Induced High-Yield Production of Zeaxanthin, Lutein, and β-Carotene by a Mutant of Chlorella zofingiensis.

PubMed

Huang, Weiping; Lin, Yan; He, Mingxia; Gong, Yuhao; Huang, Junchao

2018-01-31

Natural resources of zeaxanthin are extremely limited. A Chlorella zofingiensis mutant (CZ-bkt1), which could accumulate high amounts of zeaxanthin, was generated and characterized. CZ-bkt1 was achieved by treating the algal cells with a chemical mutagen followed by a color-based colony-screening approach. CZ-bkt1 was found to consist of a dysfunctional carotenoid ketolase, leading to the accumulation of zeaxanthin rather than to its downstream ketocarotenoid astaxanthin. Light irradiation, glucose, NaCl, and nitrogen deficiency all induced CZ-bkt1 to accumulate zeaxanthin. CZ-bkt1 accumulated zeaxanthin up to 7.00 ± 0.82 mg/g when induced by high-light irradiation and nitrogen deficiency and up to 36.79 ± 2.23 mg/L by additional feeding with glucose. Furthermore, in addition to zeaxanthin, CZ-bkt1 also accumulated high amounts of β-carotene (7.18 ± 0.72 mg/g or 34.64 ± 1.39 mg/L) and lutein (13.81 ± 1.23 mg/g or 33.97 ± 2.61 mg/L). CZ-bkt1 is the sole species up to date with the ability to accumulate high amounts of the three carotenoids that are essential for human health.

Canopy Light Interception of a Conventional and an Erect Leaf Mutant Sorghum

USDA-ARS?s Scientific Manuscript database

Two sorghum lines, an erect leafed mutant sorghum and the wild type from which the mutant was generated, were field grown in rectilinear arrays at low (23 plants per square meter) and high (10 plants per square meter) population densities. Canopy light interception, biomass accretion and yield were ...

Development of high-yield influenza B virus vaccine viruses

PubMed Central

Ping, Jihui; Lopes, Tiago J. S.; Neumann, Gabriele; Kawaoka, Yoshihiro

2016-01-01

The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates. Therefore, we screened virus libraries possessing random mutations in the six “internal” influenza B viral RNA segments [i.e., those not encoding the major viral antigens, hemagglutinin (HA) and neuraminidase NA)] for mutants that confer efficient replication. Candidate viruses that supported high yield in cell culture were tested with the HA and NA genes of eight different viruses of the Victoria and Yamagata lineages. We identified combinations of mutations that increased the titers of candidate vaccine viruses in mammalian cells used for human influenza vaccine virus propagation and in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus vaccine backbones can be used for improved vaccine virus production. PMID:27930325

Development of high-yield influenza B virus vaccine viruses.

PubMed

Ping, Jihui; Lopes, Tiago J S; Neumann, Gabriele; Kawaoka, Yoshihiro

2016-12-20

The burden of human infections with influenza A and B viruses is substantial, and the impact of influenza B virus infections can exceed that of influenza A virus infections in some seasons. Over the past few decades, viruses of two influenza B virus lineages (Victoria and Yamagata) have circulated in humans, and both lineages are now represented in influenza vaccines, as recommended by the World Health Organization. Influenza B virus vaccines for humans have been available for more than half a century, yet no systematic efforts have been undertaken to develop high-yield candidates. Therefore, we screened virus libraries possessing random mutations in the six "internal" influenza B viral RNA segments [i.e., those not encoding the major viral antigens, hemagglutinin (HA) and neuraminidase NA)] for mutants that confer efficient replication. Candidate viruses that supported high yield in cell culture were tested with the HA and NA genes of eight different viruses of the Victoria and Yamagata lineages. We identified combinations of mutations that increased the titers of candidate vaccine viruses in mammalian cells used for human influenza vaccine virus propagation and in embryonated chicken eggs, the most common propagation system for influenza viruses. These influenza B virus vaccine backbones can be used for improved vaccine virus production.

Enhanced lipid production in thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738.

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Growth and sporulation of a pyrimidine spore color mutant of Sordaria fimicola.

PubMed

el-Ani, A S

1967-04-07

A nonautonomous spore color mutant of Sordaria fimicola is a pyrimidine auxotroph that produces hyaline nonviable ascospores. Uracil, uridine, and cytidine are more effective growth factors than cytosine and thymine and, in high concentrations, render the mutant self-fertile by inducing the ascospores to resume development and maturation. Crosses with the unlinked arginine non-autonomus spore color mutant st-59 yielded the double mutant st-59 pyr that requires both arginine and a pyrimidine for growth, which indicates a lack of suppression of the pyrimidine requirement by the arginine locus.

Bioconversion of glycerol to ethanol by a mutant Enterobacter aerogenes

PubMed Central

2012-01-01

The main objective of this research is to develop, by adaptive evolution, mutant strains of Enterobacter aerogenes ATCC 13048 that are capable of withstanding high glycerol concentration as well as resisting ethanol-inhibition. The mutant will be used for high ethanol fermentation from glycerol feedstock. Ethanol production from pure (P-) and recovered (R-) glycerol using the stock was evaluated. A six-tube-subculture-generations method was used for developing the mutant. This involved subculturing the organism six consecutive times in tubes containing the same glycerol and ethanol concentrations at the same culture conditions. Then, the glycerol and/or ethanol concentration was increased and the six subculture generations were repeated. A strain capable of growing in 200 g/L glycerol and 30 g/L ethanol was obtained. The ability of this mutant, vis-à-vis the original strain, in utilizing glycerol in a high glycerol containing medium, with the concomitant ethanol yield, was assessed. Tryptic soy broth without dextrose (TSB) was used as the fermentation medium. Fermentation products were analyzed using HPLC. In a 20 g/L glycerol TSB, E. aerogenes ATCC 13048 converted 18.5 g/L P-glycerol and 17.8 g/L R-glycerol into 12 and 12.8 g/L ethanol, respectively. In a 50 g/L P-glycerol TSB, it utilized only 15.6 g/L glycerol; but the new strain used up 39 g/L, yielding 20 g/L ethanol after 120 h, an equivalence of 1.02 mol ethanol/mol-glycerol. This is the highest ethanol yield reported from glycerol bioconversion. The result of this P-glycerol fermentation can be duplicated using the R-glycerol from biodiesel production. PMID:22455837

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

PubMed

Wang, Rong; Yang, Fei; Zhang, Xiao-Qi; Wu, Dianxin; Tan, Cong; Westcott, Sharon; Broughton, Sue; Li, Chengdao; Zhang, Wenying; Xu, Yanhao

2017-01-01

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

Proteomic analysis of high yield rice variety mutated from spaceflight

NASA Astrophysics Data System (ADS)

Ma, Y.; Cheng, Z.; Wang, W.; Sun, Y.

Seeds of pure rice varieties were flown on Chinese recoverable satellite, JB-1, for a 15-day flight in 1996. Many mutant rice varieties with various phenotypes were generated after continuous selection and breeding. Among the mutants, a variety 971-5 showed a significant increase in grain yield compared to its control (971ck). In this study, proteomic analysis of both mutant variety 971-5 and control variety 971ck were carried out to investigate the changes of protein expression level in their leaves at three different growth stages (early and middle stage of tillering, and booting stage). Results showed that (1) almost all differentially expressed proteins were down-regulated in 971-5 with only one exception, (2) the percentages of differentially expressed proteins were 3.1%, 2.1% and 3.1% at the three stages, respectively, and (3) one protein showed a significant alteration in its molecular weight (MW). These data demonstrated that the space environment can alter the expression level of rice proteins both quantitatively and qualitatively.

Extractive Fermentation of Sugarcane Juice to Produce High Yield and Productivity of Bioethanol

NASA Astrophysics Data System (ADS)

Rofiqah, U.; Widjaja, T.; Altway, A.; Bramantyo, A.

2017-04-01

Ethanol production by batch fermentation requires a simple process and it is widely used. Batch fermentation produces ethanol with low yield and productivity due to the accumulation of ethanol in which poisons microorganisms in the fermenter. Extractive fermentation technique is applied to solve the microorganism inhibition problem by ethanol. Extractive fermentation technique can produce ethanol with high yield and productivity. In this process raffinate still, contains much sugar because conversion in the fermentation process is not perfect. Thus, to enhance ethanol yield and productivity, recycle system is applied by returning the raffinate from the extraction process to the fermentation process. This raffinate also contains ethanol which would inhibit the performance of microorganisms in producing ethanol during the fermentation process. Therefore, this study aims to find the optimum condition for the amount of solvent to broth ratio (S: B) and recycle to fresh feed ratio (R: F) which enter the fermenter to produce high yield and productivity. This research was carried out by experiment. In the experiment, sugarcane juice was fermented using Zymomonasmobilis mutant. The fermentation broth was extracted using amyl alcohol. The process was integrated with the recycle system by varying the recycle ratio. The highest yield and productivity is 22.3901% and 103.115 g / L.h respectively, obtained in a process that uses recycle to fresh feed ratio (R: F) of 50:50 and solvents to both ratio of 1.

Statistical optimization for enhanced yields of probiotic Bacillus coagulans and its phage resistant mutants followed by kinetic modelling of the process.

PubMed

Pandey, Kavita R; Joshi, Chetan; Vakil, Babu V

2016-01-01

Probiotics are microorganisms which when administered in adequate amounts confer health benefits to the host. A leading pharmaceutical company producing Bacillus coagulans as a probiotic was facing the problem of recurring phage attacks. Two mutants viz. B. co PIII and B. co MIII that were isolated as phage resistant mutants after UV irradiation and MMS treatment of phage sensitive B. coagulans parental culture were characterized at functional and molecular level and were noted to have undergone interesting genetic changes. The non-specific genetic alterations induced by mutagenesis can also lead to alterations in cell performance. Hence, in the current study the parental strain and the two mutants were selected for shake flask optimization. Plackett-Burman design was used to select the significant culture variables affecting biomass production. Evolutionary operation method was applied for further optimization. The study showed wide variations in the nutritional requirements of phage resistant mutants, post exposure to mutagens. An increment of 150, 134 and 152 % was observed in the biomass productions of B. coagulans (parental type) and mutants B.co PIII and B.co MIII respectively, compared to the yield from one-factor-at-a-time technique. Using Logistic and modified Leudeking-Piret equations, biomass accumulation and substrate utilization efficiency of the bioprocess were determined. The experimental data was in agreement with the results predicted by statistical analysis and modelling. The developed model may be useful for controlling the growth and substrate consumption kinetics in large scale fermentation using B. coagulans .

Ectopic expression of specific GA2 oxidase mutants promotes yield and stress tolerance in rice.

PubMed

Lo, Shuen-Fang; Ho, Tuan-Hua David; Liu, Yi-Lun; Jiang, Mirng-Jier; Hsieh, Kun-Ting; Chen, Ku-Ting; Yu, Lin-Chih; Lee, Miin-Huey; Chen, Chi-Yu; Huang, Tzu-Pi; Kojima, Mikiko; Sakakibara, Hitoshi; Chen, Liang-Jwu; Yu, Su-May

2017-07-01

A major challenge of modern agricultural biotechnology is the optimization of plant architecture for enhanced productivity, stress tolerance and water use efficiency (WUE). To optimize plant height and tillering that directly link to grain yield in cereals and are known to be tightly regulated by gibberellins (GAs), we attenuated the endogenous levels of GAs in rice via its degradation. GA 2-oxidase (GA2ox) is a key enzyme that inactivates endogenous GAs and their precursors. We identified three conserved domains in a unique class of C 20 GA2ox, GA2ox6, which is known to regulate the architecture and function of rice plants. We mutated nine specific amino acids in these conserved domains and observed a gradient of effects on plant height. Ectopic expression of some of these GA2ox6 mutants moderately lowered GA levels and reprogrammed transcriptional networks, leading to reduced plant height, more productive tillers, expanded root system, higher WUE and photosynthesis rate, and elevated abiotic and biotic stress tolerance in transgenic rice. Combinations of these beneficial traits conferred not only drought and disease tolerance but also increased grain yield by 10-30% in field trials. Our studies hold the promise of manipulating GA levels to substantially improve plant architecture, stress tolerance and grain yield in rice and possibly in other major crops. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant Arabidopsis thaliana

PubMed Central

Abreu, Maria Elizabeth; Munné-Bosch, Sergi

2009-01-01

Salicylic acid-deficient NahG transgenic lines and sid2 mutants were used to evaluate the role of this compound in the development of the short-lived, annual plant Arabidopsis thaliana, with a particular focus on the interplay between salicylic acid and other phytohormones. Low salicylic acid levels led to increased growth, as well as to smaller abscisic acid levels and reduced damage to PSII (as indicated by Fv/Fm ratios) during the reproductive stages in rosette leaves of NahG transgenic lines and sid2 mutants, compared with wild-type plants. Furthermore, salicylic acid deficiency highly influenced seed yield and composition. Seed production increased by 4.4-fold and 3.5-fold in NahG transgenic lines and sid2 mutants, respectively, compared to the wild type. Salicylic acid deficiency also improved seed composition in terms of antioxidant vitamin concentrations, seeds of salicylic acid-deficient plants showing higher levels of α- and γ-tocopherol (vitamin E) and β-carotene (pro-vitamin A) than seeds of wild-type plants. Seeds of salicylic acid-deficient plants also showed higher nitrogen concentrations than seeds of wild-type plants. It is concluded that (i) the sid2 gene, which encodes for isochorismate synthase, plays a central role in salicylic acid biosynthesis during plant development in A. thaliana, (ii) salicylic acid plays a role in the regulation of growth, senescence, and seed production, (iii) there is a cross-talk between salicylic acid and other phytohormones during plant development, and (iv) the concentrations of antioxidant vitamins in seeds may be influenced by the endogenous levels of salicylic acid in plants. PMID:19188277

A natural variant of NAL1, selected in high-yield rice breeding programs, pleiotropically increases photosynthesis rate.

PubMed

Takai, Toshiyuki; Adachi, Shunsuke; Taguchi-Shiobara, Fumio; Sanoh-Arai, Yumiko; Iwasawa, Norio; Yoshinaga, Satoshi; Hirose, Sakiko; Taniguchi, Yojiro; Yamanouchi, Utako; Wu, Jianzhong; Matsumoto, Takashi; Sugimoto, Kazuhiko; Kondo, Katsuhiko; Ikka, Takashi; Ando, Tsuyu; Kono, Izumi; Ito, Sachie; Shomura, Ayahiko; Ookawa, Taiichiro; Hirasawa, Tadashi; Yano, Masahiro; Kondo, Motohiko; Yamamoto, Toshio

2013-01-01

Improvement of leaf photosynthesis is an important strategy for greater crop productivity. Here we show that the quantitative trait locus GPS (GREEN FOR PHOTOSYNTHESIS) in rice (Oryza sativa L.) controls photosynthesis rate by regulating carboxylation efficiency. Map-based cloning revealed that GPS is identical to NAL1 (NARROW LEAF1), a gene previously reported to control lateral leaf growth. The high-photosynthesis allele of GPS was found to be a partial loss-of-function allele of NAL1. This allele increased mesophyll cell number between vascular bundles, which led to thickened leaves, and it pleiotropically enhanced photosynthesis rate without the detrimental side effects observed in previously identified nal1 mutants, such as dwarf plant stature. Furthermore, pedigree analysis suggested that rice breeders have repeatedly selected the high-photosynthesis allele in high-yield breeding programs. The identification and utilization of NAL1 (GPS) can enhance future high-yield breeding and provides a new strategy for increasing rice productivity.

A natural variant of NAL1, selected in high-yield rice breeding programs, pleiotropically increases photosynthesis rate

PubMed Central

Takai, Toshiyuki; Adachi, Shunsuke; Taguchi-Shiobara, Fumio; Sanoh-Arai, Yumiko; Iwasawa, Norio; Yoshinaga, Satoshi; Hirose, Sakiko; Taniguchi, Yojiro; Yamanouchi, Utako; Wu, Jianzhong; Matsumoto, Takashi; Sugimoto, Kazuhiko; Kondo, Katsuhiko; Ikka, Takashi; Ando, Tsuyu; Kono, Izumi; Ito, Sachie; Shomura, Ayahiko; Ookawa, Taiichiro; Hirasawa, Tadashi; Yano, Masahiro; Kondo, Motohiko; Yamamoto, Toshio

2013-01-01

Improvement of leaf photosynthesis is an important strategy for greater crop productivity. Here we show that the quantitative trait locus GPS (GREEN FOR PHOTOSYNTHESIS) in rice (Oryza sativa L.) controls photosynthesis rate by regulating carboxylation efficiency. Map-based cloning revealed that GPS is identical to NAL1 (NARROW LEAF1), a gene previously reported to control lateral leaf growth. The high-photosynthesis allele of GPS was found to be a partial loss-of-function allele of NAL1. This allele increased mesophyll cell number between vascular bundles, which led to thickened leaves, and it pleiotropically enhanced photosynthesis rate without the detrimental side effects observed in previously identified nal1 mutants, such as dwarf plant stature. Furthermore, pedigree analysis suggested that rice breeders have repeatedly selected the high-photosynthesis allele in high-yield breeding programs. The identification and utilization of NAL1 (GPS) can enhance future high-yield breeding and provides a new strategy for increasing rice productivity. PMID:23985993

Diaminopurine-Resistant Mutants of Cultured, Diploid Human Fibroblasts

PubMed Central

Rappaport, Harriet; DeMars, Robert

1973-01-01

Clones of cells resistant to 2,6-diaminopurine were detected in skin fibroblast cultures derived from 13 of 21 normal humans of both sexes from 17 unrelated families. Almost all of the cultures that yielded mutants were chosen for further study from among a total of 83 surveyed because they displayed a slight resistance to low concentrations of diaminopurine. The incidences of mutant colonies ranged between about 10-5 and 10-4 per cell surviving prior mutagenic treatment with MNNG. The incidences of spontaneous mutants were about 10-7 to 10-5 in three unrelated cultures. Most independent mutants had distinctly reduced activity of adenine phosphoribosyltransferase but some had apparently normal amounts of activity. Two mutants from unrelated boys had little or no detectable enzyme activity and were unable to effectively use exogenous adenine for growth when purine biosynthesis was blocked with azaserine. Most mutants could utilize exogenous adenine, just as most azaguanine-resistant fibroblast mutants can utilize exogenous hypoxanthine, even when their hypoxanthine-guanine phosphoribosyltransferase activity is reduced. Diverse genetic changes conferred diaminopurine resistance but their specific natures are still undefined. Gross numerical or structural chromosome abnormalities were not observed in the mutants examined so far. Since at least one gene responsible for adenine phosphoribosyltransferase activity is on autosome No. 16 our results suggest that at least some of the cultures yielding mutants were heterozygous and that alleles conferring diaminopurine resistance may be frequent enough to comprise a polymorphism. PMID:4358687

Breeding for blast-disease-resistant and high-yield Thai jasmine rice (Oryza sativa L. cv. KDML 105) mutants using low-energy ion beams

NASA Astrophysics Data System (ADS)

Mahadtanapuk, S.; Teraarusiri, W.; Phanchaisri, B.; Yu, L. D.; Anuntalabhochai, S.

