growth rate in comparison with wild type plants. The loss of function OsTPKb mutations invariably led to (semisterile rice plants. The functional disruption of OsTPKb channel has negative impact on plant growth and development. It might completely change the cell morphology of aleurone layer.

  3. Similarity in properties and mapping of three rec mutants of Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Kooistra, J.; Setlow, J.K.

    1976-07-01

    Three Rec/sup -/ mutants of Haemophilus influenzae have been studied with respect to their transformability, ultraviolet and mitomycin C sensitivities, spontaneous and ultraviolet-induced deoxyribonucleic acid breakdown, inducibility of lysogens, and the linkage of the three mutations to a streptomycin resistance marker. The data indicate that the three mutations cause the same phenotypic changes, and that they are all on the same gene. Transformability of the mutants is different when two different media are used for competence development, although transformability with the two competence methods is not different in a Rec/sup -/ strain that is mutant at another gene.

  4. Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin.

    Science.gov (United States)

    Gebru, Elias; Choi, Myung-Jin; Lee, Seung-Jin; Damte, Dereje; Park, Seung Chun

    2011-10-01

    The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC(24 h))/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC(24 h)/MIC (AUC(24 h)/MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC(24 h)/MIC ratios of 40-60. AUC(24 h)/MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.

  5. A forward genetic screen with a thalamocortical axon reporter mouse yields novel neurodevelopment mutants and a distinct emx2 mutant phenotype

    Directory of Open Access Journals (Sweden)

    Vock Vita M

    2011-01-01

    Full Text Available Abstract Background The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. Results To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs, but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. Conclusions These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the

  6. Regulation of (1,3;1,4)-beta-D-glucan synthesis in developing endosperm of barley lys mutants

    DEFF Research Database (Denmark)

    Christensen, Ulla; Scheller, Henrik Vibe

    2012-01-01

    The mechanism behind altered content of (1,3:1,4)-beta-D-glucan was investigated in developing endosperm of barley lys3 and lys5 mutants. Both types of mutants are primarily affected in starch biosynthesis, and hence effects on (1,3;1,4)-beta-D-glucan are likely to be pleiotropic. The mutant alle...

  7. Population Shift between the Open and Closed States Changes the Water Permeability of an Aquaporin Z Mutant

    DEFF Research Database (Denmark)

    Xin, Lin; Helix Nielsen, Claus; Su, Haibin;

    2012-01-01

    gate in the triple mutant with R189 as the primary steric gate in both mutant and WT AqpZ. The double gates (R189 and W43-F183) result in a high population of the closed conformation in the mutant. Occasionally an open state, with diffusive water permeability very close to that of WT AqpZ, was observed...

  8. What can long-lived mutants tell us about mechanisms causing aging and lifespan variation in natural environments?

    NARCIS (Netherlands)

    Briga, Michael; Verhulst, Simon

    2015-01-01

    Long-lived mutants of model organisms have brought remarkable progress in our understanding of aging mechanisms. However, long-lived mutants are usually maintained in optimal standardized laboratory environments (SLEs), and it is not obvious to what extent insights from long-lived mutants in SLEs ca

  9. What can long-lived mutants tell us about mechanisms causing aging and lifespan variation in natural environments?

    NARCIS (Netherlands)

    Briga, Michael; Verhulst, Simon

    2015-01-01

    Long-lived mutants of model organisms have brought remarkable progress in our understanding of aging mechanisms. However, long-lived mutants are usually maintained in optimal standardized laboratory environments (SLEs), and it is not obvious to what extent insights from long-lived mutants in SLEs

  10. 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.

  11. Enhanced lipid production in thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738.

    Science.gov (United States)

    Sachdeva, Neha; Gupta, Ravi Prakash; Mathur, Anshu Shankar; Tuli, Deepak Kumar

    2016-12-01

    The present study aimed to develop thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738 for high lipids production. For this, ethyl methane sulfonate was used, which generated two effective thermo-tolerant mutants, M18 and M24 of Chlorella pyrenoidosa NCIM 2738, capable of surviving at temperature up to 47°C and showing improved lipid and biomass yields. They showed 59.62% and 50.75% increase, respectively in lipid content compared to wild type at 30°C, which could not grow at temperature above 35°C. The novelty of this study lied in incorporation of PAM Flurometry with mutagenesis to generate thermo-tolerant mutants of C. pyrenoidosa and investigating the reasons for increased yields of mutants at cellular and photosynthetic levels with the aim to use them for commercial biodiesel production.

  12. Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1989-01-01

    R and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show...

  13. Proton induced X-ray emission and electron microscopy analysis of induced mutants of sorghum

    CSIR Research Space (South Africa)

    Mbambo, Z

    2014-01-01

    Full Text Available of elements in preferential accumulation tissues and entire changes in cellular localization. Transmission and scanning electron microscopy of the mutants resolved changes in size, shape, ultra-structure and packed cell volumes of protein- and starch bodies...

  14. In silico prediction of mutant HIV-1 proteases cleaving a target sequence

    CERN Document Server

    Jensen, Jan H; Winther, Jakob R; De Vico, Luca

    2014-01-01

    HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636 -- 1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidate...

  15. Reactivating mutant p53 using small molecules as zinc metallochaperones: awakening a sleeping giant in cancer.

    Science.gov (United States)

    Blanden, Adam R; Yu, Xin; Loh, Stewart N; Levine, Arnold J; Carpizo, Darren R

    2015-11-01

    Tumor protein p53 (TP53) is the most commonly mutated gene in human cancer. The majority of mutations are missense, and generate a defective protein that is druggable. Yet, for decades, the small-molecule restoration of wild-type (WT) p53 function in mutant p53 tumors (so-called p53 mutant 'reactivation') has been elusive to researchers. The p53 protein requires the binding of a single zinc ion for proper folding, and impairing zinc binding is a major mechanism for loss of function in missense mutant p53. Here, we describe recent work defining a new class of drugs termed zinc metallochaperones that restore WT p53 structure and function by restoring Zn(2+) to Zn(2+)-deficient mutant p53.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-01-01

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

  17. A direct screening procedure for gravitropism mutants in Arabidopsis thaliana (L. ) Heynh

    Energy Technology Data Exchange (ETDEWEB)

    Bullen, B.L.; Best, T.R.; Gregg, M.M.; Barsel, S.E.; Poff, K.L. (Michigan State Univ., East Lansing (USA))

    1990-06-01

    In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable.

  18. A mutant sialidase having trans-sialidase activity for use in production of sialylated glycans

    DEFF Research Database (Denmark)

    2014-01-01

    galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), malto-oligosaccharides (MOS), isomalto-oligosaccarides (IMO), lactulose, melibiose, maltose, glycosyl sucrose, lactosucrose and fucose. Trans-sialidated mono- and oligo- saccharides, produced with the mutant enzyme, are useful in preparing...

  19. Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2.

    Science.gov (United States)

    Hoffman, Ellen J; Turner, Katherine J; Fernandez, Joseph M; Cifuentes, Daniel; Ghosh, Marcus; Ijaz, Sundas; Jain, Roshan A; Kubo, Fumi; Bill, Brent R; Baier, Herwig; Granato, Michael; Barresi, Michael J F; Wilson, Stephen W; Rihel, Jason; State, Matthew W; Giraldez, Antonio J

    2016-02-17

    Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

  20. Isolation and characterization of temperature-sensitive mutants of cyanophage N-1.

    Science.gov (United States)

    Sarma, T A; Singh, R

    1994-02-01

    Optimal conditions for the induction of temperature-sensitive (ts) mutants of cyanophage N-1 were established after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A treatment with MNNG (400 micrograms/ml) for 2 hrs at pH 8.0 induced ts-mutants at a maximum frequency of 1.46 x 10(-3). A characterization of 10 such ts-mutants with regard to adsorption, one-step growth and temperature-shift experiments with Nostoc muscorum as host bacterium led to the identification of temperature-sensitive steps in the phage multiplication at the restrictive temperature (37 degrees C). All the mutants were found to be conditionally lethal at 37 degrees C since they resumed growth upon shifting to 28 degrees C.