2013-07-01

Low-energy ion beam was applied on mutation induction for plant breeding of blast-disease-resistant Thai jasmine rice (Oryza sativa L. cv. KDML 105). Seeds of the wild-type rice were bombarded in vacuum by nitrogen ion beam at energy of 60-80 keV to a beam fluence range of 2 × 1016-2 × 1017 ions/cm2. The ion-bombarded rice seeds were grown in soil for 2 weeks as transplanted rice in plastic pots at 1 seedling/pot. The seedlings were then screened for blast resistance by Pyricularia grisea inoculation with 106 spores/ml concentrations. The blast-resistant rice mutant was planted up to F6 generation with the consistent phenotypic variation. The high percentage of the blast-disease-resistant rice was analyzed with DNA fingerprint. The HAT-RAPD (high annealing temperature-random amplified polymorphic DNA) marker revealed the modified polymorphism fragment presenting in the mutant compared with wild type (KDML 105). The cDNA fingerprints were investigated and the polymorphism fragment was subcloned into pGEM-T easy vector and then sequenced. The sequence of this fragment was compared with those already contained in the database, and the fragment was found to be related to the Spotted leaf protein 11 (Spl11).

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

PubMed

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

2018-06-01

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

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

PubMed Central

Gomes, Cláudia; Martínez-Puchol, Sandra; Ruiz-Roldán, Lidia; Pons, Maria J.; del Valle Mendoza, Juana; Ruiz, Joaquim

2016-01-01

The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments. PMID:27667026

Diastereoselective synthesis of L: -threo-3,4-dihydroxyphenylserine by low-specific L: -threonine aldolase mutants.

PubMed

Gwon, Hui-Jeong; Baik, Sang-Ho

2010-01-01

Diastereoselectivity-enhanced mutants of L: -threonine aldolase (L: -TA) for L: -threo-3,4-dihydroxyphenylserine (L: -threo-DOPS) synthesis were isolated by error-prone PCR followed by a high-throughput screening. The most improved mutant was achieved from the mutant T3-3mm2, showing a 4-fold increase over the wild-type L: -TA. When aldol condensation activity was examined using whole cells of T3-3mm2, its de was constantly maintained at 55% during the batch reactions for 80 h, yielding 3.8 mg L: -threo-DOPS/ml.

A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133.

PubMed

Moirangthem, Lakshmipyari Devi; Bhattacharya, Sudeshna; Stensjö, Karin; Lindblad, Peter; Bhattacharya, Jyotirmoy

2014-04-01

A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H₂O₂ could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.

Copper phytoextraction in tandem with oilseed production using commercial cultivars and mutant lines of sunflower.

PubMed

Kolbas, A; Mench, M; Herzig, R; Nehnevajova, E; Bes, C M

2011-01-01

Use of sunflower (Helianthus annuus L.) for Cu phytoextraction and oilseed production on Cu-contaminated topsoils was investigated in afield trial at a former wood preservation site. Six commercial cultivars and two mutant lines were cultivated in plots with and without the addition of compost (5% w/w) and dolomitic limestone (0.2% w/w). Total soil Cu ranged from 163 to 1170 mg kg(-1). In soil solutions, Cu concentration varied between 0.16-0.93 mg L(-1). The amendment increased soil pH, reduced Cu exposure and promoted sunflower growth. Stem length, shoot and capitulum biomasses, seed yield, and shoot and leaf Cu concentrations were measured. At low total soil Cu, shoot Cu mineralomass was higher in commercial cultivars, Le., Salut, Energic, and Countri, whereas competition and shading affected morphological traits of mutants. Based on shoot yield (7 Mg DW ha(-1)) and Cu concentration, the highest removal was 59 g Cu ha(-1). At high total soil Cu, shoot Cu mineralomass peaked for mutants (e.g., 52 g Cu ha(-1) for Mutant 1 line) and cultivars Energic and Countri. Energic seed yield (3.9 Mg air-DW ha(-1)) would be sufficient to produce oil Phenotype traits and shoot Cu removal depended on sunflower types and Cu exposure.

The yield and post-yield behavior of high-density polyethylene

NASA Technical Reports Server (NTRS)

Semeliss, M. A.; Wong, R.; Tuttle, M. E.

1990-01-01

An experimental and analytical evaluation was made of the yield and post-yield behavior of high-density polyethylene, a semi-crystalline thermoplastic. Polyethylene was selected for study because it is very inexpensive and readily available in the form of thin-walled tubes. Thin-walled tubular specimens were subjected to axial loads and internal pressures, such that the specimens were subjected to a known biaxial loading. A constant octahederal shear stress rate was imposed during all tests. The measured yield and post-yield behavior was compared with predictions based on both isotropic and anisotropic models. Of particular interest was whether inelastic behavior was sensitive to the hydrostatic stress level. The major achievements and conclusions reached are discussed.

Development of DNA markers for newly identified high-oleate peanut mutants

USDA-ARS?s Scientific Manuscript database

Development of high-oleate cultivars is one of the important objectives of peanut breeding because consuming products containing high oleate can benefit human health in many aspects. By screening the entire USDA cultivated peanut collection, we have identified two new high-oleate mutants (PI 342664...

Mutants of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil as a biofactory for biodiesel production.

PubMed

Katre, Gouri; Ajmera, Namasvi; Zinjarde, Smita; RaviKumar, Ameeta

2017-10-24

Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L -1  h -1 ) as compared to the wild type (0.033 g L -1  h -1 ). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and

High yield fabrication of fluorescent nanodiamonds

PubMed Central

Boudou, Jean-Paul; Curmi, Patrick; Jelezko, Fedor; Wrachtrup, Joerg; Aubert, Pascal; Sennour, Mohamed; Balasubramanian, Gopalakrischnan; Reuter, Rolf; Thorel, Alain; Gaffet, Eric

2009-01-01

A new fabrication method to produce homogeneously fluorescent nanodiamonds with high yields is described. The powder obtained by high energy ball milling of fluorescent high pressure, high temperature diamond microcrystals was converted in a pure concentrated aqueous colloidal dispersion of highly crystalline ultrasmall nanoparticles with a mean size less than or equal to 10 nm. The whole fabrication yield of colloidal quasi-spherical nanodiamonds was several orders of magnitude higher than those previously reported starting from microdiamonds. The results open up avenues for the industrial cost-effective production of fluorescent nanodiamonds with well-controlled properties. PMID:19451687

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kressel, Lucas; Faries, Kaitlyn M.; Wander, Marc J.

2014-08-01

From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of thismore » process to < 50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. In conclusion, this change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.« less

Combining high biodiversity with high yields in tropical agroforests.

PubMed

Clough, Yann; Barkmann, Jan; Juhrbandt, Jana; Kessler, Michael; Wanger, Thomas Cherico; Anshary, Alam; Buchori, Damayanti; Cicuzza, Daniele; Darras, Kevin; Putra, Dadang Dwi; Erasmi, Stefan; Pitopang, Ramadhanil; Schmidt, Carsten; Schulze, Christian H; Seidel, Dominik; Steffan-Dewenter, Ingolf; Stenchly, Kathrin; Vidal, Stefan; Weist, Maria; Wielgoss, Arno Christian; Tscharntke, Teja

2011-05-17

Local and landscape-scale agricultural intensification is a major driver of global biodiversity loss. Controversially discussed solutions include wildlife-friendly farming or combining high-intensity farming with land-sparing for nature. Here, we integrate biodiversity and crop productivity data for smallholder cacao in Indonesia to exemplify for tropical agroforests that there is little relationship between yield and biodiversity under current management, opening substantial opportunities for wildlife-friendly management. Species richness of trees, fungi, invertebrates, and vertebrates did not decrease with yield. Moderate shade, adequate labor, and input level can be combined with a complex habitat structure to provide high biodiversity as well as high yields. Although livelihood impacts are held up as a major obstacle for wildlife-friendly farming in the tropics, our results suggest that in some situations, agroforests can be designed to optimize both biodiversity and crop production benefits without adding pressure to convert natural habitat to farmland.

Recombination Between Guanidine-resistant and Dextran Sulfate-resistant Mutants of Type 1 Poliovirus

PubMed Central

Sergiescu, Dina; Aubert-Combiescu, Andrei; Crainic, Radu

1969-01-01

Mixed infection of monkey kidney cells with two mutants of the LSc2ab strain of poliovirus, one resistant to guanidine and the other resistant to both dextran sulfate and 2-(α-hydroxybenzyl)-benzimidazole (HBB), yielded progeny in which the number of guardexr particles exceeded by a factor of 7 to 10 the expected number of similar particles occurring through spontaneous mutation; recombination would explain the fairly high excess of doubly mutant particles that was obtained. Scoring of HBB resistance in 50 guardexr clones suggested that, during recombination, resistance to dextran sulfate is not associated with HBB resistance. Images PMID:4305674

Low-energy N-ion beam biotechnology application in the induction of Thai jasmine rice mutant with improved seed storability

NASA Astrophysics Data System (ADS)

Semsang, Nuananong; Techarang, Jiranat; Yu, Liangdeng; Phanchaisri, Boonrak

2018-06-01

Low-energy heavy-ion beam is a novel biotechnology used for mutation induction in plants. We used a low-energy N-ion beam to induce mutations in Thai jasmine rice (Oryza sativa L. cv. KDML 105) to improve the yield and seed quality. Seeds of BKOS6, a Thai jasmine rice mutant previously induced by ion beams, were re-bombarded with 60-kV-accelerated N-ions (N++N2+) to fluences of 1-2 × 1016 ions/cm2. The resulting mutant, named HyKOS21, exhibited photoperiod insensitivity, semi-dwarfness, and high yield potential. Seed storability of the mutant was studied in natural and accelerated ageing conditions and compared to that of KDML 105 and six other Thai rice varieties. In both testing conditions, HyKOS21 mutant had the highest seed storability among the tested varieties. After storage in the natural condition for 18 months, HyKOS21 had a seed germination percentage nearly two times as that of the original KDML 105. Biochemical analysis showed that the lipid peroxidation level of the mutant seeds was the lowest among those of the tested varieties. Furthermore, an expression analysis of genes encoding lipoxygenase isoenzyme (lox1, lox2, and lox3) revealed that the mutant lacked expression of lox1 and lox2 and expressed only lox3 in seeds. These results may explain the improved seed longevity of the mutant after storage. This work provides further evidence of the modification of biological materials using a low-energy ion beam to produce rice mutants with improved yield and seed storability. The benefits of this technology, to create new varieties with improved values, could serve for local economic development.

Combining high biodiversity with high yields in tropical agroforests

PubMed Central

Clough, Yann; Barkmann, Jan; Juhrbandt, Jana; Kessler, Michael; Wanger, Thomas Cherico; Anshary, Alam; Buchori, Damayanti; Cicuzza, Daniele; Darras, Kevin; Putra, Dadang Dwi; Erasmi, Stefan; Pitopang, Ramadhanil; Schmidt, Carsten; Schulze, Christian H.; Seidel, Dominik; Steffan-Dewenter, Ingolf; Stenchly, Kathrin; Vidal, Stefan; Weist, Maria; Wielgoss, Arno Christian; Tscharntke, Teja

2011-01-01

Local and landscape-scale agricultural intensification is a major driver of global biodiversity loss. Controversially discussed solutions include wildlife-friendly farming or combining high-intensity farming with land-sparing for nature. Here, we integrate biodiversity and crop productivity data for smallholder cacao in Indonesia to exemplify for tropical agroforests that there is little relationship between yield and biodiversity under current management, opening substantial opportunities for wildlife-friendly management. Species richness of trees, fungi, invertebrates, and vertebrates did not decrease with yield. Moderate shade, adequate labor, and input level can be combined with a complex habitat structure to provide high biodiversity as well as high yields. Although livelihood impacts are held up as a major obstacle for wildlife-friendly farming in the tropics, our results suggest that in some situations, agroforests can be designed to optimize both biodiversity and crop production benefits without adding pressure to convert natural habitat to farmland. PMID:21536873

High temperature specifically affects the photoprotective responses of chlorophyll b-deficient wheat mutant lines.

PubMed

Brestic, Marian; Zivcak, Marek; Kunderlikova, Kristyna; Allakhverdiev, Suleyman I

2016-12-01

The effects of high temperature on CO 2 assimilation rate, processes associated with photosynthetic electron and proton transport, as well as photoprotective responses, were studied in chlorophyll b-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). Despite the low chlorophyll content and chlorophyll a-to-b ratio, the non-stressed mutant plants had the similar level of CO 2 assimilation and photosynthetic responses as WT. However, in ANK mutant plants exposed to prolonged high temperature episode (42 °C for ~10 h), we observed lower CO 2 assimilation compared to WT, especially when a high CO 2 supply was provided. In all heat-exposed plants, we found approximately the same level of PSII photoinhibition, but the decrease in content of photooxidizable PSI was higher in ANK mutant plants compared to WT. The PSI damage can be well explained by the level of overreduction of PSI acceptor side observed in plants exposed to high temperature, which was, in turn, the result of the insufficient transthylakoid proton gradient associated with low non-photochemical quenching and lack of ability to downregulate the linear electron transport to keep the reduction state of PSI acceptor side low enough. Compared to WT, the ANK mutant lines had lower capacity to drive the cyclic electron transport around PSI in moderate and high light; it confirms the protective role of cyclic electron transport for the protection of PSI against photoinhibition. Our results, however, also suggest that the inactivation of PSI in heat stress conditions can be the protective mechanism against photooxidative damage of chloroplast and cell structures.

Evaluation of Brown Midrib Sorghum Mutants as a Potential Biomass Feedstock for 2,3-Butanediol Biosynthesis.

PubMed

Guragain, Yadhu N; Srinivasa Rao, P; Vara Prasad, P V; Vadlani, Praveen V

2017-11-01

Three sorghum backgrounds [Atlas, Early Hegari (EH), and Kansas Collier (KC)] and two bmr mutants (bmr6 and bmr12) of each line were evaluated and compared for grain and biomass yield, biomass composition, and 2,3-butanediol production from biomass. The data showed that the bmr6 mutation in EH background led to a significant decrease in stover yield and increase in grain yield, whereas the stover yield was increased by 64% without affecting grain yield in KC background. The bmr mutants had 10 to 25% and 2 to 9% less lignin and structural carbohydrate contents, respectively, and 24 to 93% more non-structural sugars than their parents in all sorghum lines, except EH bmr12. The total fermentable sugars released were 22 to 36% more in bmr mutants than in parents for Atlas and KC, but not for EH. The bmr6 mutation in KC background produced the most promising feedstock, among the evaluated bmr mutants, for 2,3-butanediol production without affecting grain yield, followed by KC bmr12 and Atlas bmr6, but the bmr mutation had an adverse effect in EH background. This indicated that the genetic background of the parent line and type of bmr mutation significantly affect the biomass quality as a feedstock for biochemical production.

Ascorbate, added after irradiation, reduces the mutant yield and alters the spectrum of CD59- mutations in A(L) cells irradiated with high LET carbon ions

NASA Technical Reports Server (NTRS)

Ueno, Akiko; Vannais, Diane; Lenarczyk, Marek; Waldren, Charles A.; Chatterjee, A. (Principal Investigator)

2002-01-01

It has been reported that X-ray induced HPRT- mutation in cultured human cells is prevented by ascorbate added after irradiation. Mutation extinction is attributed to neutralization by ascorbate, of radiation-induced long-lived radicals (LLR) with half-lives of several hours. We here show that post-irradiation treatment with ascorbate (5 mM added 30 min after radiation) reduces, but does not eliminate, the induction of CD59- mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon ions (LET of 100 KeV/microm). RibCys, [2(R,S)-D-ribo-1',2',3',4'-Tetrahydroxybutyl]-thiazolidene-4(R)-ca riboxylic acid] (4 mM) gave a similar but lesser effect. The lethality of the carbon ions was not altered by these chemicals. Preliminary data are presented that ascorbate also alters the spectrum of CD59- mutations induced by the carbon beam, mainly by reducing the incidence of small mutations and mutants displaying transmissible genomic instability (TGI), while large mutations are unaffected. Our results suggest that LLR are important in initiating TGI.