  1. Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Greg D. Bell

    2002-01-01

    Full Text Available To investigate how hyperthermophilic archaea can propagate their genomes accurately, we isolated Sulfolobus acidocaldarius mutants exhibiting abnormally high rates of spontaneous mutation. Our isolation strategy involved enrichment for mutator lineages via alternating selections, followed by screening for the production of spontaneous, 5-fluoro-orotate-resistant mutants in micro-colonies. Several candidates were evaluated and found to have high frequencies of pyrE and pyrF mutation and reversion. Neither an increased efficiency of plating of mutants on selective medium, nor the creation of a genetically unstable pyrE allele, could be implicated as the cause of these high frequencies. The strains had elevated frequencies of other mutations, and exhibited certain phenotypic differences among themselves. A large increase in sensitivity to DNA-damaging agents was not observed, however. These properties generally resemble those of bacterial mutator mutants and suggest loss of functions specific to genetic accuracy.

  2. Water relations of the tos1 tomato mutant at contrasting evaporative demand

    OpenAIRE

    Albacete, Alfonso; Martínez-Ballesta, M. Carmen; Carvajal, Micaela; Autor Pérez-Alfocea, Francisco; Dodd, Ian C.; Romero-Aranda, Remedios

    2009-01-01

    The tos1 (tomato osmotically sensitive) mutant, isolated from an in vitro screen of root growth during osmotic stress, was less sensitive to exogenous ABA, but accumulated more ABA under osmotic stress than WT plants.

  3. Identification and primary genetic analysis of Arabidopsis stomatal mutants in response to multiple stresses

    Institute of Scientific and Technical Information of China (English)

    SONG Yuwei; KANG Yanli; LIU Hao; ZHAO Xiaoliang; WANG Pengtao; AN Guoyong; ZHOU Yun; MIAO Chen; SONG Chunpeng

    2006-01-01

    In response to variable environmental conditions, guard cells located in the leaf epidermis can integrate and cope with a multitude of complicated stimuli, thereby making stomata in an appropriate state. However, many signaling components in guard cell signaling remain elusive. In our laboratory,a tool for non-invasive remote infrared thermal images was used to screen an ethyl methane sulfonate-mutagenized population for Arabidopsis stomatal response mutants under multiple stresses (ABA, H2O2, CO2, etc.). More than forty "hot" or "cold"mutants were isolated (above or below 0.5℃ in contrast to normal plantlets). Identification and primary genetic analysis of these mutants show that they are monogenic recessive mutations and there exist distinct difference in stomata apertures compared to wild type. These mutants in response to various environmental stresses and hormones were comprehensively investigated, which enables us to further understand the cross-talk in different signal transduction pathways.

  4. Growth With of Alfalfa Mutant in Different Nitrogen Fertilizer and Defoliation Intensity

    Directory of Open Access Journals (Sweden)

    Widyati Slamet

    2012-10-01

    Full Text Available The research was conducted to evaluate growth  of alfalfa mutan (plant height increment, number of leaves and dry matter production in different  Nitrogen Fertilizer and defoliation intensity. The design used was randomized block design 4x2 factorial with 3 replications. The first was dosage of Nitrogen fertilizerNitrogen   (0, 30, 60 and 90 kg N / ha,  the second factor was defoliation intensity (5 and 10 cm. Variables observed alfalfa growth (plant height increment, number of leaves, the production of dry matter (DM forage. The results showed that different N fertilization did not affect the growth of alfalfa mutants. Defoliation intensity  affectedmnumber of leaves and   DM  production of alfalfa mutant. Fertilization to 90 kg N / ha has not affected the growth and   defoliation  intensity 10 cm gave better growth on alfalfa mutant.   [Keywords: alfalfa mutant; Nitrogen fertilizer; defoliation; growth

  5. Genetic Screening for Bacterial Mutants in Liquid Growth Media By Fluorescence-Activated Cell Sorting

    Science.gov (United States)

    Abuaita, Basel H.; Withey, Jeffrey H.

    2010-01-01

    Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and make screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. PMID:21094189

  6. Molecular Mechanisms Regulating Ocular Apoptosis in Zebrafish gdf6a Mutants

    DEFF Research Database (Denmark)

    Pant, Sameer D.; March, Lindsey D.; Famulski, Jakub K.

    2013-01-01

    occurs 28 hours post fertilization (hpf) in gdf6a(-/-) mutants that is mediated independently of p53 by intrinsic mechanisms involving Bax proteins. Also, gdf6a(-/-) mutants exhibit markedly increased p38 MAP kinase activation that can be inhibited to significantly reduce retinal apoptosis. A reduction...... in retinal smad1 expression was also noted in gdf6a(-/-) mutants. CONCLUSIONS. gdf6a(-/-)-induced apoptosis is characterized by the involvement of intrinsic apoptotic pathways, p38 MAP kinases, and dysregulated smad expression. Modulation of key mediators can inhibit retinal apoptosis offering potential......PURPOSE. To characterize the molecular mechanisms underlying retinal apoptosis induced by loss of Gdf6, a TGF beta ligand. METHODS. The role of Gdf6 in regulating apoptosis was studied using a zebrafish gdf6a(-/-) mutant, which encodes a truncated, nonfunctional protein. To investigate whether...

  7. Temperature-sensitive mutants of fowl plague virus: isolation and genetic characterization. [UV radiation

    Energy Technology Data Exchange (ETDEWEB)

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

    1979-01-30

    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/sup +/ 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/sub 1/, P/sub 2/, P/sub 3/, HA, NP, and NS proteins.

  8. Recovery and Biological Properties of Nitrate Non-utilizing Mutants of Rice Blast, Magnaporthe grisea

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chuan-qing; ZHOU Ming-guo

    2004-01-01

    Eleven nitrate non-utilizing (nit) mutants were recovered from six isolates of Magnaporthe grisea cultured on MM media amended with 60 g/L potassium chlorate, with a frequency of 1.42 %. Some biological properties, such as growth rate, growth biomass, cultural characters, conidial production, sexual reproduction ability, and pathogenicity were compared between nit mutants and their parent isolates. Results showed that all the nit mutants were resistant to chlorate. Some important biological properties such as the growth rate on YPSA, conidial production ability on TPSA, pathogenicity, had no significant differences between nit mutants and their parent isolates. Mating type didn't change, but perithecia production ability of fertile isolates changed significantly as compared with that of their parent isolates. Therefore, the nit can be used as a genetic marker to study the genetics such as pathogenicity, fungicide resistance in Magnaporthe grisea.

  9. Proto-oncogenic role of mutant IDH2 in leukemia initiation and maintenance

    National Research Council Canada - National Science Library

    Kats, Lev M; Reschke, Markus; Taulli, Riccardo; Pozdnyakova, Olga; Burgess, Kerri; Bhargava, Parul; Straley, Kimberly; Karnik, Rahul; Meissner, Alexander; Small, Donald; Su, Shinsan M; Yen, Katharine; Zhang, Jiangwen; Pandolfi, Pier Paolo

    2014-01-01

    ...) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML...

  10. Investigation into the resistance of lactoperoxidase tolerant Escherichia coli mutants to different forms of oxidative stress.

    Science.gov (United States)

    De Spiegeleer, Philipp; Vanoirbeek, Kristof; Lietaert, Annelies; Sermon, Jan; Aertsen, Abram; Michiels, Chris W

    2005-11-15

    Six lactoperoxidase tolerant Escherichia coli transposon mutants isolated and characterized in an earlier study, and some newly constructed double mutants, were subjected to peroxide, superoxide and hypochlorite stress, and their inactivation was compared to that of the wild type strain MG1655. Knock out mutants of waaQ and waaO, which owed their lactoperoxidase tolerance to an impaired outer membrane permeability due to a reduced porin content, also exhibited higher resistance to hypochlorite, as did a knock-out strain of lrp, encoding a regulatory protein affecting a wide range of cellular functions. Unlike the outer membrane mutants however, the lrp strain was also more resistant to t-butyl hydroperoxide, but more susceptible to the superoxide generating compound plumbagin. Finally, a lactoperoxidase tolerant knock-out strain of ulaA, involved in ascorbic acid uptake, did not show resistance to any of the other oxidants. The possible modes of action of these different oxidants are discussed.

  11. A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.

    Science.gov (United States)

    Adorno, Maddalena; Cordenonsi, Michelangelo; Montagner, Marco; Dupont, Sirio; Wong, Christine; Hann, Byron; Solari, Aldo; Bobisse, Sara; Rondina, Maria Beatrice; Guzzardo, Vincenza; Parenti, Anna R; Rosato, Antonio; Bicciato, Silvio; Balmain, Allan; Piccolo, Stefano

    2009-04-03

    TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

  12. Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor

    DEFF Research Database (Denmark)

    Poulsen, Peter; Gaber, Richard F.; Kielland-Brandt, Morten

    2008-01-01

    T639I) turned out to be hyporesponsive, i.e., it signals only at high inducer concentration. In accordance with a transporter-like mechanism for Ssy1p function we suggest that the hyper- and hyporesponsive mutant forms differ from the wild-type sensor by being more and less inclined, respectively......The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structurally...... related to amino acid permeases, but unable to transport amino acids. We isolated SSY1 mutants that constitutively activate a target promoter. Dose-response analysis showed that the mutants are hyperresponsive, requiring less inducer to give strong signaling than does the wild type. Another mutant (Ssy1p...

  13. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Hathroubi, S; Hancock, M A; Bossé, J T; Langford, P R; Tremblay, Y D N; Labrie, J; Jacques, M

    2015-10-19

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Aberrant cGMP-binding activity in non-chemotactic Dictyostelium discoideum mutants

    NARCIS (Netherlands)

    Kuwayama, Hidekazu; Viel, Gerhard T.; Ishida, Shuji; Haastert, Peter J.M. van

    1995-01-01

    The kinetics of cGMP-binding to the major cGMP-binding activity in Dictyostelium, were investigated in 10 non-chemotactic mutants (KI mutants; KI-1 similar to 10). A wild-type cell contains about 3000 binding sites with a K-d of 1.5 nM. cGMP may dissociate from these binding sites with fast (F-type)

  15. A new conditional Apc-mutant mouse model for colorectal cancer.

    OpenAIRE

    Robanus-Maandag, E C; Koelink, P J; Breukel, C; Salvatori, D. C. F.; Jagmohan-Changur, S. C.; Bosch, C. A. J.; Verspaget, H. W.; Devilee, P; Fodde, R.; Smits, R

    2010-01-01

    textabstractMutations of the adenomatous polyposis coli (APC) gene predispose individuals to familial adenomatous polyposis (FAP), characterized by multiple tumours in the large intestine. Most mouse models heterozygous for truncating mutant Apc alleles mimic FAP, however, the intestinal tumours occur mainly in the small intestine. To model large intestinal tumours, we generated a new conditional Apc-mutant allele, Apc15lox, with exon 15 flanked by loxP sites. Similar survival of Apc1638N/15l...

  16. Molecular characterization of Tn5-induced symbiotic (Fix-) mutants of Rhizobium meliloti.

    OpenAIRE

    Zimmerman, J L; Szeto, W W; Ausubel, F M

    1983-01-01

    To investigate the expression of specific symbiotic genes during the development of nitrogen-fixing root nodules, we conducted a systematic analysis of nodule-specific proteins and RNAs produced after the inoculation of alfalfa roots with a series of Rhizobium meliloti mutants generated by site-directed transposon Tn5 mutagenesis. The mutagenized region of the Rhizobium genome covered approximately 10 kilobases and included the region encoding the nitrogenase polypeptides. All mutant strains ...

  17. The toc132toc120 heterozygote mutant of Arabidopsis thaliana accumulates reduced levels of hexadecatrienoic acid.

    Science.gov (United States)

    Afitlhile, Meshack; Duffield-Duncan, Kayla; Fry, Morgan; Workman, Samantha; Hum-Musser, Sue; Hildebrand, David

    2015-11-01

    A null and heterozygous mutant for the Arabidopsis thaliana TOC132 and TOC120 genes accumulates increased levels of 16:0 and decreased 16:3, suggesting altered homeostasis in fatty acid synthesis. The FAD5 gene encodes a plastid desaturase that catalyzes the first step in the synthesis of 16:3 in monogalactosyldiacylglycerol (MGDG). In non-acclimated toc132toc120+/- mutant plants, the FAD5 gene was repressed and this correlated with decreased levels of 16:3. In cold-acclimated mutant however, the FAD5 gene was upregulated and there was a small increase in 16:3 levels relative to the non-acclimated mutant plants. The MGD1 gene was expressed at control levels and the mutant accumulated levels of MGDG that were similar to the wild type. In the mutant however, MGDG had decreased 16:3 levels, suggesting that the activity of FAD5 desaturase was compromised. In the mutant, the FAD2 and FAD3 genes were downregulated but levels of 18:3-PC were increased, suggesting posttranscriptional regulation for the ER-localized fatty acid desaturases. The Toc120 or Toc159 receptor is likely to compensate for a defective Toc132 receptor. In the cold-acclimated mutant, the TOC159 gene was repressed ca. 300-fold, whereas the TOC120 gene was repressed 7-fold relative to the non-acclimated wild type. Thus, the TOC159 gene is more sensitive to cold-stress and might not compensate for defect in the TOC132 gene under these conditions. Overall, these data show that a mutation in the TOC132 gene results in decreased 16:3 levels, indicating the need for an intact Toc132/Toc120 receptor, presumably to facilitate the import of the FAD5 preprotein into chloroplasts.

  18. Towards a "Golden Standard" for computing globin stability: Stability and structure sensitivity of myoglobin mutants

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta

    2015-01-01

    calculations. The trend accuracy of the methods decreased as I-Mutant 2.0 (R=0.64-0.65), SDM (R=0.57-0.60), POPMUSIC2.1 (R=0.54-0.57), I-Mutant 3.0 (R=0.53-0.55), mCSM (R=0.35-0.47), and CUPSAT (R=0.25-0.48). The mean signed errors increased as SDMMean...

  19. Herpes simplex virus type 1 ribonucleotide reductase null mutants induce lesions in guinea pigs.

    Science.gov (United States)

    Turk, S R; Kik, N A; Birch, G M; Chiego, D J; Shipman, C

    1989-12-01

    Two herpes simplex virus type 1 ribonucleotide reductase null mutants, hrR3 and ICP6 delta, produced cutaneous lesions in guinea pigs as severe as those of wild-type strains. The lesions induced by hrR3 resulted from in vivo replication of the mutant virus, suggesting that this virus-encoded enzyme is nonessential for virus replication in guinea pigs.