Genetics of Ustilago violacea. I. Carotenoid mutants and carotenogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garber, E.D.; Baird, M.L.; Chapman, D.J.

1975-12-01

Wild-type strains of Ustilago violacea produce pink colonies on laboratory medium and yield white, orange, pumpkin, and yellow colonies after uv mutagenesis. The wild-type strains contain neurosporene and lycopene; one orange mutant, $gamma$-carotene; and one yellow mutant, $beta$-carotene. One white mutant had no detectable carotenoids. Diploid colonies heterozygous for wild type and orange, pumpkin, yellow, or white are phenotypically wild type. Diploid colonies heterozygous for yellow and orange are also phenotypically wild type. Diploid colonies heterozygous for white and orange; white and yellow; and white, yellow, and orange are phenotypically light orange, light yellow, and orange- yellow, respectively. The whitemore » mutants give a circular complementation map; the color mutants fit a linear complementation map. We propose a multienzyme of four identical dehydrogenases and one or two identical cyclases for carotenogenesis in this species. The white and color mutants represent structural mutations altering the conformation of the dehydrogenase or cyclase, respectively. Furthermore, cyclases may or may not aggregate in association with the dehydrogenase aggregate to form the multienzyme aggregate responsible for the color mutants. (auth)« less

Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study.

PubMed

Ma, Yingfang; Yang, Haiquan; Chen, Xianzhong; Sun, Bo; Du, Guocheng; Zhou, Zhemin; Song, Jiangning; Fan, You; Shen, Wei

2015-10-01

In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae.

PubMed

Pagliardini, Julien; Hubmann, Georg; Alfenore, Sandrine; Nevoigt, Elke; Bideaux, Carine; Guillouet, Stephane E

2013-03-28

Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions. Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further developing robust strains with improved

Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

PubMed

Muthusamy, Annamalai; Jayabalan, Narayanasamy

2013-01-01

The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

Transcriptome sequencing and metabolic pathways of astaxanthin accumulated in Haematococcus pluvialis mutant under 15% CO2.

PubMed

Cheng, Jun; Li, Ke; Zhu, Yanxia; Yang, Weijuan; Zhou, Junhu; Cen, Kefa

2017-03-01

Transcriptome sequencing and annotation was performed on Haematococcus pluvialis mutant red cells induced with high light under 15% CO 2 to demonstrate why astaxanthin yield of the mutant was 1.7 times higher than that of a wild strain. It was found that 56% of 1947 differentially expressed genes were upregulated in mutant cells. Most significant differences were found in unigenes related to photosynthesis, carotenoid biosynthesis and fatty acid biosynthesis pathways. The pyruvate kinase increased by 3.5-fold in mutant cells. Thus, more pyruvate, which was beneficial to carotenoids and fatty acid biosynthesis, was generated. Phytoene synthase, zeta-carotene desaturase, lycopene beta-cyclase involved in β-carotene biosynthesis in mutant cells were upregulated by 10.4-, 4.4-, and 5.8-fold, respectively. Beta-carotene 3-hydroxylase catalyzing conversion of β-carotene into astaxanthin was upregulated by 18.4-fold. The fatty acid biosynthesis was promoted because of the upregulation of acetyl-CoA synthetase and acetyl-CoA carboxylase, thus increasing astaxanthin esterification and accumulation in mutant cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice[OPEN

PubMed Central

Wang, Wei; Hu, Bin; Liu, Yongqiang; Che, Ronghui; Hu, Yingchun; Zhang, Zhihua; Wang, Hongru; Li, Hua; Jiang, Zhimin; Zhang, Zhengli; Gao, Xiaokai; Qiu, Yahong; Meng, Xiangbing; Liu, Yongxin; Bai, Yang; Liang, Yan; Wang, Yiqin; Zhang, Lianhe; Li, Legong; Sodmergen; Jing, Haichun

2018-01-01

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice (Oryza sativa) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana. Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security. PMID:29475937

Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil.

PubMed

Pham, Anh-Tung; Shannon, J Grover; Bilyeu, Kristin D

2012-08-01

High oleic acid soybeans were produced by combining mutant FAD2-1A and FAD2-1B genes. Despite having a high oleic acid content, the linolenic acid content of these soybeans was in the range of 4-6 %, which may be high enough to cause oxidative instability of the oil. Therefore, a study was conducted to incorporate one or two mutant FAD3 genes into the high oleic acid background to further reduce the linolenic acid content. As a result, soybean lines with high oleic acid and low linolenic acid (HOLL) content were produced using different sources of mutant FAD2-1A genes. While oleic acid content of these HOLL lines was stable across two testing environments, the reduction of linolenic acid content varied depending on the number of mutant FAD3 genes combined with mutant FAD2-1 genes, on the severity of mutation in the FAD2-1A gene, and on the testing environment. Combination of two mutant FAD2-1 genes and one mutant FAD3 gene resulted in less than 2 % linolenic acid content in Portageville, Missouri (MO) while four mutant genes were needed to achieve the same linolenic acid in Columbia, MO. This study generated non-transgenic soybeans with the highest oleic acid content and lowest linolenic acid content reported to date, offering a unique alternative to produce a fatty acid profile similar to olive oil.

High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection

PubMed Central

Matsunaga, James; Haake, David A.

2016-01-01

Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants. We infected hamsters with a pool of 42 mutants (input pool), which included control mutants with insertions in four genes previously analyzed by virulence testing (loa22, ligB, flaA1, and lic20111) and 23 mutants with disrupted signal transduction genes. We quantified the mutants in different tissues (blood, kidney and liver) at 4 days post-challenge by high-throughput sequencing and compared the frequencies of mutants recovered from tissues to their frequencies in the input pool. Control mutants that were less fit in the Tn-Seq experiment were attenuated for virulence when tested separately in the hamster model of lethal leptospirosis. Control mutants with unaltered fitness were as virulent as the wild-type strain. We identified two mutants with the transposon inserted in the same putative adenylate/guanylate cyclase gene (lic12327) that had reduced in vivo fitness in blood, kidney and liver. Both lic12327 mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and lic12327 mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of L. interrogans transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing. PMID

The Mechanism of Starch Over-Accumulation in Chlamydomonas reinhardtii High-Starch Mutants Identified by Comparative Transcriptome Analysis

PubMed Central

Koo, Kwang M.; Jung, Sera; Lee, Beom S.; Kim, Jin-Baek; Jo, Yeong D.; Choi, Hong-Il; Kang, Si-Yong; Chung, Gook-H.; Jeong, Won-Joong; Ahn, Joon-Woo

2017-01-01

The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii. PMID:28588557

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice.

PubMed

Wang, Wei; Hu, Bin; Yuan, Dingyang; Liu, Yongqiang; Che, Ronghui; Hu, Yingchun; Ou, Shujun; Liu, Yongxin; Zhang, Zhihua; Wang, Hongru; Li, Hua; Jiang, Zhimin; Zhang, Zhengli; Gao, Xiaokai; Qiu, Yahong; Meng, Xiangbing; Liu, Yongxin; Bai, Yang; Liang, Yan; Wang, Yiqin; Zhang, Lianhe; Li, Legong; Sodmergen; Jing, Haichun; Li, Jiayang; Chu, Chengcai

2018-03-01

Nitrogen (N) is a major driving force for crop yield improvement, but application of high levels of N delays flowering, prolonging maturation and thus increasing the risk of yield losses. Therefore, traits that enable utilization of high levels of N without delaying maturation will be highly desirable for crop breeding. Here, we show that OsNRT1.1A (OsNPF6.3), a member of the rice ( Oryza sativa ) nitrate transporter 1/peptide transporter family, is involved in regulating N utilization and flowering, providing a target to produce high yield and early maturation simultaneously. OsNRT.1A has functionally diverged from previously reported NRT1.1 genes in plants and functions in upregulating the expression of N utilization-related genes not only for nitrate but also for ammonium, as well as flowering-related genes. Relative to the wild type, osnrt1.1a mutants exhibited reduced N utilization and late flowering. By contrast, overexpression of OsNRT1.1A in rice greatly improved N utilization and grain yield, and maturation time was also significantly shortened. These effects were further confirmed in different rice backgrounds and also in Arabidopsis thaliana Our study paves a path for the use of a single gene to dramatically increase yield and shorten maturation time for crops, outcomes that promise to substantially increase world food security. © 2018 American Society of Plant Biologists. All rights reserved.

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast.

PubMed

Seo, Hogyu David; Lee, Daeyoup

2018-05-15

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

High-biomass C4 grasses-Filling the yield gap.

PubMed

Mullet, John E

2017-08-01

A significant increase in agricultural productivity will be required by 2050 to meet the needs of an expanding and rapidly developing world population, without allocating more land and water resources to agriculture, and despite slowing rates of grain yield improvement. This review examines the proposition that high-biomass C 4 grasses could help fill the yield gap. High-biomass C 4 grasses exhibit high yield due to C 4 photosynthesis, long growth duration, and efficient capture and utilization of light, water, and nutrients. These C 4 grasses exhibit high levels of drought tolerance during their long vegetative growth phase ideal for crops grown in water-limited regions of agricultural production. The stems of some high-biomass C 4 grasses can accumulate high levels of non-structural carbohydrates that could be engineered to enhance biomass yield and utility as feedstocks for animals and biofuels production. The regulatory pathway that delays flowering of high-biomass C 4 grasses in long days has been elucidated enabling production and deployment of hybrids. Crop and landscape-scale modeling predict that utilization of high-biomass C 4 grass crops on land and in regions where water resources limit grain crop yield could increase agricultural productivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Listeria monocytogenes mutants with altered growth phenotypes at refrigeration temperature and high salt concentrations.

PubMed

Burall, Laurel S; Laksanalamai, Pongpan; Datta, Atin R

2012-02-01

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes.

Listeria monocytogenes Mutants with Altered Growth Phenotypes at Refrigeration Temperature and High Salt Concentrations

PubMed Central

Burall, Laurel S.; Laksanalamai, Pongpan

2012-01-01

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes. PMID:22179239

Rubisco mutants of Chlamydomonas reinhardtii display divergent photosynthetic parameters and lipid allocation.

PubMed

Esquível, M G; Matos, A R; Marques Silva, J

2017-07-01

Photosynthesis and lipid allocation were investigated in Rubisco small subunit mutants of the microalga Chlamydomonas reinhardtii. Comparative analyses were undertaken with cells grown photoheterotrophically under sulphur-replete or sulphur-depleted conditions. The Y67A Rubisco mutant, which has previously demonstrated a pronounced reduction in Rubisco levels and higher hydrogen production rates than the wild type, also shows the following divergences in photosynthetic phenotype and lipid allocation: (i) low Fv/Fm (maximum photochemical efficiency), (ii) low effective quantum yield of photosystem II (ΦPSII), (iii) low effectiveness at protection against high light intensities, (iv) a higher level of total lipids per pigment and (v) changes in the relative proportions of different fatty acids, with a marked decrease in unsaturated fatty acids (FAs). The most abundant thylakoid membrane lipid, monogalactosyldiacylglycerol, decreased in amount, while the neutral lipid/polar lipid ratio increased in the mutant. The low amount and activity of the mutated Rubisco Y67A enzyme seems to have an adverse effect on photosynthesis and causes changes in carbon allocation in terms of membrane fatty acid composition and storage lipid accumulation. Our results suggest that Rubisco mutants of Chlamydomonas might be useful in biodiesel production.

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells

NASA Technical Reports Server (NTRS)

Ueno, A. M.; Vannais, D. B.; Gustafson, D. L.; Wong, J. C.; Waldren, C. A.

1996-01-01

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

Identification and Characterization of an Arabidopsis thaliana Mutant lbt With High Tolerance to Boron Deficiency

PubMed Central

Huai, Zexun; Peng, Lishun; Wang, Sheliang; Zhao, Hua; Shi, Lei; Xu, Fangsen

2018-01-01

Boron (B) is an essential micronutrient of plants. In the present study, we characterized an Arabidopsis mutant lbt with significant low-boron tolerance that was identified based on our previous mapping of QTL for B efficiency in Arabidopsis. Multiple nutrient-deficiency analyses point out that lbt mutant is insensitive to only B-limitation stress. Compared with wild-type Col-0, the fresh weight, leaf area, root length and root elongation rate of lbt mutant were significantly improved under B deficiency during vegetative growth. lbt mutant also showed the improvements in plant height, branches and inflorescences compared with Col-0 during the reproductive stage under B limitation. Ultrastructure analysis of the leaves showed that starch accumulation in lbt mutant was significantly diminished compared with Col-0. Furthermore, there were no significant differences in the expression of transporter-related genes and B concentrations between Col-0 and lbt mutant under both normal B and low-B conditions. These results suggest that lbt mutant has a lower B demand than Col-0. Genetic analysis suggests that the low-B tolerant phenotype of lbt mutant is under the control of a monogenic recessive gene. Based on the high-density SNP linkage genetic map, only one QTL for low-B tolerance was mapped on chromosome 4 between 10.4 and 14.8 Mb. No any reported B-relative genes exist in the QTL interval, suggesting that a gene with unknown function controls the tolerance of lbt to B limitation. Taken together, lbt is a low-B tolerant mutant that does not depend on the uptake or transport of B and is controlled by a monogenic recessive gene mapped on chromosome 4, and cloning and functional analysis of LBT gene are expected to reveal novel mechanisms for plant resistance to B deficiency.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

PubMed

Chung, K-R; Ehrenshaft, M; Wetzel, D K; Daub, M E

2003-11-01

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

PubMed

Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

2011-09-01

The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Specific interaction of mutant p53 with regions of matrix attachment region DNA elements (MARs) with a high potential for base-unpairing

PubMed Central

Will, Katrin; Warnecke, Gabriele; Wiesmüller, Lisa; Deppert, Wolfgang

1998-01-01

Mutant, but not wild-type p53 binds with high affinity to a variety of MAR-DNA elements (MARs), suggesting that MAR-binding of mutant p53 relates to the dominant-oncogenic activities proposed for mutant p53. MARs recognized by mutant p53 share AT richness and contain variations of an AATATATTT “DNA-unwinding motif,” which enhances the structural dynamics of chromatin and promotes regional DNA base-unpairing. Mutant p53 specifically interacted with MAR-derived oligonucleotides carrying such unwinding motifs, catalyzing DNA strand separation when this motif was located within a structurally labile sequence environment. Addition of GC-clamps to the respective MAR-oligonucleotides or introducing mutations into the unwinding motif strongly reduced DNA strand separation, but supported the formation of tight complexes between mutant p53 and such oligonucleotides. We conclude that the specific interaction of mutant p53 with regions of MAR-DNA with a high potential for base-unpairing provides the basis for the high-affinity binding of mutant p53 to MAR-DNA. PMID:9811860

The metabolic costs of improving ethanol yield by reducing glycerol formation capacity under anaerobic conditions in Saccharomyces cerevisiae

PubMed Central

2013-01-01

Background Finely regulating the carbon flux through the glycerol pathway by regulating the expression of the rate controlling enzyme, glycerol-3-phosphate dehydrogenase (GPDH), has been a promising approach to redirect carbon from glycerol to ethanol and thereby increasing the ethanol yield in ethanol production. Here, strains engineered in the promoter of GPD1 and deleted in GPD2 were used to investigate the possibility of reducing glycerol production of Saccharomyces cerevisiae without jeopardising its ability to cope with process stress during ethanol production. For this purpose, the mutant strains TEFmut7 and TEFmut2 with different GPD1 residual expression were studied in Very High Ethanol Performance (VHEP) fed-batch process under anaerobic conditions. Results Both strains showed a drastic reduction of the glycerol yield by 44 and 61% while the ethanol yield improved by 2 and 7% respectively. TEFmut2 strain showing the highest ethanol yield was accompanied by a 28% reduction of the biomass yield. The modulation of the glycerol formation led to profound redox and energetic changes resulting in a reduction of the ATP yield (YATP) and a modulation of the production of organic acids (acetate, pyruvate and succinate). Those metabolic rearrangements resulted in a loss of ethanol and stress tolerance of the mutants, contrarily to what was previously observed under aerobiosis. Conclusions This work demonstrates the potential of fine-tuned pathway engineering, particularly when a compromise has to be found between high product yield on one hand and acceptable growth, productivity and stress resistance on the other hand. Previous study showed that, contrarily to anaerobiosis, the resulting gain in ethanol yield was accompanied with no loss of ethanol tolerance under aerobiosis. Moreover those mutants were still able to produce up to 90 gl-1 ethanol in an anaerobic SSF process. Fine tuning metabolic strategy may then open encouraging possibilities for further

Regulatory Mutants at the his1 Locus of Yeast

PubMed Central

Lax, Carol; Fogel, Seymour; Cramer, Carole

1979-01-01

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1–7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auoxtrophic parental alleles. The double mutants, HIS1–7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1–7 background, the mutant regulatory site (HIS1–e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine. PMID:385447

Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction

PubMed Central

Basit, Nada; Wechsler, Harry

2011-01-01

Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1). This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets. PMID:22007208

Analysis of the trade-off between high crop yield and low yield instability at the global scale

NASA Astrophysics Data System (ADS)

Ben-Ari, Tamara; Makowski, David

2016-10-01

Yield dynamics of major crops species vary remarkably among continents. Worldwide distribution of cropland influences both the expected levels and the interannual variability of global yields. An expansion of cultivated land in the most productive areas could theoretically increase global production, but also increase global yield instability if the most productive regions are characterized by high interannual yield variability. In this letter, we use portfolio analysis to quantify the tradeoff between the expected values and the interannual variance of global yield. We compute optimal frontiers for four crop species i.e., maize, rice, soybean and wheat and show how the distribution of cropland among large world regions can be optimized to either increase expected global crop production or decrease its interannual variability. We also show that a preferential allocation of cropland in the most productive regions can increase global expected yield at the expense of yield stability. Theoretically, optimizing the distribution of a small fraction of total cultivated areas can help find a good compromise between low instability and high crop yields at the global scale.