  20. Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

    Science.gov (United States)

    Oda, Michael N.; Forte, Trudy M.

    2007-05-29

    Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

  1. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

    OpenAIRE

    Ouyang Xiaosen; Liang Qiuli; Schneider Lonnie; Zhang Jianhua

    2011-01-01

    Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease a...

  2. The phenotype of a phospholipase C (plc-1) mutant in a filamentous fungus, Neurospora crassa.

    Science.gov (United States)

    Lew, Roger R; Giblon, Rachel E; Lorenti, Miranda S H

    2015-09-01

    In the filamentous fungus Neurospora crassa, phospholipase C may play a role in hyphal extension at the growing tips as part of a growth-sensing mechanism that activates calcium release from internal stores to mediate continued expansion of the hyphal tip. One candidate for a tip-localized phospholipase C is PLC-1. We characterized morphology and growth characteristics of a knockout mutant (KO plc-1) and a RIP mutated strain (RIP plc-1) (missense mutations and a nonsense mutation render the gene product non-functional). Growth and hyphal cytology of wildtype and KO plc-1 were similar, but the RIP plc-1 mutant grew slower and exhibited abnormal membrane structures at the hyphal tip, imaged using the fluorescence dye FM4-64. To test for causes of the slower growth of the RIP plc-1 mutant, we examined its physiological poise compared to wildtype and the KO plc-1 mutant. The electrical properties of all three strains and the electrogenic contribution of the plasma membrane H(+)-ATPase (identified by cyanide inhibition) were the same. Responses to high osmolarity were also similar. However, the RIP plc-1 mutant had a significantly lower turgor, a possible cause of its slower growth. While growth of all three strains was inhibited by the phospholipase C inhibitor 3-nitrocoumarin, the RIP plc-1 mutant did not exhibit hyphal bursting after addition of the inhibitor, observed in both wildtype and the KO plc-1 mutant. Although the plc-1 gene is not obligatory for tip growth, the phenotype of the RIP plc-1 mutant - abnormal tip cytology, lower turgor and resistance to inhibitor-induced hyphal bursting - suggest it does play a role in tip growth. The expression of a dysfunctional plc-1 gene may cause a shift to alternative mechanism(s) of growth sensing in hyphal extension. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Infection of the ferret stomach by isogenic flagellar mutant strains of Helicobacter mustelae.

    OpenAIRE

    Andrutis, K A; Fox, J G; Schauer, D B; Marini, R P; Li, X.; L. Yan; Josenhans, C; Suerbaum, S

    1997-01-01

    Helicobacter mustelae, like Helicobacter pylori, possesses two flagellin proteins, FlaA and FlaB. Isogenic mutant strains of H. mustelae have been constructed by disruption of the flaA or flaB gene with a kanamycin resistance cassette or by introduction of both a kanamycin and a chloramphenicol resistance gene to produce a double mutant. To determine whether one or both flagellin proteins are necessary for colonization and persistence of infection with H. mustelae, 19 ferrets, specific pathog...

  4. Probability of fixation of an advantageous mutant in a viral quasispecies.

    OpenAIRE

    Wilke, Claus O.

    2003-01-01

    The probability that an advantageous mutant rises to fixation in a viral quasispecies is investigated in the framework of multi-type branching processes. Whether fixation is possible depends on the overall growth rate of the quasispecies that will form if invasion is successful, rather than on the individual fitness of the invading mutant. The exact fixation probability can only be calculated if the fitnesses of all potential members of the invading quasispecies are known. Quasispecies fixati...

  5. A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology.

    Science.gov (United States)

    Lewis, Derrick L; Notey, Jaspreet S; Chandrayan, Sanjeev K; Loder, Andrew J; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2015-03-01

    A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.

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

    OpenAIRE

    Hironori eKawakami; Eiji eOhashi; Toshiki eTsurimoto; Tsutomu eKatayama

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as ...

  7. Tetrahydrobiopterin non-responsiveness in dihydropteridine reductase deficiency is associated with the presence of mutant protein.

    Science.gov (United States)

    Cotton, R G; Jennings, I; Bracco, G; Ponzone, A; Guardamagna, O

    1986-01-01

    Correlation of the response to a load of tetrahydrobiopterin (BH4) in dihydropterin reductase (DHPR) deficient patients to the type of mutation in these patients has led to the conclusion that 4 patients without mutant DHPR molecules in their cells respond to the BH4 load, whereas 3 patients with mutant DHPR in their cells do not respond. Intravenous injection of BH4 in 1 of the cases not responding to BH4 again showed no response.

  8. Imipenem- and meropenem-resistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins.

    Science.gov (United States)

    Raimondi, A; Traverso, A; Nikaido, H

    1991-01-01

    Carbapenems such as imipenem and meropenem are not rapidly hydrolyzed by commonly occurring beta-lactamases. Nevertheless, it was possible, by mutagenesis and selection, to isolate mutant strains of Enterobacter cloacae and Proteus rettgeri that are highly resistant to meropenem and imipenem. Two alterations were noted in the E. cloacae mutants. First, the mutant strains appeared to be strongly derepressed in the production of beta-lactamases, which reached a very high level when the strains were grown in the presence of imipenem. Second, these mutants were deficient in the production of nonspecific porins, as judged by the pattern of outer membrane proteins as well as by reconstitution assays of permeability. As with most porin-deficient mutants, their cultures were unstable, and their cultivation in the absence of carbapenems rapidly led to an overgrowth of porin-producing revertants. Analysis of the data suggests that the synergism between the lowered outer membrane permeability and the slow but significant hydrolysis of carbapenems by the overproduced enzymes can explain the resistance phenotypes quantitatively, although the possibility of alteration of the target cannot be excluded at present. With P. rettgeri mutants, there was no indication of further derepression of beta-lactamase, but the enzyme hydrolyzed imipenem much more efficiently than the E. cloacae enzyme did. In addition, the major porin was absent in one mutant strain. These results suggest that a major factor for the carbapenem resistance of these enteric bacteria is the porin deficiency, and this conclusion forms a contrast to the situation in Pseudomonas aeruginosa, in which the most prevalent class of imipenem-resistant mutants appears to lack the specific channel protein D2 yet retains the major nonspecific porin F. Images PMID:1656855

  9. The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

    Science.gov (United States)

    Ahne, F; Merlos-Lange, A M; Lang, B F; Wolf, K

    1984-09-01

    The three mutator strains ana (r)-8, ana (r)-14, and diu (r)-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

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

    Science.gov (United States)

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

    2016-05-16

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

  11. Photooxidative stress stimulates illegitimate recombination and mutability in carotenoid-less mutants of Rubrivivax gelatinosus.

    Science.gov (United States)

    Ouchane, S; Picaud, M; Vernotte, C; Astier, C

    1997-08-01

    Carotenoids are essential to protection against photooxidative damage in photosynthetic and non-photosynthetic organisms. In a previous study, we reported the disruption of crtD and crtC carotenoid genes in the purple bacterium Rubrivivax gelatinosus, resulting in mutants that synthesized carotenoid intermediates. Here, carotenoid-less mutants have been constructed by disruption of the crtB gene. To study the biological role of carotenoids in photoprotection, the wild-type and the three carotenoid mutants were grown under different conditions. When exposed to photooxidative stress, only the carotenoid-less strains (crtB-) gave rise with a high frequency to four classes of mutants. In the first class, carotenoid biosynthesis was partially restored. The second class corresponded to photosynthetic-deficient mutants. The third class corresponded to mutants in which the LHI antenna level was decreased. In the fourth class, synthesis of the photosynthetic apparatus was inhibited only in aerobiosis. Molecular analyses indicated that the oxidative stress induced mutations and illegitimate recombination. Illegitimate recombination events produced either functional or non-functional chimeric genes. The R. gelatinosus crtB- strain could be very useful for studies of the SOS response and of illegitimate recombination induced by oxidants in bacteria.