High-molecular-weight glutenin subunit-deficient mutants induced by ion beam and the effects of Glu-1 loci deletion on wheat quality properties.

PubMed

Zhang, Lujun; Chen, Qiufang; Su, Mingjie; Yan, Biao; Zhang, Xiangqi; Jiao, Zhen

2016-03-15

High-molecular-weight glutenin subunits (HMW-GSs) play a critical role in determining the viscoelastic properties of wheat. Mutations induced by ion beam radiation have been applied to improve the yield and quality of crop. In this study, HMW-GS-deficient mutant lines were selected and the effects of Glu-1 loci deletion on wheat quality properties were illustrated according to the analysis of dry seeds of common wheat (Triticum aestivum L.) Xiaoyan 81 treated with a nitrogen ion beam. Three HMW-GS-deficient mutant lines were obtained and then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Large-chromosome-fragment deletion resulted in specific deficiencies, and the deleted region sizes were determined using molecular markers. Agronomic characters, quantity and proportion of glutenins and dough microstructure of the deletion lines all proved to be quite different from those of wild-type Xiaoyan 81. Analysis of quality properties suggested that GluA1(-) had superior property parameters, while GluB1(-) and GluD1(-) both showed a significant decrease in quality properties compared with Xiaoyan 81. The effects of the three Glu-1 loci on flour and dough quality-related parameters should be Glu-D1 > Glu-B1 > Glu-A1. Ion beam radiation can be used as a mutagen to create new crop mutants. © 2015 Society of Chemical Industry.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

PubMed

Zhang, Huidan; Lu, Dong; Li, Xin; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2018-05-02

Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L - 1 , which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

High-yield positron systems for linear colliders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clendenin, J.E.

1989-04-01

Linear colliders, such as the SLC, are among those accelerators for which a high-yield positron source operating at the repetition rate of the accelerator is desired. The SLC, having electron energies up to 50 GeV, presents the possibility of generating positron bunches with useful charge even exceeding that of the initial electron bunch. The exact positron yield to be obtained depends on the particular capture, transport and damping system employed. Using 31 GeV electrons impinging on a W-type converter phase-space at the target to the acceptance of the capture rf section, the SLC source is capable of producing, for everymore » electron, up to two positrons within the acceptance of the positron damping ring. The design of this source and the performance of the positron system as built are described. Also, future prospects and limitations for high-yield positron systems are discussed. 11 refs., 5 figs., 3 tabs.« less

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

PubMed Central

SENGUPTA, GARGI; PALIT, P.

2004-01-01

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

Strontium and barium iodide high light yield scintillators

NASA Astrophysics Data System (ADS)

Cherepy, Nerine J.; Hull, Giulia; Drobshoff, Alexander D.; Payne, Stephen A.; van Loef, Edgar; Wilson, Cody M.; Shah, Kanai S.; Roy, Utpal N.; Burger, Arnold; Boatner, Lynn A.; Choong, Woon-Seng; Moses, William W.

2008-02-01

Europium-doped strontium and barium iodide are found to be readily growable by the Bridgman method and to produce high scintillation light yields. SrI2(Eu ) emits into the Eu2+ band, centered at 435nm, with a decay time of 1.2μs and a light yield of ˜90000photons/MeV. It offers energy resolution better than 4% full width at half maximum at 662keV, and exhibits excellent light yield proportionality. BaI2(Eu ) produces >30000photons/MeV into the Eu2+ band at 420nm (<1μs decay). An additional broad impurity-mediated recombination band is present at 550nm (>3μs decay), unless high-purity feedstock is used.

Reducing pressure on natural forests through high-yield forestry

Treesearch

W.T. Gladstone; F. Thomas Ledig

1990-01-01

High-yield forestry can make a valuable contribution to the conservation and sustained use of forest ecosystems. Despite the pressing reasons for conserving forest resources, population growth creates pressures for exploiting them. Unless needs for forest products, export credits, and local employment can be met by new devices, such as high-yield forestry, these...

Increasing the Triacylglycerol Content in Dunaliella tertiolecta through Isolation of Starch-Deficient Mutants.

PubMed

Sirikhachornkit, Anchalee; Vuttipongchaikij, Supachai; Suttangkakul, Anongpat; Yokthongwattana, Kittisak; Juntawong, Piyada; Pokethitiyook, Prayad; Kangvansaichol, Kunn; Meetam, Metha

2016-05-28

The production cost of biodiesel from microalgae is still not competitive, compared with that of petroleum fuels. The genetic improvement of microalgal strains to increase triacylglycerol (TAG) accumulation is one way to reduce production costs. One of the most promising approaches is the isolation of starch-deficient mutants, which have been reported to successfully increase TAG yields. To date, such a stable mutant is not available in an oleaginous marine microalga, despite several advantages of using marine species for biodiesel production. Algae in the genus Dunaliella are known to tolerate high salt concentration and other environmental stresses. In addition, the cultivation processes for large-scale outdoor commercialization have been well established for this genus. In this study, Dunaliella tertiolecta was used to screen for starch-deficient mutants, using an iodine vapor-staining method. Four out of 20,016 UV-mutagenized strains showed a substantial reduction of starch content. A significantly higher TAG content, up to 3-fold of the wild-type level, was observed in three of the mutants upon induction by nitrogen depletion. The carotenoid production and growth characteristics of these mutants, under both normal and oxidative stress conditions, were not compromised, suggesting that these processes are not necessarily affected by starch deficiency. The results from this work open up new possibilities for exploring Dunaliella for biodiesel production.

Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

DOE PAGES

Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; ...

2016-08-24

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

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

PubMed

Norman, Kaitlyn L; Kumar, Anuj

2016-03-01

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

Butanol production by a Clostridium beijerinckii mutant with high ferulic acid tolerance.

PubMed

Liu, Jun; Guo, Ting; Wang, Dong; Xu, Jiahui; Ying, Hanjie

2016-09-01

A mutant strain of Clostridium beijerinckii, with high tolerance to ferulic acid, was generated using atmospheric pressure glow discharge and high-throughput screening of C. beijerinckii NCIMB 8052. The mutant strain M11 produced 7.24 g/L of butanol when grown in P2 medium containing 30 g/L of glucose and 0.5 g/L of ferulic acid, which is comparable to the production from non-ferulic acid cultures (8.11 g/L of butanol). When 0.8 g/L of ferulic acid was introduced into the P2 medium, C. beijerinckii M11 grew well and produced 4.91 g/L of butanol. Both cell growth and butanol production of C. beijerinckii M11 were seriously inhibited when 0.9 g/L of ferulic acid was added into the P2 medium. Furthermore, C. beijerinckii M11 could produce 6.13 g/L of butanol using non-detoxified hemicellulosic hydrolysate from diluted sulfuric acid-treated corn fiber (SAHHC) as the carbon source. These results demonstrate that C. beijerinckii M11 has a high ferulic acid tolerance and is able to use non-detoxified SAHHC for butanol production. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Restoring de novo Coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous Coenzyme Q supplementation

PubMed Central

Gomez, Fernando; Saiki, Ryoichi; Chin, Randall; Srinivasan, Chandra; Clarke, Catherine F.

2012-01-01

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 E. coli, but their second generation homozygous progeny do not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5′ and 3′, respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q10. Here we show that the Q9 content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q9 content, indicating

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation.

PubMed

Gomez, Fernando; Saiki, Ryoichi; Chin, Randall; Srinivasan, Chandra; Clarke, Catherine F

2012-09-10

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9

[Study on High-yield Cultivation Measures for Arctii Fructus].

PubMed

Liu, Shi-yong; Jiang, Xiao-bo; Wang, Tao; Sun, Ji-ye; Hu, Shang-qin; Zhang, Li

2015-02-01

To find out the high yield cultivation measures for Arctii Fructus. Completely randomized block experiment design method was used in the field planting, to analyze the effect of different cultivation way on agronomic characters, phenological phase,quality and quantity of Arctii Fructus. Arctium lappa planted on August 28 had the best results of plant height, thousand seeds weight and yield. The highest yield of Arctii Fructus was got at the density of 1,482 plants/667 m2. Arctiin content was in an increase trend with the planting time delay and planting density increasing. The plant height, thousand seeds weight, yield and arctiin content by split application of fertilizer were significantly higher than that by one-time fertilization. Compared with open field Arctium lappa, plant height, yield, arctiin content and relative water content of plastic film mulching Arctium lappa was higher by 7.74%, 10.87%, 6.38% and 24.20%, respectively. In the topping Arctium lappa, the yield was increased by 11.09%, with 39. 89% less branching number. Early planting time and topping shortened the growth cycle of Arctium lappa plant. The high-yield cultivation measures of Arctii Fructus are: around August 28 to sowing, planting density of 1 482 plants/667 m2, split application of fertilizer for four times, covering film on surface of the soil and topping in bolting.

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G

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 responsivenessmore » 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.« less

Highly Branched Phenotype of the Petunia dad1-1 Mutant Is Reversed by Grafting.

PubMed Central

Napoli, C.

1996-01-01

The recessive dad1-1 allele conditions a highly branched growth habit resulting from a proliferation of first- and second-order branches. Unlike the wild-type parent, which has lateral branching delayed until the third or fourth leaf node distal to the cotyledons, dad1-1 initiates lateral branching from each cotyledon axil. In addition to initiating lateral branching sooner than the wild type, dad1-1 sustains branching through more nodes on the main shoot axis than the wild type. In keeping with a propensity for branching at basal nodes, dad1-1 produces second-order branches at the proximal-most nodes on first-order branches and small shoots from accessory buds at basal nodes on the main shoot axis. Additional traits associated with the mutation are late flowering, adventitious root formation, shortened internodes, and mild leaf chlorosis. Graft studies show that a dad1-1 scion, when grafted onto wild-type stock, is converted to a phenotype resembling the wild type. Furthermore, a small wild-type interstock fragment inserted between a mutant root stock and a mutant scion is sufficient to convert the dad1-1 scion from mutant to a near wild-type appearance. The recessive dad1-1 phenotype combines traits associated with cytokinin overexpression, auxin overexpression, and gibberellin limitation, which suggests a complex interaction of hormones in establishing the mutant phenotype. PMID:12226274

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

PubMed

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

2011-12-08

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

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

PubMed Central

2011-01-01

Background Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. PMID:22152063

Malic acid production by chemically induced Aspergillus niger MTCC 281 mutant from crude glycerol.

PubMed

Iyyappan, J; Bharathiraja, B; Baskar, G; Jayamuthunagai, J; Barathkumar, S; Anna Shiny, R

2018-03-01

In the present investigation, crude glycerol derived from transesterification process was utilized to produce the commercially-valuable malic acid. A combined resistant on methanol and malic acid strain of Aspergillus niger MTCC 281 mutant was generated in solid medium containing methanol (1-5%) and malic acid (40-80 g/L) by the adaptation process for 22 weeks. The ability of induced Aspergillus niger MTCC 281 mutant to utilize crude glycerol and pure glycerol to produce malic acid was studied. The yield of malic acid was increased with 4.45 folds compared with that of parent strain from crude glycerol. The highest concentration of malic acid from crude glycerol by using beneficial mutant was found to be 77.38 ± 0.51 g/L after 192 h at 25 °C. This present study specified that crude glycerol by-product from biodiesel production could be used for producing high amount of malic acid without any pretreatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor.

PubMed

Sakaguchi, Kouta; Katoh, Toshihiko; Yamamoto, Kenji

2016-11-01

Glycan conversion of glycoprotein via the transglycosylation activity of endo-β-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

High Energy Explosive Yield Enhancer Using Microencapsulation.

DTIC Science & Technology

The invention consists of a class of high energy explosive yield enhancers created through the use of microencapsulation techniques. The... microcapsules consist of combinations of highly reactive oxidizers that are encapsulated in either passivated inorganic fuels or inert materials and inorganic...fuels. Depending on the application, the availability of the various oxidizers and fuels within the microcapsules can be customized to increase the

Co-culturing of Lactobacillus paracasei subsp. paracasei with a Lactobacillus delbrueckii subsp. delbrueckii mutant to make high cell density for increased lactate productivity from cassava bagasse hydrolysate.

PubMed

John, Rojan Pappy; Nampoothiri, K Madhavan

2011-03-01

To increase the productivity of lactic acid, a co-culture of lactobacilli was made by mixing 1:1 ratio of Lactobacillus paracasei subsp. paracasei and a fast growing L. delbrueckii subsp. delbrueckii mutant. The culture was embedded on to polyurethane foam (PUF) cubes as a biofilm and used for fermentation. In order to prevent the cell leakage, the PUF cubes were further entrapped in calcium cross-linked alginate. The maximum lactic acid production using a high cell density free culture was >38 g l(-1) from ~40 g l(-1) of reducing sugar within 12 h of fermentation. Using PUF biofilms, the same yield of lactic acid attained after 24 h. When the cubes were further coated with alginate it took 36 h for the maximum yield. Even though, the productivity is slightly lesser with the alginate coating, cell leakage was decreased and cubes were reused without much decrease in production in repeated batches. Using a conventional control inoculum (3%, w/v), it took 120 h to yield same amount of lactic acid.

Characterization of sarcoplasmic reticulum Ca{sup 2+} ATPase nucleotide binding domain mutants using NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myint, Wazo; Gong, Qingguo; Ahn, Jinwoo

2011-02-04

Research highlights: {yields} Structural consequence by substitution mutations on the isolated SERCA-nucleotide binding (SERCA-N) domain was studied. {yields} The study fills a gap between the previous clinical, physiological, and biochemical data and the molecular basis of SERCA-N. {yields} The E412G mutation, known to be seen in patients with Darier's disease, was found to maintain the active conformation but exhibit reduced protein stability. -- Abstract: Sarcoplasmic reticulum Ca{sup 2+} ATPase (SERCA) is essential for muscle function by transporting Ca{sup 2+} from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolatedmore » SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. {sup 15}N-{sup 1}H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant ({approx}2.5 mM) was similar to that of WT ({approx}3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.« less

High-resolution analysis of locomotor activity rhythms in disconnected, a visual-system mutant of Drosophila melanogaster.

PubMed

Dowse, H B; Dushay, M S; Hall, J C; Ringo, J M

1989-07-01

Free-running locomotor activity and eclosion rhythms of Drosophila melanogaster, mutant at the disconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushay et al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard the disco mutants are much like flies expressing mutant alleles of the period gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted in disco flies as a result of the neural lesion.

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes

PubMed Central

Mok, Calvin A.; Au, Vinci; Thompson, Owen A.; Edgley, Mark L.; Gevirtzman, Louis; Yochem, John; Lowry, Joshua; Memar, Nadin; Wallenfang, Matthew R.; Rasoloson, Dominique; Bowerman, Bruce; Schnabel, Ralf; Seydoux, Geraldine; Moerman, Donald G.; Waterston, Robert H.

2017-01-01

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans. Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2. We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures. PMID:28827289

Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress.

PubMed

Sánchez-Corrionero, Álvaro; Sánchez-Vicente, Inmaculada; González-Pérez, Sergio; Corrales, Ascensión; Krieger-Liszkay, Anja; Lorenzo, Óscar; Arellano, Juan B

2017-09-01

The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen ( 1 O 2 ) and activating 1 O 2 -mediated cell death. Thylakoids of aba1 produced twice as much 1 O 2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1 O 2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1 O 2 generation in aba1. Up-regulation of the 1 O 2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1 O 2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1 O 2 -mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1 O 2 -overproducing mutants of Arabidopsis for investigating 1 O 2 -mediated cell death. Copyright © 2017 Elsevier GmbH. All rights reserved.

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †

PubMed Central

Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J.; Shearman, Claire A.; Santos, Helena

2011-01-01

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds. PMID:21841021

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

PubMed

Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

2015-02-01

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Durand, Fabien; Stines-Chaumeil, Claire; Flexer, Victoria

2010-11-26

Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to amore » better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.« less

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants

NASA Technical Reports Server (NTRS)

Davenport, K. D.; Williams, K. E.; Ullmann, B. D.; Gustin, M. C.; McIntire, L. V. (Principal Investigator)

1999-01-01

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants.

PubMed Central

Davenport, K D; Williams, K E; Ullmann, B D; Gustin, M C

1999-01-01

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype. PMID:10545444

Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

NASA Astrophysics Data System (ADS)

Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

2011-01-01

The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

[Construction and stress tolerance of trehalase mutant in Saccharomyces cerevisiae].

PubMed

Lv, Ye; Xiao, Dongguang; He, Dongqin; Guo, Xuewu

2008-10-01

Accumulation of trehalose is critical in improving the stress tolerance of Saccharomyces cerevisiae. Two enzymes are capable of hydrolyzing trehalose: a neutral trehalase (NTH1) and an acidic trehalase (ATH1). We constructed trehalase disruption mutants to provide a basis for future commercial application. To retain the accumulation of trehalose in yeast cell, we constructed diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1) and double mutants (Deltaath1Deltanth1) by using gene disruption. We tested mutants'trehalose content and their tolerance to freezing, heat, high-sugar and ethanol concentrations. These trehalase disruption mutants were further confirmed by PCR amplification and southern blot. All mutant strains accumulated higher levels of cellular trehalose and grew to a higher cell density than the isogenic parent strain. In addition, the levels of trehalose in these mutants correlated with increased tolerance to freezing, heat, high-sugar and ethanol concentration. The improved tolerance of trehalase mutants may make them useful in commercial applications, including baking and brewing protein.

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

NASA Astrophysics Data System (ADS)

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

2003-10-01

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

High-resolution mapping of yield curve shape and evolution for high porosity sandstones

NASA Astrophysics Data System (ADS)

Bedford, J. D.; Faulkner, D.; Wheeler, J.; Leclere, H.

2017-12-01

The onset of permanent inelastic deformation for porous rock is typically defined by a yield curve plotted in P-Q space, where P is the effective mean stress and Q is the differential stress. Sandstones usually have broadly elliptical shaped yield curves, with the low pressure side of the ellipse associated with localized brittle faulting (dilation) and the high pressure side with distributed ductile deformation (compaction). However recent works have shown that these curves might not be perfectly elliptical and that significant evolution in shape occurs with continued deformation. We therefore use a novel stress-probing methodology to map in high-resolution the yield curve shape for Boise and Idaho Gray sandstones (36-38% porosity) and also investigate curve evolution with increasing deformation. The data reveal yield curves with a much flatter geometry than previously recorded for porous sandstone and that the compactive side of the curve is partly comprised of a near vertical limb. The yield curve evolution is found to be strongly dependent on the nature of inelastic strain. Samples that were compacted under a deviatoric load, with a component of inelastic shear strain, were found to have yield curves with peaks that are approximately 50% higher than similar porosity samples that were hydrostatically compacted (i.e. purely volumetric strain). The difference in yield curve evolution along the different loading paths is attributed to mechanical anisotropy that develops during deviatoric loading by the closure of preferentially orientated fractures. Increased shear strain also leads to the formation of a plateau at the peak of the yield curve as samples deform along the deviatoric loading path. These results have important implications for understanding how the strength of porous rock evolves along different stress paths, including during fluid extraction from hydrocarbon reservoirs where the stress state is rarely isotropic.

Mutant Transcriptome Sequencing Provides Insights into Pod Development in Peanut (Arachis hypogaea L.)

PubMed Central

Wan, Liyun; Li, Bei; Lei, Yong; Yan, Liying; Ren, Xiaoping; Chen, Yuning; Dai, Xiaofeng; Jiang, Huifang; Zhang, Juncheng; Guo, Wei; Chen, Ao; Liao, Boshou

2017-01-01

Pod size is the major yield component and a key target trait that is selected for in peanut breeding. However, although numerous quantitative trait loci (QTLs) for peanut pod size have been described, the molecular mechanisms underlying the development of this characteristic remain elusive. A peanut mutant with a narrower pod was developed in this study using ethyl methanesulfonate (EMS) mutagenesis and designated as the “pod width” mutant line (pw). The fresh pod weight of pw was only about 40% of that seen in the wild-type (WT) Zhonghua16, while the hull and seed filling of the mutant both also developed at earlier stages. Pods from both pw and WT lines were sampled 20, 40, and 60 days after flowering (DAF) and used for RNA-Seq analysis; the results revealed highly differentially expressed lignin metabolic pathway genes at all three stages, but especially at DAF 20 and DAF 40. At the same time, expression of genes related to auxin signal transduction was found to be significantly repressed during the pw early pod developmental stage. A genome-wide comparative analysis of expression profiles revealed 260 differentially expressed genes (DEGs) across all three stages, and two candidate genes, c26901_g1 (CAD) and c37339_g1 (ACS), responsible for pod width were identified by integrating expression patterns and function annotation of the common DEGs within the three stages. Taken together, the information provided in this study illuminates the processes underlying peanut pod development, and will facilitate further identification of causal genes and the development of improved peanut varieties with higher yields. PMID:29170673

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes.

PubMed

Mok, Calvin A; Au, Vinci; Thompson, Owen A; Edgley, Mark L; Gevirtzman, Louis; Yochem, John; Lowry, Joshua; Memar, Nadin; Wallenfang, Matthew R; Rasoloson, Dominique; Bowerman, Bruce; Schnabel, Ralf; Seydoux, Geraldine; Moerman, Donald G; Waterston, Robert H

2017-10-01

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C . elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2 We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures. Copyright © 2017 by the Genetics Society of America.

Genetic variations in ARE1 mediate grain yield by modulating nitrogen utilization in rice.

PubMed

Wang, Qing; Nian, Jinqiang; Xie, Xianzhi; Yu, Hong; Zhang, Jian; Bai, Jiaoteng; Dong, Guojun; Hu, Jiang; Bai, Bo; Chen, Lichao; Xie, Qingjun; Feng, Jian; Yang, Xiaolu; Peng, Juli; Chen, Fan; Qian, Qian; Li, Jiayang; Zuo, Jianru

2018-02-21

In crops, nitrogen directly determines productivity and biomass. However, the improvement of nitrogen utilization efficiency (NUE) is still a major challenge in modern agriculture. Here, we report the characterization of are1, a genetic suppressor of a rice fd-gogat mutant defective in nitrogen assimilation. ARE1 is a highly conserved gene, encoding a chloroplast-localized protein. Loss-of-function mutations in ARE1 cause delayed senescence and result in 10-20% grain yield increases, hence enhance NUE under nitrogen-limiting conditions. Analysis of a panel of 2155 rice varieties reveals that 18% indica and 48% aus accessions carry small insertions in the ARE1 promoter, which result in a reduction in ARE1 expression and an increase in grain yield under nitrogen-limiting conditions. We propose that ARE1 is a key mediator of NUE and represents a promising target for breeding high-yield cultivars under nitrogen-limiting condition.

A quantitative method for defining high-arched palate using the Tcof1(+/-) mutant mouse as a model.

PubMed

Conley, Zachary R; Hague, Molly; Kurosaka, Hiroshi; Dixon, Jill; Dixon, Michael J; Trainor, Paul A

2016-07-15

The palate functions as the roof of the mouth in mammals, separating the oral and nasal cavities. Its complex embryonic development and assembly poses unique susceptibilities to intrinsic and extrinsic disruptions. Such disruptions may cause failure of the developing palatal shelves to fuse along the midline resulting in a cleft. In other cases the palate may fuse at an arch, resulting in a vaulted oral cavity, termed high-arched palate. There are many models available for studying the pathogenesis of cleft palate but a relative paucity for high-arched palate. One condition exhibiting either cleft palate or high-arched palate is Treacher Collins syndrome, a congenital disorder characterized by numerous craniofacial anomalies. We quantitatively analyzed palatal perturbations in the Tcof1(+/-) mouse model of Treacher Collins syndrome, which phenocopies the condition in humans. We discovered that 46% of Tcof1(+/-) mutant embryos and new born pups exhibit either soft clefts or full clefts. In addition, 17% of Tcof1(+/-) mutants were found to exhibit high-arched palate, defined as two sigma above the corresponding wild-type population mean for height and angular based arch measurements. Furthermore, palatal shelf length and shelf width were decreased in all Tcof1(+/-) mutant embryos and pups compared to controls. Interestingly, these phenotypes were subsequently ameliorated through genetic inhibition of p53. The results of our study therefore provide a simple, reproducible and quantitative method for investigating models of high-arched palate. Copyright © 2015 Elsevier Inc. All rights reserved.

[Characteristics of phosphorus uptake and use efficiency of rice with high yield and high phosphorus use efficiency].

PubMed

Li, Li; Zhang, Xi-Zhou; Li, Tinx-Xuan; Yu, Hai-Ying; Ji, Lin; Chen, Guang-Deng

2014-07-01

A total of twenty seven middle maturing rice varieties as parent materials were divided into four types based on P use efficiency for grain yield in 2011 by field experiment with normal phosphorus (P) application. The rice variety with high yield and high P efficiency was identified by pot experiment with normal and low P applications, and the contribution rates of various P efficiencies to yield were investigated in 2012. There were significant genotype differences in yield and P efficiency of the test materials. GRLu17/AiTTP//Lu17_2 (QR20) was identified as a variety with high yield and high P efficiency, and its yields at the low and normal rates of P application were 1.96 and 1.92 times of that of Yuxiang B, respectively. The contribution rate of P accumulation to yield was greater than that of P grain production efficiency and P harvest index across field and pot experiments. The contribution rates of P accumulation and P grain production efficiency to yield were not significantly different under the normal P condition, whereas obvious differences were observed under the low P condition (66.5% and 26.6%). The minimal contribution to yield was P harvest index (11.8%). Under the normal P condition, the contribution rates of P accumulation to yield and P harvest index were the highest at the jointing-heading stage, which were 93.4% and 85.7%, respectively. In addition, the contribution rate of P accumulation to grain production efficiency was 41.8%. Under the low P condition, the maximal contribution rates of P accumulation to yield and grain production efficiency were observed at the tillering-jointing stage, which were 56.9% and 20.1% respectively. Furthermore, the contribution rate of P accumulation to P harvest index was 16.0%. The yield, P accumulation, and P harvest index of QR20 significantly increased under the normal P condition by 20.6%, 18.1% and 18.2% respectively compared with that in the low P condition. The rank of the contribution rates of P

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

PubMed

Deng, Lili; Qi, Zongtai; Zou, Binjie; Wu, Haiping; Huang, Huan; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

2012-07-03

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

PubMed

Kim, Yonghwan; Koo, Bong-Seong; Lee, Hyeon-Cheol; Yoon, Youngdae

2015-03-01

Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

Pyrimidine sulfonylacetanilides with improved potency against key mutant viruses of HIV-1 by specific targeting of a highly conserved residue.

PubMed

Wan, Zheng-Yong; Yao, Jin; Mao, Tian-Qi; Wang, Xin-Long; Wang, Hai-Feng; Chen, Wen-Xue; Yin, Hong; Chen, Fen-Er; De Clercq, Erik; Daelemans, Dirk; Pannecouque, Christophe

2015-09-18

Based on molecular simulation, the etravirine-VRX-480773 hybrids previously disclosed by our group were optimized to yield novel pyrimidine sulfonylacetanilides 8 with improved activity against a panel of seven clinically relevant single and double mutant strains of HIV-1. The improvement in potency in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits the reverse transcriptase (RT), and represents a remarkable step forward in the development of anti-HIV drugs. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

High pressure intensification of cassava resistant starch (RS3) yields.

PubMed

Lertwanawatana, Proyphon; Frazier, Richard A; Niranjan, Keshavan

2015-08-15

Cassava starch, typically, has resistant starch type 3 (RS3) content of 2.4%. This paper shows that the RS3 yields can be substantially enhanced by debranching cassava starch using pullulanase followed by high pressure or cyclic high-pressure annealing. RS3 yield of 41.3% was obtained when annealing was carried out at 400MPa/60°C for 15 min, whereas it took nearly 8h to obtain the same yield under conventional atmospheric annealing at 60°C. The yield of RS3 could be further significantly increased by annealing under 400 MPa/60°C pressure for 15 min followed by resting at atmospheric pressure for 3h 45 min, and repeating this cycle for up to six times. Microstructural surface analysis of the product under a scanning electron microscope showed an increasingly rigid density of the crystalline structure formed, confirming higher RS3 content. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhanced transpiration rate in the high pigment 1 tomato mutant and its physiological significance.

PubMed

Carvalho, R F; Aidar, S T; Azevedo, R A; Dodd, I C; Peres, L E P

2011-05-01

Tomato high pigment (hp) mutants represent an interesting horticultural resource due to their enhanced accumulation of carotenoids, flavonoids and vitamin C. Since hp mutants are known for their exaggerated light responses, the molecules accumulated are likely to be antioxidants, recruited to deal with light and others stresses. Further phenotypes displayed by hp mutations are reduced growth and an apparent disturbance in water loss. Here, we examined the impact of the hp1 mutation and its near isogenic line cv Micro-Tom (MT) on stomatal conductance (gs), transpiration (E), CO(2) assimilation (A) and water use efficiency (WUE). Detached hp1 leaves lost water more rapidly than control leaves, but this behaviour was reversed by exogenous abscisic acid (ABA), indicating the ability of hp1 to respond to this hormone. Although attached hp1 leaves had enhanced gs, E and A compared to control leaves, genotypic differences were lost when water was withheld. Both instantaneous leaf-level WUE and long-term whole plant WUE did not differ between hp1 and MT. Our results indicate a link between exaggerated light response and water loss in hp1, which has important implications for the use of this mutant in both basic and horticultural research. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Added-values of high spatiotemporal remote sensing data in crop yield estimation

NASA Astrophysics Data System (ADS)

Gao, F.; Anderson, M. C.

2017-12-01

Timely and accurate estimation of crop yield before harvest is critical for food market and administrative planning. Remote sensing derived parameters have been used for estimating crop yield by using either empirical or crop growth models. The uses of remote sensing vegetation index (VI) in crop yield modeling have been typically evaluated at regional and country scales using coarse spatial resolution (a few hundred to kilo-meters) data or assessed over a small region at field level using moderate resolution spatial resolution data (10-100m). Both data sources have shown great potential in capturing spatial and temporal variability in crop yield. However, the added value of data with both high spatial and temporal resolution data has not been evaluated due to the lack of such data source with routine, global coverage. In recent years, more moderate resolution data have become freely available and data fusion approaches that combine data acquired from different spatial and temporal resolutions have been developed. These make the monitoring crop condition and estimating crop yield at field scale become possible. Here we investigate the added value of the high spatial and temporal VI for describing variability of crop yield. The explanatory ability of crop yield based on high spatial and temporal resolution remote sensing data was evaluated in a rain-fed agricultural area in the U.S. Corn Belt. Results show that the fused Landsat-MODIS (high spatial and temporal) VI explains yield variability better than single data source (Landsat or MODIS alone), with EVI2 performing slightly better than NDVI. The maximum VI describes yield variability better than cumulative VI. Even though VI is effective in explaining yield variability within season, the inter-annual variability is more complex and need additional information (e.g. weather, water use and management). Our findings augment the importance of high spatiotemporal remote sensing data and supports new moderate

Spatial constraints govern competition of mutant clones in human epidermis.

PubMed

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

2017-10-24

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

New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates

NASA Astrophysics Data System (ADS)

Li, Guangyue; Yao, Peiyuan; Cong, Peiqian; Ren, Jie; Wang, Lei; Feng, Jinhui; Lau, Peter C. K.; Wu, Qiaqing; Zhu, Dunming

2016-05-01

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.

New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates

PubMed Central

Li, Guangyue; Yao, Peiyuan; Cong, Peiqian; Ren, Jie; Wang, Lei; Feng, Jinhui; Lau, Peter C.K.; Wu, Qiaqing; Zhu, Dunming

2016-01-01

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines. PMID:27138090

Xylitol production by a Pichia stipitis D-xylulokinase mutant

Treesearch

Yong-Su Jin; Jose Cruz; Thomas W. Jeffries

2005-01-01

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-Ä1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-Ä1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g...

Sharing mutants and experimental information prepublication using FgMutantDB

USDA-ARS?s Scientific Manuscript database

There has been no central location for storing generated mutants of Fusarium graminearum or for data associated with these mutants. Instead researchers relied on several independent, non-integrated databases. FgMutantDB was designed as a simple spreadsheet that is accessible globally on the web th...

Optimization of L-lactic Acid Production of Rhizopus Oryzae Mutant RLC41-6 by Ion Beam Implantation at Low-Energy

NASA Astrophysics Data System (ADS)

Zhou, Xiuhong; Ge, Chunmei; Yao, Jianming; Pan, Renrui; Yu, Zengliang

2005-10-01

In order to obtain an industrial strain with a higher L(+)-lactic acid yield, the strain Rhizopus oryzae RF3608 was mutated by means of nitrogen ion beam implantation and the mutant strain RLC41-6 was isolated. Under optimal conditions the yield of L(+)-lactic acid produced in a shake-flask reached 133 g/L-137 g/L after 36 h cultivation, indicating that the conversion rate based on glucose was as high as 88%-91% and the productivity was 3.75 g/L.h. It was almost a 115% increase in lactic acid production compared with the original strain RF3608.

Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene.

PubMed

Pourzardosht, Navid; Rasaee, Mohammad Javad

2017-06-01

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana r -R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA.

High-yielding continuous-flow synthesis of antimalarial drug hydroxychloroquine

PubMed Central

Telang, Nakul S; Kong, Caleb J; Verghese, Jenson; Gilliland III, Stanley E; Ahmad, Saeed; Dominey, Raymond N

2018-01-01

Numerous synthetic methods for the continuous preparation of fine chemicals and active pharmaceutical ingredients (API’s) have been reported in recent years resulting in a dramatic improvement in process efficiencies. Herein we report a highly efficient continuous synthesis of the antimalarial drug hydroxychloroquine (HCQ). Key improvements in the new process include the elimination of protecting groups with an overall yield improvement of 52% over the current commercial process. The continuous process employs a combination of packed bed reactors with continuous stirred tank reactors for the direct conversion of the starting materials to the product. This high-yielding, multigram-scale continuous synthesis provides an opportunity to achieve increase global access to hydroxychloroquine for treatment of malaria. PMID:29623120

A quantitative method for defining high-arched palate using the Tcof1+/− mutant mouse as a model

PubMed Central

Conley, Zachary R.; Hague, Molly; Kurosaka, Hiroshi; Dixon, Jill; Dixon, Michael J.; Trainor, Paul A.