  12. New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

    Science.gov (United States)

    Pérez-Amador, M A; Lidder, P; Johnson, M A; Landgraf, J; Wisman, E; Green, P J

    2001-12-01

    In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

  13. Characterization of a novel gravitropic mutant of morning glory, weeping2

    Science.gov (United States)

    Kitazawa, Daisuke; Miyazawa, Yutaka; Fujii, Nobuharu; Nitasaka, Eiji; Takahashi, Hideyuki

    2008-09-01

    In higher plants, gravity is a major environmental cue that governs growth orientation, a phenomenon termed gravitropism. It has been suggested that gravity also affects other aspects of morphogenesis, such as circumnutation and winding movements. Previously, we showed that these aspects of plant growth morphology require amyloplast sedimentation inside gravisensing endodermal cells. However, the molecular mechanism of the graviresponse and its relationship to circumnutation and winding remains obscure. Here, we have characterized a novel shoot gravitropic mutant of morning glory, weeping2 ( we2). In the we2 mutant, the gravitropic response of the stem was absent, and hypocotyls exhibited a severely reduced gravitropic response, whereas roots showed normal gravitropism. In agreement with our previous studies, we found that we2 mutant has defects in shoot circumnutation and winding. Histological analysis showed that we2 mutant forms abnormal endodermal cells. We identified a mutation in the morning glory homolog of SHORT-ROOT ( PnSHR1) that was genetically linked to the agravitropic phenotype of we2 mutant, and which may underlie the abnormal differentiation of endodermal cells in this plant. These results suggest that the phenotype of we2 mutant is due to a mutation of PnSHR1, and that PnSHR1 regulates gravimorphogenesis, including circumnutation and winding movements, in morning glory.

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

    Science.gov (United States)

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

    2016-01-01

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

  15. ID4 regulates transcriptional activity of wild type and mutant p53 via K373 acetylation.

    Science.gov (United States)

    Morton, Derrick J; Patel, Divya; Joshi, Jugal; Hunt, Aisha; Knowell, Ashley E; Chaudhary, Jaideep

    2017-01-10

    Given that mutated p53 (50% of all human cancers) is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant (P223L, V274F, R175H, R273H), acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

  16. Potential Mitochondrial Isocitrate Dehydrogenase R140Q Mutant Inhibitor from Traditional Chinese Medicine against Cancers

    Directory of Open Access Journals (Sweden)

    Wen-Yuan Lee

    2014-01-01

    Full Text Available A recent research of cancer has indicated that the mutant of isocitrate dehydrogenase 1 and 2 (IDH1 and 2 genes will induce various cancers, including chondrosarcoma, cholangiocarcinomas, and acute myelogenous leukemia due to the effect of point mutations in the active-site arginine residues of isocitrate dehydrogenase (IDH, such as IDH1/R132, IDH2/R140, and IDH2/R172. As the inhibition for those tumor-associated mutant IDH proteins may induce differentiation of those cancer cells, these tumor-associated mutant IDH proteins can be treated as a drug target proteins for a differentiation therapy against cancers. In this study, we aim to identify the potent TCM compounds from the TCM Database@Taiwan as lead compounds of IDH2 R140Q mutant inhibitor. Comparing to the IDH2 R140Q mutant protein inhibitor, AGI-6780, the top two TCM compounds, precatorine and abrine, have higher binding affinities with target protein in docking simulation. After MD simulation, the top two TCM compounds remain as the same docking poses under dynamic conditions. In addition, precatorine is extracted from Abrus precatorius L., which represents the cytotoxic and proapoptotic effects for breast cancer and several tumor lines. Hence, we propose the TCM compounds, precatorine and abrine, as potential candidates as lead compounds for further study in drug development process with the IDH2 R140Q mutant protein against cancer.

  17. Potential mitochondrial isocitrate dehydrogenase R140Q mutant inhibitor from traditional Chinese medicine against cancers.

    Science.gov (United States)

    Lee, Wen-Yuan; Chen, Kuan-Chung; Chen, Hsin-Yi; Chen, Calvin Yu-Chian

    2014-01-01

    A recent research of cancer has indicated that the mutant of isocitrate dehydrogenase 1 and 2 (IDH1 and 2) genes will induce various cancers, including chondrosarcoma, cholangiocarcinomas, and acute myelogenous leukemia due to the effect of point mutations in the active-site arginine residues of isocitrate dehydrogenase (IDH), such as IDH1/R132, IDH2/R140, and IDH2/R172. As the inhibition for those tumor-associated mutant IDH proteins may induce differentiation of those cancer cells, these tumor-associated mutant IDH proteins can be treated as a drug target proteins for a differentiation therapy against cancers. In this study, we aim to identify the potent TCM compounds from the TCM Database@Taiwan as lead compounds of IDH2 R140Q mutant inhibitor. Comparing to the IDH2 R140Q mutant protein inhibitor, AGI-6780, the top two TCM compounds, precatorine and abrine, have higher binding affinities with target protein in docking simulation. After MD simulation, the top two TCM compounds remain as the same docking poses under dynamic conditions. In addition, precatorine is extracted from Abrus precatorius L., which represents the cytotoxic and proapoptotic effects for breast cancer and several tumor lines. Hence, we propose the TCM compounds, precatorine and abrine, as potential candidates as lead compounds for further study in drug development process with the IDH2 R140Q mutant protein against cancer.

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

    Science.gov (United States)

    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.

  19. Temperature-sensitive mutants of Actinobacillus pleuropneumoniae induce protection in mice.

    Science.gov (United States)

    Byrd, W; Hooke, A M

    1997-01-01

    Temperature-sensitive mutants of Actinobacillus pleuropneumoniae 4074, serotype 1, were isolated after treatment with nitrosoguanidine and enrichment with penicillin and D-cycloserine. Of the four temperature-sensitive mutants evaluated in mice, one (A-1) had a tight phenotype (i.e., it ceased replication immediately after transfer to the nonpermissive temperature [37 degrees C]) and three (1-2, 4-1, and 12-1) were coasters that continued replication for up to three generations after transfer to 37 degrees C. The reversion frequencies ranged from 10(-6) to 10(-9), and cutoff temperatures ranged from 33 to 35 degrees C. No major changes were detected in the biochemical profiles; agglutination reactions; electrophoretic profiles of the lipopolysaccharides, outer membrane proteins, and hemolysin proteins; hemolytic titers; or CAMP factor reactions of the mutants and the wild-type bacteria. Groups of 3- to 5-week-old, female ICR mice were immunized intranasally with three doses of 3.5 x 10(6) CFU of the mutants over 3 weeks and subsequently challenged intranasally with 5 50% lethal doses of the parental wild-type. Protection was induced by both the tight and the coaster mutants, with the 4-1 and 12-1 coasters eliciting greater protection (67 and 82%, respectively) than that induced by the A-1 tight mutant (57%). Intranasal immunization with both phenotypes induced serum antibody responses against the surface antigens and the hemolysin protein. PMID:9169752

  20. A rice mutant displaying a heterochronically elongated internode carries a 100 kb deletion

    Institute of Scientific and Technical Information of China (English)

    Mika Hayashi-Tsugane; Masahiko Maekawa; Qian Qian; Hirokazu Kobayashi; Shigeru Iida; Kazuo Tsugane

    2011-01-01

    We have isolated a recessive rice mutant,designated as indeterminate growth(ing),which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms.Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion,which corresponds to a 103 kb region in the Nipponbare genome,containing nine annotated genes on chromosome 3.Of these annotated genes,the SLRI gene encoding a DELLA protein is the only one that is well characterized in its function,and its null mutation,which is caused by a single base deletion in the middle of the intronless SLR1 gene,confers a slender phenotype that bears close resemblance to the ing mutant phenotype.The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene,and the ing mutant appears to be the first characterized mutant having the entire SLRI sequence deleted.Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene,whose dysfunction must result in a lethal phenotype.

  1. Sequestration of Glyceraldehyde-3-phosphate Dehydrogenase to Aggregates Formed by Mutant Huntingtin

    Institute of Scientific and Technical Information of China (English)

    Junchao WU; Fang LIN; Zhenghong QIN

    2007-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to interact with proteins containing the polyglutamine (polyQ) domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline (polyP) domains to the co-localization of mutant huntingtin (htt) and GAPDH. Overexpression of N-terminal htt (1-969 amino acids) with 100Q and 46Q (htt1-969-100Q and httl-969-46Q, mutant htt) in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of htt1-969-18Q (wild-type htt). The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyp region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyp domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington's disease.

  2. Screening of Toxin Mutant of Dickeya zeae and Its Biological Characters

    Institute of Scientific and Technical Information of China (English)

    Jingyi ZHANG; Yutao WANG; Yanchang LI; Qiongguang LIU

    2013-01-01

    [Objective] This study aimed to screen toxin mutant of Dickeya zeae (Er-winia chrysanthemi pv. zeae) and investigate its biological characters. [Method] We obtained a toxin mutant strain D. zeae Ech7-3-42 by using acridine orange as a mutagenic agent and compared their biological characteristics and virulence between the toxin mutant and wild strain. [Result] There was no significant difference in pectin lyase, protease, cellulase and the production of extracellular polysaccharide and lipopolysaccharide, but significant difference in toxin biological activities and vir-ulence. Ech7-3-42 mutant did not produce toxin, as wel as the loss of virulence on rice and HR on tobacco, but did not lose the ability to soft rot on potato. Mutant strain Ech7-3-42 can infect rice root and then enriched in the root neck and stalk, but it could not cause rice foot rot. Dickeya zeae (wild and mutant strain) could be detected by PCR in the root neck and below the 1-2 cm long stem area, but could not be detected in the leaves. [Conclusion] We believed that toxin may be one of the important factors for D. zeae virulence on rice.

  3. Decreased uv mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, L.; Hinkle, D.; Prakash, S.

    1978-01-01

    A DNA replication mutant of yeast, cdc8, was found to decrease uv-induced reversion of lys2-1, arg4-17, tryl and ural. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following uv irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since uv-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.

  4. [Phenotype analysis and mutant gene location of ventral yellow mouse (VY(Slac))].

    Science.gov (United States)

    Shi, Mei-Lian; Xu, Ping; Yin, Xiao-Shu; Yang, Wei-Wei; Gu, Mei-Er; Yu, Li-Ping; Liu, Gui-Jie; Wu, Bao-Jin

    2012-06-01

    The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)×D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene.

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

    Institute of Scientific and Technical Information of China (English)

    HUANG Xiao-qun; WANG Ping-rong; ZHAO Hai-xin; DENG Xiao-jian

    2008-01-01

    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 generetions 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 shon 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 now chlorophyll-deficit mutant gene and tentatively named as chl11(t).

  6. [Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus].

    Science.gov (United States)

    Zaretskaia, M Sh; Nefelova, M V; Baratova, L A; Polin, A N

    1984-12-01

    The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.

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

    Science.gov (United States)

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

    2015-01-01

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

  8. Gr and hp-1 tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening.

    Science.gov (United States)

    Chialva, Matteo; Zouari, Inès; Salvioli, Alessandra; Novero, Mara; Vrebalov, Julia; Giovannoni, James J; Bonfante, Paola

    2016-07-01

    Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

  9. Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem 1

    Science.gov (United States)

    Yang, Cheng; Signer, Ethan R.; Hirsch, Ann M.

    1992-01-01

    Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed. ImagesFigure 3Figure 1Figure 2Figure 4 PMID:16668605

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

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

    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.

  11. Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.

    1977-01-01

    The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32/sup 0/C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32/sup 0/C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32/sup 0/C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

  12. Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants

    Indian Academy of Sciences (India)

    M Selvaraj; A Govindan; A Seshadri; B Dubey; U Varshney; M Vijayan

    2013-12-01

    Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.

  13. 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.

  14. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

    Directory of Open Access Journals (Sweden)

    Guocai Li

    Full Text Available Neisseria gonorrhoeae (N. gonorrhoeae outer membrane protein reduction modifiable protein (Rmp has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

  15. Antibodies with higher bactericidal activity induced by a Neisseria gonorrhoeae Rmp deletion mutant strain.

    Science.gov (United States)

    Li, Guocai; Xie, Rushan; Zhu, Xiaoping; Mao, Yanli; Liu, Shuangxi; Jiao, Hongmei; Yan, Hua; Xiong, Kun; Ji, Mingchun

    2014-01-01

    Neisseria gonorrhoeae (N. gonorrhoeae) outer membrane protein reduction modifiable protein (Rmp) has strong immunogenicity. However, anti-Rmp antibodies block rather than preserve the antibacterial effects of protective antibodies, which hampers the development of vaccines for gonococcal infections. We herein constructed an Rmp deletion mutant strain of N. gonorrhoeae by gene homologous recombination. The 261-460 nucleotide residues of Rmp gene amplified from N. gonorrhoeae WHO-A strain were replaced with a kanamycin-resistant Kan gene amplified from pET-28a. The resultant hybridized DNA was transformed into N. gonorrhoeae WHO-A strain. PCR was used to screen the colonies in which wild-type Rmp gene was replaced with a mutant gene fragment. Western blotting revealed that the Rmp deletion mutant strain did not express Rmp protein. Rmp deletion did not alter the morphological and Gram staining properties of the mutant strain that grew slightly more slowly than the wild-type one. Rmp gene mutated stably throughout 25 generations of passage. Antibody-mediated complement-dependent cytotoxicity assay indicated that the antibodies induced by the mutant strain had evidently higher bactericidal activities than those induced by the wild-type strain. Further modification of the Rmp deletion mutant strain is still required in the development of novel live attenuated vaccines for gonorrhea by Opa genes deletion or screening of phenotypic variant strains that do not express Opa proteins.

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

    Science.gov (United States)

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

    2016-06-01

    The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Mutant TDP-43 within motor neurons drives disease onset but not progression in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Ditsworth, Dara; Maldonado, Marcus; McAlonis-Downes, Melissa; Sun, Shuying; Seelman, Amanda; Drenner, Kevin; Arnold, Eveline; Ling, Shuo-Chien; Pizzo, Donald; Ravits, John; Cleveland, Don W; Da Cruz, Sandrine

    2017-06-01

    Mutations in TDP-43 cause amyotrophic lateral sclerosis (ALS), a fatal paralytic disease characterized by degeneration and premature death of motor neurons. The contribution of mutant TDP-43-mediated damage within motor neurons was evaluated using mice expressing a conditional allele of an ALS-causing TDP-43 mutant (Q331K) whose broad expression throughout the central nervous system mimics endogenous TDP-43. TDP-43(Q331K) mice develop age- and mutant-dependent motor deficits from degeneration and death of motor neurons. Cre-recombinase-mediated excision of the TDP-43(Q331K) gene from motor neurons is shown to delay onset of motor symptoms and appearance of TDP-43-mediated aberrant nuclear morphology, and abrogate subsequent death of motor neurons. However, reduction of mutant TDP-43 selectively in motor neurons did not prevent age-dependent degeneration of axons and neuromuscular junction loss, nor did it attenuate astrogliosis or microgliosis. Thus, disease mechanism is non-cell autonomous with mutant TDP-43 expressed in motor neurons determining disease onset but progression defined by mutant acting within other cell types.