2016-01-01

The palate functions as the roof of the mouth in mammals, separating the oral and nasal cavities. Its complex embryonic development and assembly poses unique susceptibilities to intrinsic and extrinsic disruptions. Such disruptions may cause failure of the developing palatal shelves to fuse along the midline resulting in a cleft. In other cases the palate may fuse at an arch, resulting in a vaulted oral cavity, termed high-arched palate. There are many models available for studying the pathogenesis of cleft palate but a relative paucity for high-arched palate. One condition exhibiting either cleft palate or high-arched palate is Treacher Collins syndrome, a congenital disorder characterized by numerous craniofacial anomalies. We quantitatively analyzed palatal perturbations in the Tcof1+/− mouse model of Treacher Collins syndrome, which phenocopies the condition in humans. We discovered that 46% of Tcof1+/− mutant embryos and new born pups exhibit either soft clefts or full clefts. In addition, 17% of Tcof1+/− mutants were found to exhibit high-arched palate, defined as two sigma above the corresponding wild-type population mean for height and angular based arch measurements. Furthermore, palatal shelf length and shelf width were decreased in all Tcof1+/− mutant embryos and pups compared to controls. Interestingly, these phenotypes were subsequently ameliorated through genetic inhibition of p53. The results of our study therefore provide a simple, reproducible and quantitative method for investigating models of high-arched palate. PMID:26772999

Unexpectedly low nitrogen acquisition and absence of root architecture adaptation to nitrate supply in a Medicago truncatula highly branched root mutant

PubMed Central

Bourion, Virginie

2014-01-01

To complement N2 fixation through symbiosis, legumes can efficiently acquire soil mineral N through adapted root architecture. However, root architecture adaptation to mineral N availability has been little studied in legumes. Therefore, this study investigated the effect of nitrate availability on root architecture in Medicago truncatula and assessed the N-uptake potential of a new highly branched root mutant, TR185. The effects of varying nitrate supply on both root architecture and N uptake were characterized in the mutant and in the wild type. Surprisingly, the root architecture of the mutant was not modified by variation in nitrate supply. Moreover, despite its highly branched root architecture, TR185 had a permanently N-starved phenotype. A transcriptome analysis was performed to identify genes differentially expressed between the two genotypes. This analysis revealed differential responses related to the nitrate acquisition pathway and confirmed that N starvation occurred in TR185. Changes in amino acid content and expression of genes involved in the phenylpropanoid pathway were associated with differences in root architecture between the mutant and the wild type. PMID:24706718

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

PubMed Central

Gubin, Matthew M.; Zhang, Xiuli; Schuster, Heiko; Caron, Etienne; Ward, Jeffrey P.; Noguchi, Takuro; Ivanova, Yulia; Hundal, Jasreet; Arthur, Cora D.; Krebber, Willem-Jan; Mulder, Gwenn E.; Toebes, Mireille; Vesely, Matthew D.; Lam, Samuel S.K.; Korman, Alan J.; Allison, James P.; Freeman, Gordon J.; Sharpe, Arlene H.; Pearce, Erika L.; Schumacher, Ton N.; Aebersold, Ruedi; Rammensee, Hans-Georg; Melief, Cornelis J. M.; Mardis, Elaine R.; Gillanders, William E.; Artyomov, Maxim N.; Schreiber, Robert D.

2014-01-01

The immune system plays key roles in determining the fate of developing cancers by not only functioning as a tumour promoter facilitating cellular transformation, promoting tumour growth and sculpting tumour cell immunogenicity1–6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer induced immunosuppression. In many individuals, immunosuppression is mediated by Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) and Programmed Death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal antibody (mAb) based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10–13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following αPD-1 and/or αCTLA-4 therapy of mice bearing progressively growing sarcomas and show that therapeutic synthetic long peptide (SLP) vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, mutant tumour antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with αPD-1- and/or αCTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens (TSMA) are not only important targets of checkpoint blockade therapy but also can be

Breeding of a sake yeast mutant with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio.

PubMed

Takahashi, Toshinari; Ohara, Yusuke; Sueno, Kazuo

2017-06-01

Sake yeast produces a fruity flavor known as ginjo-ko-which is mainly attributable to ethyl caproate and isoamyl acetate-during fermentation in sake brewing. The production of these flavor components is inhibited by unsaturated fatty acids derived from the outer layer of rice as raw material. We isolated three mutants (hec2, hec3, and hec6) with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio from a cerulenin-resistant mutant derived from the hia1 strain, which shows enhanced isoamyl acetate productivity. The hec2 mutant had the homozygous FAS2 mutation Gly1250Ser, which is known to confer high ethyl caproate productivity. When the homozygous FAS2 mutation Gly1250Ser was introduced into strain hia1, ethyl caproate productivity was increased but neither this nor intracellular caproic acid content approached the levels observed in the hec2 mutant, indicating that a novel mutation was responsible for the high ethyl caproate productivity. We also found that the expression of EEB1 encoding acyl-coenzyme A:ethanol O-acyltransferase (AEATase) and enzymatic activity were increased in the hec2 mutant. These results suggest that the upregulation of EEB1 expression and AEATase activity may also have contributed to the enhancement of ethyl caproate synthesis from ethanol and caproyl-CoA. Our findings are useful for the brewing of sake with improved flavor due to high levels of isoamyl acetate and ethyl caproate. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Separating heat stress from moisture stress: analyzing yield response to high temperature in irrigated maize

NASA Astrophysics Data System (ADS)

Carter, Elizabeth K.; Melkonian, Jeff; Riha, Susan J.; Shaw, Stephen B.

2016-09-01

Several recent studies have indicated that high air temperatures are limiting maize (Zea mays L.) yields in the US Corn Belt and project significant yield losses with expected increases in growing season temperatures. Further work has suggested that high air temperatures are indicative of high evaporative demand, and that decreases in maize yields which correlate to high temperatures and vapor pressure deficits (VPD) likely reflect underlying soil moisture limitations. It remains unclear whether direct high temperature impacts on yields, independent of moisture stress, can be observed under current temperature regimes. Given that projected high temperature and moisture may not co-vary the same way as they have historically, quantitative analyzes of direct temperature impacts are critical for accurate yield projections and targeted mitigation strategies under shifting temperature regimes. To evaluate yield response to above optimum temperatures independent of soil moisture stress, we analyzed climate impacts on irrigated maize yields obtained from the National Corn Growers Association (NCGA) corn yield contests for Nebraska, Kansas and Missouri. In irrigated maize, we found no evidence of a direct negative impact on yield by daytime air temperature, calculated canopy temperature, or VPD when analyzed seasonally. Solar radiation was the primary yield-limiting climate variable. Our analyses suggested that elevated night temperature impacted yield by increasing rates of phenological development. High temperatures during grain-fill significantly interacted with yields, but this effect was often beneficial and included evidence of acquired thermo-tolerance. Furthermore, genetics and management—information uniquely available in the NCGA contest data—explained more yield variability than climate, and significantly modified crop response to climate. Thermo-acclimation, improved genetics and changes to management practices have the potential to partially or completely

Breeding high-yielding drought-tolerant rice: genetic variations and conventional and molecular approaches

PubMed Central

Kumar, Arvind; Dixit, Shalabh; Ram, T.; Yadaw, R. B.; Mishra, K. K.; Mandal, N. P.

2014-01-01

The increased occurrence and severity of drought stress have led to a high yield decline in rice in recent years in drought-affected areas. Drought research at the International Rice Research Institute (IRRI) over the past decade has concentrated on direct selection for grain yield under drought. This approach has led to the successful development and release of 17 high-yielding drought-tolerant rice varieties in South Asia, Southeast Asia, and Africa. In addition to this, 14 quantitative trait loci (QTLs) showing a large effect against high-yielding drought-susceptible popular varieties were identified using grain yield as a selection criterion. Six of these (qDTY 1.1, qDTY 2.2, qDTY 3.1, qDTY 3.2, qDTY 6.1, and qDTY 12.1) showed an effect against two or more high-yielding genetic backgrounds in both the lowland and upland ecosystem, indicating their usefulness in increasing the grain yield of rice under drought. The yield of popular rice varieties IR64 and Vandana has been successfully improved through a well-planned marker-assisted backcross breeding approach, and QTL introgression in several other popular varieties is in progress. The identification of large-effect QTLs for grain yield under drought and the higher yield increase under drought obtained through the use of these QTLs (which has not been reported in other cereals) indicate that rice, because of its continuous cultivation in two diverse ecosystems (upland, drought tolerant, and lowland, drought susceptible), has benefited from the existence of larger genetic variability than in other cereals. This can be successfully exploited using marker-assisted breeding. PMID:25205576

Mapping Crop Yield and Sow Date Using High Resolution Imagery

NASA Astrophysics Data System (ADS)

Royal, K.

2015-12-01

Keitasha Royal, Meha Jain, Ph.D., David Lobell, Ph.D Mapping Crop Yield and Sow Date Using High Resolution ImageryThe use of satellite imagery in agriculture is becoming increasingly more significant and valuable. Due to the emergence of new satellites, such as Skybox, these satellites provide higher resolution imagery (e.g 1m) therefore improving the ability to map smallholder agriculture. For the smallholder farm dominated area of northern India, Skybox high-resolution satellite imagery can aid in understanding how to improve farm yields. In particular, we are interested in mapping winter wheat in India, as this region produces approximately 80% of the country's wheat crop, which is important given that wheat is a staple crop that provides approximately 20% of household calories. In northeast India, the combination of increased heat stress, limited irrigation access, and the difficulty for farmers to access advanced farming technologies results in farmers only producing about 50% of their potential crop yield. The use of satellite imagery can aid in understanding wheat yields through time and help identify ways to increase crop yields in the wheat belt of India. To translate Skybox satellite data into meaningful information about wheat fields, we examine vegetation indices, such as the normalized difference vegetation index (NDVI), to measure the "greenness" of plants to help determine the health of the crops. We test our ability to predict crop characteristics, like sow date and yield, using vegetation indices of 59 fields for which we have field data in Bihar, India.

Indolyl aryl sulfones (IASs): development of highly potent NNRTIs active against wt-HIV-1 and clinically relevant drug resistant mutants.

PubMed

Silvestri, Romano; Artico, Marino

2005-01-01

Indolyl aryl sulfones (IASs) are a potent class of NNRTIs developed from L-737,126, a lead agent discovered by Merck AG. IAS derivatives are endowed with inhibitory activities against wt HIV-1 in the low nanomolar concentration range. Introduction of two methyl groups at positions 3 and 5 of the phenyl ring of the aryl sulfonyl moiety furnished IAS derivatives such as 5-chloro- or 5-bromo-3-[(3,5-dimethylphenyl)sulfonyl]indole-2-carboxyamide, which showed very potent and selective anti-HIV-1 activity against some mutants carrying NNRTI resistant mutations at positions 103 and 181 of the reverse transcriptase. IAS derivatives bearing 2-hydroxyethylcarboxyamide or 2-hydroxyethylcarboxyhydrazide groups at position 2 of the indole nucleus were more active than L-737,126 against the K103N-Y181C double mutant. A great improvement of antiviral activity against wt HIV-1 and resistant mutants was obtained by coupling 1-3 simple amino acids, such as glycine and alanine, in sequence, with the 3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carbonyl moiety. The transformation of the chain terminus into amide or hydrazide, produced short peptides with high selectivity and potent activity against wt HIV-1, and the viral mutants Y181C, K103N-Y181C and EFV(R). IAS having two halogen atoms at the indole showed potent inhibitory activity against the Y181C and the EFV(R) resistant mutant strains. In particular, the introduction of a fluorine atom at position 4 of the indole ring notably contributed to improve the antiviral activities against both wt and the related resistant mutants. 5-Nitro-IASs were highly active against wt HIV-1 and exhibited low cytotoxicity. Experimental data highlighted the class IAS derivatives as promising candidates for clinical trials.

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei

PubMed Central

Chen, Ji-Hong; Li, Wen-Jian; Liu, Jing; Hu, Wei; Xiao, Guo-Qing; Dong, Miao-Yin; Wang, Yu-Chen

2015-01-01

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme. PMID:26656155

Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis.

PubMed

Ma, Kedong; Ruan, Zhiyong; Shui, Zongxia; Wang, Yanwei; Hu, Guoquan; He, Mingxiong

2016-03-01

The aim of present study was to develop a process for open ethanol fermentation from food waste using an acid-tolerant mutant of Zymomonas mobilis (ZMA7-2). The mutant showed strong tolerance to acid condition of food waste hydrolysate and high ethanol production performance. By optimizing fermentation parameters, ethanol fermentation with initial glucose concentration of 200 g/L, pH value around 4.0, inoculum size of 10% and without nutrient addition was considered as best conditions. Moreover, the potential of bench scales fermentation and cell reusability was also examined. The fermentation in bench scales (44 h) was faster than flask scale (48 h), and the maximum ethanol concentration and ethanol yield (99.78 g/L, 0.50 g/g) higher than that of flask scale (98.31 g/L, 0.49 g/g). In addition, the stable cell growth and ethanol production profile in five cycles successive fermentation was observed, indicating the mutant was suitable for industrial ethanol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Methods for high yield production of terpenes

DOEpatents

Kutchan, Toni; Higashi, Yasuhiro; Feng, Xiaohong

2017-01-03

Provided are enhanced high yield production systems for producing terpenes in plants via the expression of fusion proteins comprising various combinations of geranyl diphosphate synthase large and small subunits and limonene synthases. Also provided are engineered oilseed plants that accumulate monoterpene and sesquiterpene hydrocarbons in their seeds, as well as methods for producing such plants, providing a system for rapidly engineering oilseed crop production platforms for terpene-based biofuels.

High yield neutron generator based on a high-current gasdynamic electron cyclotron resonance ion source

DOE Office of Scientific and Technical Information (OSTI.GOV)

Skalyga, V.; Sidorov, A.; Lobachevsky State University of Nizhny Novgorod

2015-09-07

In present paper, an approach for high yield compact D-D neutron generator based on a high current gasdynamic electron cyclotron resonance ion source is suggested. Results on dense pulsed deuteron beam production with current up to 500 mA and current density up to 750 mA/cm{sup 2} are demonstrated. Neutron yield from D{sub 2}O and TiD{sub 2} targets was measured in case of its bombardment by pulsed 300 mA D{sup +} beam with 45 keV energy. Neutron yield density at target surface of 10{sup 9} s{sup −1} cm{sup −2} was detected with a system of two {sup 3}He proportional counters. Estimations based on obtained experimental resultsmore » show that neutron yield from a high quality TiD{sub 2} target bombarded by D{sup +} beam demonstrated in present work accelerated to 100 keV could reach 6 × 10{sup 10} s{sup −1} cm{sup −2}. It is discussed that compact neutron generator with such characteristics could be perspective for a number of applications like boron neutron capture therapy, security systems based on neutron scanning, and neutronography.« less

Flying-patch patch-clamp study of G22E-MscL mutant under high hydrostatic pressure.

PubMed

Petrov, Evgeny; Rohde, Paul R; Martinac, Boris

2011-04-06

High hydrostatic pressure (HHP) present in natural environments impacts on cell membrane biophysical properties and protein quaternary structure. We have investigated the effect of high hydrostatic pressure on G22E-MscL, a spontaneously opening mutant of Escherichia coli MscL, the bacterial mechanosensitive channel of large conductance. Patch-clamp technique combined with a flying-patch device and hydraulic setup allowed the study of the effects of HHP up to 90 MPa (as near the bottom of the Marianas Trench) on the MscL mutant channel reconstituted into liposome membranes, in addition to recording in situ from the mutant channels expressed in E. coli giant spheroplasts. In general, against thermodynamic predictions, hydrostatic pressure in the range of 0.1-90 MPa increased channel open probability by favoring the open state of the channel. Furthermore, hydrostatic pressure affected the channel kinetics, as manifested by the propensity of the channel to gate at subconducting levels with an increase in pressure. We propose that the presence of water molecules around the hydrophobic gate of the G22E MscL channel induce hydration of the hydrophobic lock under HHP causing frequent channel openings and preventing the channel closure in the absence of membrane tension. Furthermore, our study indicates that HHP can be used as a valuable experimental approach toward better understanding of the gating mechanism in complex channels such as MscL. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

DOE PAGES

Sattler, Scott E.; Saballos, Ana; Xin, Zhanguo; ...

2014-09-02

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum ( Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib ( bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6,and bmr12, and, in addition, six bmr mutants were identified that were notmore » allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.« less

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

PubMed Central

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

2014-01-01

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

High-yield maize with large net energy yield and small global warming intensity

PubMed Central

Grassini, Patricio; Cassman, Kenneth G.

2012-01-01

Addressing concerns about future food supply and climate change requires management practices that maximize productivity per unit of arable land while reducing negative environmental impact. On-farm data were evaluated to assess energy balance and greenhouse gas (GHG) emissions of irrigated maize in Nebraska that received large nitrogen (N) fertilizer (183 kg of N⋅ha−1) and irrigation water inputs (272 mm or 2,720 m3 ha−1). Although energy inputs (30 GJ⋅ha−1) were larger than those reported for US maize systems in previous studies, irrigated maize in central Nebraska achieved higher grain and net energy yields (13.2 Mg⋅ha−1 and 159 GJ⋅ha−1, respectively) and lower GHG-emission intensity (231 kg of CO2e⋅Mg−1 of grain). Greater input-use efficiencies, especially for N fertilizer, were responsible for better performance of these irrigated systems, compared with much lower-yielding, mostly rainfed maize systems in previous studies. Large variation in energy inputs and GHG emissions across irrigated fields in the present study resulted from differences in applied irrigation water amount and imbalances between applied N inputs and crop N demand, indicating potential to further improve environmental performance through better management of these inputs. Observed variation in N-use efficiency, at any level of applied N inputs, suggests that an N-balance approach may be more appropriate for estimating soil N2O emissions than the Intergovernmental Panel on Climate Change approach based on a fixed proportion of applied N. Negative correlation between GHG-emission intensity and net energy yield supports the proposition that achieving high yields, large positive energy balance, and low GHG emissions in intensive cropping systems are not conflicting goals. PMID:22232684

Unexpectedly low nitrogen acquisition and absence of root architecture adaptation to nitrate supply in a Medicago truncatula highly branched root mutant.