  18. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    Science.gov (United States)

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.; Shilov, Aleksandr A.; Krylov, Petr S. [D.I. Ivanovsky Institute of Virology, 123098 Moscow (Russian Federation); Ilyushina, Natalia A., E-mail: Natalia.Ilyushina@fda.hhs.gov [FDA CDER, 29 Lincoln Drive, Bethesda, MD 20892 (United States); Kaverin, Nikolai V., E-mail: nik.kaverin@gmail.com [D.I. Ivanovsky Institute of Virology, 123098 Moscow (Russian Federation)

    2013-12-15

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

  20. Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini.

    Science.gov (United States)

    McMenamin, Sarah K; Minchin, James E N; Gordon, Tiffany N; Rawls, John F; Parichy, David M

    2013-04-01

    Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity.

  1. Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

    Institute of Scientific and Technical Information of China (English)

    Zhe HU; Ping LI; Jinfang MA; Yunlong WANG; Xinyu WANG; Chongying WANG

    2008-01-01

    An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.

  2. Pollen development and tube growth are affected in the symbiotic mutant of Lotus japonicus, crinkle.

    Science.gov (United States)

    Tansengco, Myra L; Imaizumi-Anraku, Haruko; Yoshikawa, Makoto; Takagi, Shingo; Kawaguchi, Masayoshi; Hayashi, Makoto; Murooka, Yoshikatsu

    2004-05-01

    The symbiotic mutant of Lotus japonicus, crinkle (crk), exhibits abnormal nodulation and other alterations in the root hairs, trichomes, and seedpods. Defective nodulation in crk mutant is due to the arrested infection thread growth from the epidermis into the cortex. Here, we describe that crk is also affected in male fertility that causes the production of small pods with few seeds. Under in vitro conditions, pollen germination and tube growth were markedly reduced in the crk mutant. A swollen tip phenotype with disorganized filamentous actin (F-actin) was observed in the mutant pollen tubes after prolonged in vitro culture. During pollen development, the striking difference noted in the mutant was the small size of the microspores that remained spherical. Histological examination of ovule development, as well as outcrosses of the mutant as female to wild type as male, showed no evidence of abnormality in the female gametophyte development. Based on these findings, the Crk gene, aside from its role in the infection process during nodulation, is also involved in male gametophyte development and function. Therefore, this gene represents a connection between nodule symbiosis, polar tip growth, and other plant developmental processes.

  3. Comparison of the behavior of epiphytic fitness mutants of pseudomonas syringae under controlled and field conditions

    Energy Technology Data Exchange (ETDEWEB)

    Beattie, G.A.; Lindow, S.E. (Univ. of California, Berkeley, CA (United States))

    1994-10-01

    The epiphytic fitness of four Tn5 of Pseudomonas syringae that exhibited reduced epiphytic fitness in the laboratory was evaluated under field conditions. The mutants differed more from the parental strain under field conditions than under laboratory conditions, in their survival immediately following inoculation onto bean leaves and in the size of the epiphytic populations that they established at near-wild type rates, while the others exhibited reduced survival only in the warmest, driest conditions tested and grew epiphytically at reduced rates or, in the case of one mutant, not at all. The presence of the parental strain, B728a, did not influence the survival or growth of three of the mutants under field conditions; however, one mutant, an auxotroph, established larger populations in the presence of B728a than in its absence, possibly because of cross-feeding by By28a in planta. Experiments with B728a demonstrated that established epiphytic populations survived exposure of leaves to dry conditions better than newly inoculated cells did and that epiphytic survival was not dependent on the cell density in the inoculum. Three of the mutants behaved similarly to two nonpathogenic strains of P. syringae, suggesting that the mutants may be altered in traits that are missing or poorly expressed in naturally occurring nonpathogenic epiphytes. 58 refs., 5 figs., 3 tabs.

  4. Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants

    Directory of Open Access Journals (Sweden)

    Lisa Brenan

    2016-10-01

    Full Text Available Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84% missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology.

  5. Comparative docking studies of CYP1b1 and its PCG-associated mutant forms

    Indian Academy of Sciences (India)

    Malkaram Sridhar Achary; Hampapathalu Adimurthy Nagarajaram

    2008-12-01

    Molecular docking has been used to compare and contrast the binding modes of oestradiol with the wild-type and some disease-associated mutant forms of the human CYP1b1 protein. The receptor structures used for docking were derived from molecular dynamics simulations of homology-modelled structures. Earlier studies involving molecular dynamics and principal component analysis indicated that mutations could have a disruptive effect on function, by destabilizing the native properties of the functionally important regions, especially those of the haem-binding and substrate-binding regions, which constitute the site of catalytic activity of the enzyme. In order to gain more insights into the possible differences in substrate-binding and catalysis between the wild-type and mutant proteins, molecular docking studies were carried out. Mutants showed altered protein–ligand interactions compared with the wild-type as a consequence of changes in the geometry of the substrate-binding region and in the position of haem relative to the active site. An important difference in ligand–protein interactions between the wild-type and mutants is the presence of stacking interaction with phenyl residues in the wild-type, which is either completely absent or considerably weaker in mutants. The present study revealed essential differences in the interactions between ligand and protein in wild-type and disease mutants, and helped in understanding the deleterious nature of disease mutations at the level of molecular function.

  6. Nodulation gene mutants of Mesorhizobium loti R7A-nodZ and nolL mutants have host-specific phenotypes on Lotus spp.

    Science.gov (United States)

    Rodpothong, Patsarin; Sullivan, John T; Songsrirote, Kriangsak; Sumpton, David; Cheung, Kenneth W J-T; Thomas-Oates, Jane; Radutoiu, Simona; Stougaard, Jens; Ronson, Clive W

    2009-12-01

    Rhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent on Nod-factor structure and, hence, on at least some of the nodulation genes that encode Nod-factor production. Here, we report the effects of mutations in Mesorhizobium loti R7A nodulation genes on nodulation of four Lotus spp. and on Nod-factor structure. Most mutants, including a DeltanodSDeltanolO double mutant that produced Nod factors lacking the carbamoyl and possibly N-methyl groups on the nonreducing terminal residue, were unaffected for nodulation. R7ADeltanodZ and R7ADeltanolL mutants that produced Nod factors without the (acetyl)fucose on the reducing terminal residue had a host-specific phenotype, forming mainly uninfected nodule primordia on Lotus filicaulis and L. corniculatus and effective nodules with a delay on L. japonicus. The mutants also showed significantly reduced infection thread formation and Nin gene induction. In planta complementation experiments further suggested that the acetylfucose was important for balanced signaling in response to Nod factor by the L. japonicus NFR1/NFR5 receptors. Overall the results reveal differences in the sensitivity of plant perception with respect to signaling leading to root hair deformation and nodule primordium development versus infection thread formation and rhizobial entry.