PubMed

Bourion, Virginie; Martin, Chantal; de Larambergue, Henri; Jacquin, Françoise; Aubert, Grégoire; Martin-Magniette, Marie-Laure; Balzergue, Sandrine; Lescure, Geoffroy; Citerne, Sylvie; Lepetit, Marc; Munier-Jolain, Nathalie; Salon, Christophe; Duc, Gérard

2014-06-01

To complement N2 fixation through symbiosis, legumes can efficiently acquire soil mineral N through adapted root architecture. However, root architecture adaptation to mineral N availability has been little studied in legumes. Therefore, this study investigated the effect of nitrate availability on root architecture in Medicago truncatula and assessed the N-uptake potential of a new highly branched root mutant, TR185. The effects of varying nitrate supply on both root architecture and N uptake were characterized in the mutant and in the wild type. Surprisingly, the root architecture of the mutant was not modified by variation in nitrate supply. Moreover, despite its highly branched root architecture, TR185 had a permanently N-starved phenotype. A transcriptome analysis was performed to identify genes differentially expressed between the two genotypes. This analysis revealed differential responses related to the nitrate acquisition pathway and confirmed that N starvation occurred in TR185. Changes in amino acid content and expression of genes involved in the phenylpropanoid pathway were associated with differences in root architecture between the mutant and the wild type. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production.

PubMed

Zhang, Xiaojie; Lu, Chenyang; Bai, Linquan

2017-09-01

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization, fast growth, abundant precursors and energy supply, and a pronounced gene expression. Streptomyces albus BK3-25 is a high-yield industrial strain producing type-I polyketide salinomycin, with a unique ability of bean oil utilization. Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein. Firstly, introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and salinomycin, and subsequent deletion of the salinomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors, including malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA. Secondly, the energy and reducing force were measured, and the improved accumulation of ATP and NADPH was observed in the mutant. Furthermore, the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases, and a strong constitutive promoter was identified, providing a useful tool for further engineered gene expression. Finally, the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor, and the heterologous production of actinorhodin was obtained. This work clearly indicated the potential of the high-yield salinomycin producer as a surrogate host for heterologous production of polyketides, although more genetic manipulation should be conducted to streamline its performance.

High precision and high yield fabrication of dense nanoparticle arrays onto DNA origami at statistically independent binding sites

NASA Astrophysics Data System (ADS)

Takabayashi, Sadao; Klein, William P.; Onodera, Craig; Rapp, Blake; Flores-Estrada, Juan; Lindau, Elias; Snowball, Lejmarc; Sam, Joseph T.; Padilla, Jennifer E.; Lee, Jeunghoon; Knowlton, William B.; Graugnard, Elton; Yurke, Bernard; Kuang, Wan; Hughes, William L.

2014-10-01

High precision, high yield, and high density self-assembly of nanoparticles into arrays is essential for nanophotonics. Spatial deviations as small as a few nanometers can alter the properties of near-field coupled optical nanostructures. Several studies have reported assemblies of few nanoparticle structures with controlled spacing using DNA nanostructures with variable yield. Here, we report multi-tether design strategies and attachment yields for homo- and hetero-nanoparticle arrays templated by DNA origami nanotubes. Nanoparticle attachment yield via DNA hybridization is comparable with streptavidin-biotin binding. Independent of the number of binding sites, >97% site-occupation was achieved with four tethers and 99.2% site-occupation is theoretically possible with five tethers. The interparticle distance was within 2 nm of all design specifications and the nanoparticle spatial deviations decreased with interparticle spacing. Modified geometric, binomial, and trinomial distributions indicate that site-bridging, steric hindrance, and electrostatic repulsion were not dominant barriers to self-assembly and both tethers and binding sites were statistically independent at high particle densities.High precision, high yield, and high density self-assembly of nanoparticles into arrays is essential for nanophotonics. Spatial deviations as small as a few nanometers can alter the properties of near-field coupled optical nanostructures. Several studies have reported assemblies of few nanoparticle structures with controlled spacing using DNA nanostructures with variable yield. Here, we report multi-tether design strategies and attachment yields for homo- and hetero-nanoparticle arrays templated by DNA origami nanotubes. Nanoparticle attachment yield via DNA hybridization is comparable with streptavidin-biotin binding. Independent of the number of binding sites, >97% site-occupation was achieved with four tethers and 99.2% site-occupation is theoretically possible with five

High-Gum-Yielding Slash Pines Survive and Grow Well

Treesearch

S.V. Kossuth; W.J. Peters; C.R. Gansell

1982-01-01

Plantings in Georgia and Florida were established with slash pine seedlings of three genetic types: selections improved for high gum yield (IHGY), selections with improved growth and form (IGF), and commercial stock (CS). Under adverse environmental conditions in Florida, IHGY survived best at age 3, CS next best, and IGF poorest.Survival was more than twice as high in...

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana

PubMed Central

2013-01-01

Background Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance. Results Although lignin is a key polymer providing the strength necessary for the plant’s ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield. Conclusions Although the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

Difference in C3-C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1.

PubMed

Fu, Yanfen; Beck, David A C; Lidstrom, Mary E

2016-07-19

Two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. (13)C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. The major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, (13)C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.

Optimization of L(+)-Lactic Acid Production from Xylose with Rhizopus Oryzae Mutant RLC41-6 Breeding by Low-Energy Ion Implantation

NASA Astrophysics Data System (ADS)

Yang, Yingge; Fan, Yonghong; Li, Wen; Wang, Dongmei; Wu, Yuejin; Zheng, Zhiming; Yu, Zengliang

2007-10-01

In order to obtain an industrial strain with a higher L(+)-lactic acid yield, the strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion beam implantation and the mutant strain Rhizopus oryzae RLC41-6 was obtained. An experimental finding was made in surprise that Rhizopus oryzae mutant RLC41-6 is not only an L(+)-lactic acid producer from corn starch but also an efficient producer of L(+)-lactic acid from xylose. Under optimal conditions, the production of L(+)-lactic acid from 100 g/L xylose reached 77.39 g/L after 144 h fed-batch fermentation. A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment.

Physiological Basis and Transcriptional Profiling of Three Salt-Tolerant Mutant Lines of Rice

PubMed Central

Domingo, Concha; Lalanne, Eric; Catalá, María M.; Pla, Eva; Reig-Valiente, Juan L.; Talón, Manuel

2016-01-01

Salinity is a complex trait that affects growth and productivity in many crops, including rice. Mutation induction, a useful tool to generate salt tolerant plants, enables the analysis of plants with similar genetic background, facilitating the understanding of the salt tolerance mechanisms. In this work, we generated three salt tolerant mutant lines by irradiation of a salt-sensitive cultivar plants and screened M2 plants at seedling stage in the presence of high salinity. These three lines, SaT20, SaS62, and SaT58, showed different responses to salinity, but exhibited similar phenotype to wild type plants, except SaT20 that displayed shorter height when grown in the absence of salt. Under salt conditions, all three mutants and the parental line showed similar reduction in yield, although relevant differences in other physiological parameters, such as Na+ accumulation in healthy leaves of SaT20, were registered. Microarray analyses of gene expression profiles in roots revealed the occurrence of common and specific responses in the mutants. The three mutants showed up-regulation of responsive genes, the activation of oxido-reduction process and the inhibition of ion transport. The participation of jasmonate in the plant response to salt was evident by down-regulation of a gene coding for a jasmonate O-methyltransferase. Genes dealing with lipid transport and metabolism were, in general, up-regulated except in SaS62, that also exhibited down-regulation of genes involved in ion transport and Ca2+ signal transduction. The two most tolerant varieties, SaS62 and SaT20, displayed lower levels of transcripts involved in K+ uptake. The physiological study and the description of the expression analysis evidenced that the three lines showed different responses to salt: SaT20 showed a high Na+ content in leaves, SaS62 presented an inhibition of lipid metabolism and ion transport and SaT58 differs in both features in the response to salinity. The analysis of these salt

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

PubMed

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

2016-01-01

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

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

PubMed Central

Burge, Boyce W.; Pfefferkorn, E. R.

1967-01-01

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes. PMID:5630228

[Biosorption ability of mutants of Rhodotorula mucilaginosa UCM Y-1776].

PubMed

Mamieieva, O H; Kasatkina, T P; Lavrinchuk, V Ia

2007-01-01

Twenty stable mutants with various coloration intensity have been allocated in carotene-synthesizing natural strain Rhodotorula mucilaginosa UCM Y-1776 (wild type) after nitrosoguanidine action. Two brightly orange mutants 4L and 11 and one non-pigmented mutant 2 were chosen for the further researches. The ultraviolet was inefficient as a mutagen. Resistance to high concentration of copper ions (up to 200 mg/g), high sorption ability (Qmax = 9.1 mmol/g) was characteristic of R. mucilaginosa UCM Y-1776. Concentration of copper ions 50 mg/l was toxic for mutants 4L, 11 and 2, which sorption ability was lower in comparison with carotene pigmented R. mucilaginosa UCM Y-1776. It was shown, for the first time that there was a direct dependence between the presence of carotenoid pigments, resistance to high concentration of copper ions and sorption ability for yeast R. mucilaginosa UCM Y-1776.

Chemo-selective high yield microwave assisted reaction turns cellulose to green chemicals.

PubMed

Hassanzadeh, Salman; Aminlashgari, Nina; Hakkarainen, Minna

2014-11-04

Exceptionally high cellulose liquefaction yields, up to 87% as calculated from the amount of solid residue, were obtained under mild conditions by utilizing the synergistic effect of microwave radiation and acid catalysis. The effect of processing conditions on degradation products was fingerprinted by rapid laser desorption ionization-mass spectrometry (LDI-MS) method. The reaction was chemo-tunable, enabling production of glucose (Glc) or levulinic acid (LeA) at significantly high selectivity and yields, the relative molar yields being up to 50 and 69%, respectively. A turning point from pure depolymerization to glucose to further degradation to levulinic acid and formic acid was observed at approximately 50% liquefaction or above 140 °C. This was accompanied by the formation of small amounts of solid spherical carbonized residues. The reaction was monitored by multiple analytical techniques. The high yields were connected to the ability of the process to break the strong secondary interactions in cellulose. The developed method has great potential for future production of green platform chemicals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and characterization of poly (dihydroxybiphenyl borate) with high char yield for high-performance thermosetting resins

NASA Astrophysics Data System (ADS)

Wang, Shujuan; Xing, Xiaolong; Li, Jian; Jing, Xinli

2018-01-01

The objective of the current work is to synthesize novel boron-containing polymers with excellent thermal resistance, and reveal the structure and the reason for the high char yield. Thus, poly (dihydroxybiphenyl borate) (PDDB) with a more rigid molecular chain, was successfully synthesized using 4,4‧-dihydroxybiphenyl and boric acid. Structural characterizations of the prepared PDDB were performed via NMR, FTIR, XPS, and XRD analyses. The results reveal that PDDB consists of aromatic, Phsbnd Osbnd B and Bsbnd Osbnd B structures as well as a small number of boron hydroxyl and phenolic hydroxyl groups. PDDB shows good solubility in strong polar solvents, which is of great importance for the modification of thermosetting resins. TGA combined with DSC were employed to evaluate the thermal properties of PDDB, and increases in the glass transition temperature (Tg) and char yield were observed with increased boron content. Tg and char yield of PDDB (800 °C, nitrogen atmosphere) reached up to 219 °C and 66.5%, respectively. PDDB was extensively characterized during pyrolysis to reveal the high char yield of PDDB. As briefly discussed, the boron oxide and boron carbide that formed during pyrolysis play a crucial role in the high char yield of PDDB, which reduces the release of volatile carbon dioxide and carbon. This research suggests that PDDB has great potential as a novel modified agent for the improvement of the comprehensive performance of thermosetting resins to broaden their applicability in the field of advanced composites.

Enhancing the Production of D-Mannitol by an Artificial Mutant of Penicillium sp. T2-M10.

PubMed

Duan, Rongting; Li, Hongtao; Li, Hongyu; Tang, Linhuan; Zhou, Hao; Yang, Xueqiong; Yang, Yabin; Ding, Zhongtao

2018-05-26

D-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient D-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial D-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of D-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24 g/L glucose for 9 days. The results showed that the production of D-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that D-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a D-mannitol-producing strain.

Rational design of high-yield and superior-quality rice.

PubMed

Zeng, Dali; Tian, Zhixi; Rao, Yuchun; Dong, Guojun; Yang, Yaolong; Huang, Lichao; Leng, Yujia; Xu, Jie; Sun, Chuan; Zhang, Guangheng; Hu, Jiang; Zhu, Li; Gao, Zhenyu; Hu, Xingming; Guo, Longbiao; Xiong, Guosheng; Wang, Yonghong; Li, Jiayang; Qian, Qian

2017-03-20

Rice (Oryza sativa L.) is a staple food for more than half of the world's population. To meet the ever-increasing demand for food, because of population growth and improved living standards, world rice production needs to double by 2030 1 . The development of new elite rice varieties with high yield and superior quality is challenging for traditional breeding approaches, and new strategies need to be developed. Here, we report the successful development of new elite varieties by pyramiding major genes that significantly contribute to grain quality and yield from three parents over five years. The new varieties exhibit higher yield potential and better grain quality than their parental varieties and the China's leading super-hybrid rice, Liang-you-pai-jiu (LYP9 or Pei-ai 64S/93-11). Our results demonstrate that rational design is a powerful strategy for meeting the challenges of future crop breeding, particularly in pyramiding multiple complex traits.

CHO-cell mutant with a defect in cytokinesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, L.H.; Lindl, P.A.

1976-01-01

In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie, 34 percent) of polyploid cells at the permissive temperature of 34/sup 0/C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5/sup 0/ and 39.5/sup 0/. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completionmore » of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivity.« less

Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

PubMed Central

Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

1995-01-01

An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517

Bremsstrahlung Dose Yield for High-Intensity Short-Pulse Laser–Solid Experiments

DOE PAGES

Liang, Taiee; Bauer, Johannes M.; Liu, James C.; ...

2016-12-01

A bremsstrahlung source term has been developed by the Radiation Protection (RP) group at SLAC National Accelerator Laboratory for high-intensity short-pulse laser–solid experiments between 10 17 and 10 22 W cm –2. This source term couples the particle-in-cell plasma code EPOCH and the radiation transport code FLUKA to estimate the bremsstrahlung dose yield from laser–solid interactions. EPOCH characterizes the energy distribution, angular distribution, and laser-to-electron conversion efficiency of the hot electrons from laser–solid interactions, and FLUKA utilizes this hot electron source term to calculate a bremsstrahlung dose yield (mSv per J of laser energy on target). The goal of thismore » paper is to provide RP guidelines and hazard analysis for high-intensity laser facilities. In conclusion, a comparison of the calculated bremsstrahlung dose yields to radiation measurement data is also made.« less

Bremsstrahlung Dose Yield for High-Intensity Short-Pulse Laser–Solid Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Taiee; Bauer, Johannes M.; Liu, James C.

A bremsstrahlung source term has been developed by the Radiation Protection (RP) group at SLAC National Accelerator Laboratory for high-intensity short-pulse laser–solid experiments between 10 17 and 10 22 W cm –2. This source term couples the particle-in-cell plasma code EPOCH and the radiation transport code FLUKA to estimate the bremsstrahlung dose yield from laser–solid interactions. EPOCH characterizes the energy distribution, angular distribution, and laser-to-electron conversion efficiency of the hot electrons from laser–solid interactions, and FLUKA utilizes this hot electron source term to calculate a bremsstrahlung dose yield (mSv per J of laser energy on target). The goal of thismore » paper is to provide RP guidelines and hazard analysis for high-intensity laser facilities. In conclusion, a comparison of the calculated bremsstrahlung dose yields to radiation measurement data is also made.« less

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

PubMed

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

2012-08-01

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

Physical basis for altered stem elongation rates in internode length mutants of Pisum

NASA Technical Reports Server (NTRS)

Behringer, F. J.; Cosgrove, D. J.; Reid, J. B.; Davies, P. J.

1990-01-01

Biophysical parameters related to gibberellin (GA)-dependent stem elongation were examined in dark-grown stem-length genotypes of Pisum sativum L. The rate of internode expansion in these genotypes is altered due to recessive mutations which affect either the endogenous levels of, or response to, GA. The GA deficient dwarf L181 (ls), two GA insensitive semierectoides dwarfs NGB5865 and NGB5862 (lka and lkb, respectively) and the slender' line L197 (la crys), which is tall regardless of GA content, were compared to the wild-type tall cultivar, Torsdag. Osmotic pressure, estimated by vapor pressure osmometry, and turgor pressure, measured directly with a pressure probe, did not correlate with the differences in growth rate among the genotypes. Mechanical wall properties of frozen-thawed tissue were measured using a constant force assay. GA deficiency resulted in increased wall stiffness judged both on the basis of plastic compliance and plastic extensibility normalized for equal stem circumference. Plastic compliance was not reduced in the GA insensitive dwarfs, though lka reduced circumference-normalized plasticity. In contrast, in vivo wall relaxation, determined by the pressure-block technique, differed among genotypes in a manner which did correlate with extension rates. The wall yield threshold was 1 bar or less in the tall lines, but ranged from 3 to 6 bars in the dwarf genotypes. The results with the ls mutant indicate that GA enhances stem elongation by both decreasing the wall yield threshold and increasing the wall yield coefficient. In the GA-insensitive mutants, lka and lkb, the wall yield threshold is substantially elevated. Plants possessing lka may also possess a reduced wall yield coefficient.

Characterization of a heat-tolerant Chlorella sp. GD mutant with enhanced photosynthetic CO2 fixation efficiency and its implication as lactic acid fermentation feedstock.