  7. Systematic structure-function analysis of androgen receptor Leu 701 mutants explains the properties of the prostate cancer mutant L701H

    NARCIS (Netherlands)

    D.J. van de Wijngaart (Dennis); M. Molier; S.J. Lusher (Scott); R. Hersmus (Remko); G.W. Jenster (Guido); J. Trapman (Hans); H.J. Dubbink (Erik Jan)

    2010-01-01

    textabstractOne mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). Weinvestigated the unique properties of theARL701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function analysis. Most amino acid substi

  8. Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis1[W][OPEN

    Science.gov (United States)

    Fukushima, Atsushi; Kusano, Miyako; Mejia, Ramon Francisco; Iwasa, Mami; Kobayashi, Makoto; Hayashi, Naomi; Watanabe-Takahashi, Akiko; Narisawa, Tomoko; Tohge, Takayuki; Hur, Manhoi; Wurtele, Eve Syrkin; Nikolau, Basil J.; Saito, Kazuki

    2014-01-01

    Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/. PMID:24828308

  9. Construction and screening of a multi-point site- specific mutant library of subtilisin E with a set of oligonucleotides

    Institute of Scientific and Technical Information of China (English)

    肖谷田; 谢毅; 张婕; 孙筱清; 吴小舟; 陈小央; 王启松

    1997-01-01

    A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.

  10. Preparation and characteri-zation of some surface nega-tively charged residue mu-tants of cytochrome b5

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome b5 (cyt b5), in which the negatively charged residues around the heme exposed edge of cyt b5 were replaced by hydrophobic amino acid alanine. Double-site mutants, triple-site mutants and even quadruple-site mutants were obtained. DNA sequencing and molecular weight measurements of the mutant proteins both confirmed that these site-directed muta- genesises were successfully performed. Spectroelectrochemistry of these mutant proteins revealed that the apparent redox potentials of these mutant proteins caused a positive shift of 2-10 mV. The global structure of these mutant proteins did not show much difference from that of the wild type cyt b5, providing a solid base for the further study on the roles of the proteins' surface charges.

  11. Biochemical characterization of N-methyl N' -nitro-N-nitrosoguanidine-induced cadmium resistant mutants of Aspergillus niger

    Indian Academy of Sciences (India)

    Samar Kumar Pal; Tapan Kumar Das

    2005-12-01

    Two cadmium resistant mutants (Cd1 and Cd2) of Aspergillus niger, among the six isolated by mutagenization with N-methyl N′-nitro-N-nitrosoguanidine (MNNG) at pH 6.4 were selected for the study. Analysis of lipid composition of the mutants and the wildtype indicated that total lipid as well as individual lipids of the cadmium resistant mutants were changed as compared with that of the wildtype. The increased activities of metal-lothionein and reduced activities of D-xylose isomerase and L-phenylalanine ammonia lyase in cell free extract of the cadmium resistant mutants suggested that mutants could allow high concentration of cadmium salt as compared with that of the wildtype. The respiratory activity and intracellular as well as extracellular Cd2+ concentration of the mutants reflected the high tolerance of the Cd mutants to cadmium ion.

  12. Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger.

    Science.gov (United States)

    Sealy-Lewis, H M; Fairhurst, V

    1998-07-01

    Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

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

    Science.gov (United States)

    Sheridan, W F; Neuffer, M G

    1980-08-01

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

  14. 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

  15. Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants1[W][OA

    Science.gov (United States)

    Matsumoto, Shouhei; Kumasaki, Saori; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Takashi; Hoson, Takayuki

    2010-01-01

    We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth

  16. Gravity-induced modifications to development in hypocotyls of Arabidopsis tubulin mutants.

    Science.gov (United States)

    Matsumoto, Shouhei; Kumasaki, Saori; Soga, Kouichi; Wakabayashi, Kazuyuki; Hashimoto, Takashi; Hoson, Takayuki

    2010-02-01

    We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Delta), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300 g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal

  17. Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants.

    Science.gov (United States)

    Jäger, S; Lewis, J W; Zvyaga, T A; Szundi, I; Sakmar, T P; Kliger, D S

    1997-02-25

    Time-resolved absorption difference spectra of COS-cell expressed rhodopsin and rhodopsin mutants (E113D, E113A/A117E, and G90D), solubilized in detergent, were collected from 20 ns to 510 ms after laser photolysis with 7 ns pulses (lambda(max) = 477 nm). The data were analyzed using a global exponential fitting procedure following singular value decomposition (SVD). Over the entire time range excellent agreement was achieved between results for COS-cell and rod outer segment rhodopsin both in kinetics and in the lambda(max) values of the intermediates. The Schiff base counterion mutant E113D showed strong similarities to rhodopsin up to lumi, following the established scheme: batho bsi --> lumi. Including late delay times (past 1 micros), the mutant E113D lumi decayed to metarhodopsin II (MII), showing that the detergent strongly favors MII over metarhodopsin I (MI). However, a back-reaction from MII to lumi was observed that was not seen for rhodopsin. The kinetic schemes for the mutants E113A/A117E and G90D were significantly different from that of rhodopsin. In both mutants batho decay into an equilibrium with bsi was too fast to resolve ( lumi MI-like MII-like. However, the back-reaction from MI-like to lumi was not seen in G90D. MI-like spectral intermediates absorbing around 460 nm appeared in both mutants. They have been shown to be the transducin-activating species (R*). These data, interpreted in the context of previous NMR, FTIR, and Raman data, are consistent with a picture in which the kinetics of batho decay is dependent on a protein-induced perturbation near C12-C13 of the retinal chromophore. The lambda(max) values of the bsi and lumi intermediates in the mutant pigments are interpreted in terms of movement of the Schiff base relative to its counterion.

  18. Construction and characterization of double mutants in nitrogenase of Klebsiella pneumoniae

    Institute of Scientific and Technical Information of China (English)

    ZHAO Dehua; LI Jilun

    2004-01-01

    Two mutants in nitrogenase of Klebsiella pneumoniae are constructed by site-directed mutagenesis and gene replacement procedure, which express the nitrogenases with Lysine and Glutamine substituting for α-Glutamine 190and α-Histidine 194 respectively (Kp-Q α190 K and Kp-H α194 Q). The above two substitutions are respectively introduced into a nifV mutant (expressing a citrate-containing nitrogenase) and sequentially two double mutants are obtained (Kp-Q α190 K-nifV- and Kp-H α194 Q-nifV-). All four mutants exhibit strict Nif- phenotype under the N2-fixation condition and fail to grow diazotrophically. Altered nitrogneases are effeetively depressed and the C2H2 reduction analysis shows that the double substitutions in Kp-Q α190K-nifV abolish cell C2H2 reduction activity, but Kp-H α194Q-nifV cells maintain a C2H2 reduction activity at 10% of that of wild type. Whole cell C2D2 reduction by all four mutants in comparison to the wild type and nifV mutant is also detected. The results show that only single α-Gln194 substitution does not perturb the stereospecificity of protonation of C2D2. These results indicate that the α- Glutamine 190 and its combination with homocitrate are essential to the catalytic activity of nitrogenase and it is proposed that α-Glutamine 190 and its combination with homocitrate are involved in the proton and/or electron transfer to FeMoco. The nitrogenases from these double mutants will be useful in further analysis of the entry of the proton and/or electron to FeMoco and the substrate binding sites.

  19. Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

    Directory of Open Access Journals (Sweden)

    Raquel Gordo-Gilart

    Full Text Available Multidrug resistance protein 3 (MDR3, ABCB4 is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.

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

    Science.gov (United States)

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

    2014-03-01

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