PubMed

Lee, Tse-Min; Tseng, Yu-Fei; Cheng, Chieh-Lun; Chen, Yi-Chuan; Lin, Chih-Sheng; Su, Hsiang-Yen; Chow, Te-Jin; Chen, Chun-Yen; Chang, Jo-Shu

2017-01-01

Fermentative production of lactic acid from algae-based carbohydrates devoid of lignin has attracted great attention for its potential as a suitable alternative substrate compared to lignocellulosic biomass. A Chlorella sp. GD mutant with enhanced thermo-tolerance was obtained by mutagenesis using N -methyl- N '-nitro- N -nitrosoguanidine to overcome outdoor high-temperature inhibition and it was used as a feedstock for fermentative lactic acid production. The indoor experiments showed that biomass, reducing sugar content, photosynthetic O 2 evolution rate, photosystem II activity ( F v / F m and F v '/ F m '), and chlorophyll content increased as temperature, light intensity, and CO 2 concentration increased. The mutant showed similar DIC affinity and initial slope of photosynthetic light response curve (α) as that of the wild type but had higher dissolved inorganic carbon (DIC) utilization capacity and maximum photosynthesis rate ( P max ). Moreover, the PSII activity ( F v '/ F m ') in the mutant remained normal without acclimation process after being transferred to photobioreactor. This suggests that efficient utilization of incident high light and enhanced carbon fixation with its subsequent flux to carbohydrates accumulation in the mutant contributes to higher sugar and biomass productivity under enriched CO 2 condition. The mutant was cultured outdoors in a photobioreactor with 6% CO 2 aeration in hot summer season in southern Taiwan. The harvested biomass was subjected to separate hydrolysis and fermentation (SHF) for lactic acid production with carbohydrate concentration equivalent to 20 g/L glucose using the lactic acid-producing bacterium Lactobacillus plantarum 23. The conversion rate and yield of lactic acid were 80% and 0.43 g/g Chlorella biomass, respectively. These results demonstrated that the thermo-tolerant Chlorella mutant with high photosynthetic efficiency and biomass productivity under hot outdoor condition is an efficient fermentative

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

PubMed

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

2014-03-01

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

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

PubMed

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

2017-01-31

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

High yield neutron generators using the DD reaction

NASA Astrophysics Data System (ADS)

Vainionpaa, J. H.; Harris, J. L.; Piestrup, M. A.; Gary, C. K.; Williams, D. L.; Apodaca, M. D.; Cremer, J. T.; Ji, Qing; Ludewigt, B. A.; Jones, G.

2013-04-01

A product line of high yield neutron generators has been developed at Adelphi technology inc. The generators use the D-D fusion reaction and are driven by an ion beam supplied by a microwave ion source. Yields of up to 5 × 109 n/s have been achieved, which are comparable to those obtained using the more efficient D-T reaction. The microwave-driven plasma uses the electron cyclotron resonance (ECR) to produce a high plasma density for high current and high atomic ion species. These generators have an actively pumped vacuum system that allows operation at reduced pressure in the target chamber, increasing the overall system reliability. Since no radioactive tritium is used, the generators can be easily serviced, and components can be easily replaced, providing essentially an unlimited lifetime. Fast neutron source size can be adjusted by selecting the aperture and target geometries according to customer specifications. Pulsed and continuous operation has been demonstrated. Minimum pulse lengths of 50 μs have been achieved. Since the generators are easily serviceable, they offer a long lifetime neutron generator for laboratories and commercial systems requiring continuous operation. Several of the generators have been enclosed in radiation shielding/moderator structures designed for customer specifications. These generators have been proven to be useful for prompt gamma neutron activation analysis (PGNAA), neutron activation analysis (NAA) and fast neutron radiography. Thus these generators make excellent fast, epithermal and thermal neutron sources for laboratories and industrial applications that require neutrons with safe operation, small footprint, low cost and small regulatory burden.

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

PubMed

Züst, Roland; Dong, Hongping; Li, Xiao-Feng; Chang, David C; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R; Ellis, Esther M; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Diversity and stability study on rice mutants induced in space environment.

PubMed

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

2008-03-01

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 8(th) and 9(th) 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 8(th) and 9(th) generations, suggesting that they can be used as bio markers 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.

Maternal synthesis of abscisic acid controls seed development and yield in Nicotiana plumbaginifolia.

PubMed

Frey, Anne; Godin, Béatrice; Bonnet, Magda; Sotta, Bruno; Marion-Poll, Annie

2004-04-01

The role of maternally derived abscisic acid (ABA) during seed development has been studied using ABA-deficient mutants of Nicotiana plumbaginifolia Viviani. ABA deficiency induced seed abortion, resulting in reduced seed yield, and delayed growth of the remaining embryos. Mutant grafting onto wild-type stocks and reciprocal crosses indicated that maternal ABA, synthesized in maternal vegetative tissues and translocated to the seed, promoted early seed development and growth. Moreover ABA deficiency delayed both seed coat pigmentation and capsule dehiscence. Mutant grafting did not restore these phenotypes, indicating that ABA synthesized in the seed coat and capsule envelope may have a positive effect on capsule and testa maturation. Together these results shed light on the positive role of maternal ABA during N. plumbaginifolia seed development.

High-resolution, regional-scale crop yield simulations for the Southwestern United States

NASA Astrophysics Data System (ADS)

Stack, D. H.; Kafatos, M.; Medvigy, D.; El-Askary, H. M.; Hatzopoulos, N.; Kim, J.; Kim, S.; Prasad, A. K.; Tremback, C.; Walko, R. L.; Asrar, G. R.

2012-12-01

Over the past few decades, there have been many process-based crop models developed with the goal of better understanding the impacts of climate, soils, and management decisions on crop yields. These models simulate the growth and development of crops in response to environmental drivers. Traditionally, process-based crop models have been run at the individual farm level for yield optimization and management scenario testing. Few previous studies have used these models over broader geographic regions, largely due to the lack of gridded high-resolution meteorological and soil datasets required as inputs for these data intensive process-based models. In particular, assessment of regional-scale yield variability due to climate change requires high-resolution, regional-scale, climate projections, and such projections have been unavailable until recently. The goal of this study was to create a framework for extending the Agricultural Production Systems sIMulator (APSIM) crop model for use at regional scales and analyze spatial and temporal yield changes in the Southwestern United States (CA, AZ, and NV). Using the scripting language Python, an automated pipeline was developed to link Regional Climate Model (RCM) output with the APSIM crop model, thus creating a one-way nested modeling framework. This framework was used to combine climate, soil, land use, and agricultural management datasets in order to better understand the relationship between climate variability and crop yield at the regional-scale. Three different RCMs were used to drive APSIM: OLAM, RAMS, and WRF. Preliminary results suggest that, depending on the model inputs, there is some variability between simulated RCM driven maize yields and historical yields obtained from the United States Department of Agriculture (USDA). Furthermore, these simulations showed strong non-linear correlations between yield and meteorological drivers, with critical threshold values for some of the inputs (e.g. minimum and

Structure of a highly stable mutant of human fibroblast growth factor 1.

PubMed

Szlachcic, Anna; Zakrzewska, Małgorzata; Krowarsch, Daniel; Os, Vibeke; Helland, Ronny; Smalås, Arne O; Otlewski, Jacek

2009-01-01

Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).

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

PubMed Central

Schein, Stanley J.

1976-01-01

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

Circulation of Pneumocystis dihydropteroate synthase mutants in France.

PubMed

Le Gal, Solène; Damiani, Céline; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Quinio, Dorothée; Moalic, Elodie; Saliou, Philippe; Berthou, Christian; Le Meur, Yann; Totet, Anne; Nevez, Gilles

2012-10-01

Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation <30 days, iii) infection incubation is assumed to last about 2 months, the results provide evidence of mutant circulation from Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France. Copyright © 2012 Elsevier Inc. All rights reserved.

Ultra-broadband nonlinear saturable absorption of high-yield MoS2 nanosheets

NASA Astrophysics Data System (ADS)

Wei, Rongfei; Zhang, Hang; Hu, Zhongliang; Qiao, Tian; He, Xin; Guo, Qiangbing; Tian, Xiangling; Chen, Zhi; Qiu, Jianrong

2016-07-01

High-yield MoS2 nanosheets with strong nonlinear optical (NLO) responses in a broad near-infrared range were synthesized by a facile hydrothermal method. The observation of saturable absorption, which was excited by the light with photon energy smaller than the gap energy of MoS2, can be attributed to the enhancement of the hybridization between the Mo d-orbital and S p-orbital by the oxygen incorporation into MoS2. High-yield MoS2 nanosheets with high modulation depth and large saturable intensity generated a stable, passively Q-switched fiber laser pulse at 1.56 μm. The high output power of 1.08 mW can be attained under a very low pump power of 30.87 mW. Compared to recently reported passively Q-switched fiber lasers utilizing exfoliated MoS2 nanosheets, the efficiency of the laser for our passive Q-switching operation is larger and reaches 3.50%. This research may extend the understanding on the NLO properties of MoS2 and indicate the feasibility of the high-yield MoS2 nanosheets to passively Q-switched fiber laser effectively at low pump strengths.

Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

PubMed

Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

2013-08-01

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.

p53 deficiency alters the yield and spectrum of radiation-induced lacZ mutants in the brain of transgenic mice

NASA Technical Reports Server (NTRS)

Chang, P. Y.; Kanazawa, N.; Lutze-Mann, L.; Winegar, R. A.

2001-01-01

Exposure to heavy particle radiation in the galacto-cosmic environment poses a significant risk in space exploration and the evaluation of radiation-induced genetic damage in tissues, especially in the central nervous system, is an important consideration in long-term manned space missions. We used a plasmid-based transgenic mouse model system, with the pUR288 lacZ transgene integrated in the genome of every cell of C57Bl/6(lacZ) mice, to evaluate the genetic damage induced by iron particle radiation. In order to examine the importance of genetic background on the radiation sensitivity of individuals, we cross-bred p53 wild-type lacZ transgenic mice with p53 nullizygous mice, producing lacZ transgenic mice that were either hemizygous or nullizygous for the p53 tumor suppressor gene. Animals were exposed to an acute dose of 1 Gy of iron particles and the lacZ mutation frequency (MF) in the brain was measured at time intervals from 1 to 16 weeks post-irradiation. Our results suggest that iron particles induced an increase in lacZ MF (2.4-fold increase in p53+/+ mice, 1.3-fold increase in p53+/- mice and 2.1-fold increase in p53-/- mice) and that this induction is both temporally regulated and p53 genotype dependent. Characterization of mutants based on their restriction patterns showed that the majority of the mutants arising spontaneously are derived from point mutations or small deletions in all three genotypes. Radiation induced alterations in the spectrum of deletion mutants and reorganization of the genome, as evidenced by the selection of mutants containing mouse genomic DNA. These observations are unique in that mutations in brain tissue after particle radiation exposure have never before been reported owing to technical limitations in most other mutation assays.

Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

PubMed

Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

2017-05-01

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Isolation of Phaffia rhodozyma Mutants with Increased Astaxanthin Content

PubMed Central

An, Gil-Hwan; Schuman, Donald B.; Johnson, Eric A.

1989-01-01

Plating of the astaxanthin-producing yeast Phaffia rhodozyma onto yeast-malt agar containing 50 μM antimycin A gave rise to colonies of unusual morphology, characterized by a nonpigmented lower smooth surface that developed highly pigmented vertical papillae after 1 to 2 months. Isolation and purification of the pigmented papillae, followed by testing for pigment production in shake flasks, demonstrated that several antimycin isolates were increased two- to fivefold in astaxanthin content compared with the parental natural isolate (UCD-FST 67-385). One of the antimycin strains (ant-1) and a nitrosoguanidine derivative of ant-1 (ant-1-4) produced considerably more astaxanthin than the parent (ant-1 had 800 to 900 μg/g; ant-1-4 had 900 to 1,300 μg/g; and 67-385 had 300 to 450 μg/g). The mutant strains were compared physiologically with the parent. The antimycin mutants grew slower on ammonia, glutamate, or glutamine as nitrogen sources compared with the natural isolate and also had lower cell yields on several carbon sources. Although isolated on antimycin plates, they were found to be more susceptible to antimycin A, apparently owing to the spatial separation of the papillae from the agar. They were also more susceptible than the parent to the respiratory inhibitor thenoyltrifluoroacetone and were slightly more susceptible to cyanide, but did not differ from the natural isolate in susceptibility to azide. The antimycin-derived strains were also killed faster than the parent by hydrogen peroxide. The carotenoid compositions of the parent and the antimycin-derived strains were similar to those previously determined in the type strain (UCD-FST 67-210) except that two carotenoids not previously found in the type strain were present in increased quantities in the antimycin mutants and phoenicoxanthin was a minor component. The chemical properties of the unknown carotenoids suggested that the strains isolated on antimycin agar tended to oxygenate and desaturate

Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass1[OPEN

PubMed Central

Özparpucu, Merve

2018-01-01

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars due to the presence of lignin. To render lignocellulosic biomass a suitable feedstock for the bio-based economy, plants can be engineered to have decreased amounts of lignin. However, engineered plants with the lowest amounts of lignin exhibit collapsed vessels and yield penalties. Previous efforts were not able to fully overcome this phenotype without settling in sugar yield upon saccharification. Here, we reintroduced CINNAMOYL-COENZYME A REDUCTASE1 (CCR1) expression specifically in the protoxylem and metaxylem vessel cells of Arabidopsis (Arabidopsis thaliana) ccr1 mutants. The resulting ccr1 ProSNBE:CCR1 lines had overcome the vascular collapse and had a total stem biomass yield that was increased up to 59% as compared with the wild type. Raman analysis showed that monolignols synthesized in the vessels also contribute to the lignification of neighboring xylary fibers. The cell wall composition and metabolome of ccr1 ProSNBE:CCR1 still exhibited many similarities to those of ccr1 mutants, regardless of their yield increase. In contrast to a recent report, the yield penalty of ccr1 mutants was not caused by ferulic acid accumulation but was (largely) the consequence of collapsed vessels. Finally, ccr1 ProSNBE:CCR1 plants had a 4-fold increase in total sugar yield when compared with wild-type plants. PMID:29158331

Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7

PubMed Central

David, Laure C.; Dechorgnat, Julie; Ferrario-Méry, Sylvie

2014-01-01

NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation. PMID:24532452

Predawn respiration rates during flowering are highly predictive of yield response in Gossypium hirsutum when yield variability is water-induced.

PubMed

Snider, John L; Chastain, Daryl R; Meeks, Calvin D; Collins, Guy D; Sorensen, Ronald B; Byrd, Seth A; Perry, Calvin D

2015-07-01

Respiratory carbon evolution by leaves under abiotic stress is implicated as a major limitation to crop productivity; however, respiration rates of fully expanded leaves are positively associated with plant growth rates. Given the substantial sensitivity of plant growth to drought, it was hypothesized that predawn respiration rates (RPD) would be (1) more sensitive to drought than photosynthetic processes and (2) highly predictive of water-induced yield variability in Gossypium hirsutum. Two studies (at Tifton and Camilla Georgia) addressed these hypotheses. At Tifton, drought was imposed beginning at the onset of flowering (first flower) and continuing for three weeks (peak bloom) followed by a recovery period, and predawn water potential (ΨPD), RPD, net photosynthesis (AN) and maximum quantum yield of photosystem II (Fv/Fm) were measured throughout the study period. At Camilla, plants were exposed to five different irrigation regimes throughout the growing season, and average ΨPD and RPD were determined between first flower and peak bloom for all treatments. For both sites, fiber yield was assessed at crop maturity. The relationships between ΨPD, RPD and yield were assessed via non-linear regression. It was concluded for field-grown G. hirsutum that (1) RPD is exceptionally sensitive to progressive drought (more so than AN or Fv/Fm) and (2) average RPD from first flower to peak bloom is highly predictive of water-induced yield variability. Copyright © 2015 Elsevier GmbH. All rights reserved.

Laser heating challenges of high yield MagLIF targets

NASA Astrophysics Data System (ADS)

Slutz, Stephen; Sefkow, Adam; Vesey, Roger

2014-10-01

The MagLIF (Magnetized Liner Inertial Fusion) concept is predicted by numerical simulation to produce fusion yields of about 100 kJ, when driven by 25 MA from the existing Z accelerator [S. A. Slutz et al. Phys. Plasmas 17, 056303 (2010)] and much higher yields with future accelerators delivering higher currents [Slutz and Vesey PRL 108, 025003 (2012)]. The fuel must be heated before compression to obtain significant fusion yields due to the relatively slow implosion velocities (~ 100 km/s) of magnetically driven liners. Lasers provide a convenient means to accomplish this pre-compressional heating of the fusion fuel, but there are challenges. The laser must penetrate a foil covering the laser entrance hole and deposit 20-30 kJ within the ~1 cm length of the liner in fuel at 6-12 mg/cc. Such high densities could result in beam scattering due to refraction and laser plasma interactions. Numerical simulations of the laser heating process are presented, which indicate that energies as high as 30 kJ could be deposited in the fuel by using two laser pulses of different wavelengths. Simulations of this process will be presented as well of results for a MagLIF design for a potential new machine delivering 50 MA of current. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy's National Nuclear Security Administration under Contract DE-AC04-94AL85000.

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

DOE PAGES

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

2016-07-19

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Difference in C3–C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Yanfen; Beck, David A. C.; Lidstrom, Mary E.

In this study, two variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values. As a result, the major differences between the two variants occurred at the formate node as well as within C3-C4 inter-conversion pathways.more » Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction. In this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3-C4 inter-conversion strategies were the major response to the trade-off.« less

Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source.

PubMed Central

Garzón, A; Li, J; Flores, A; Casadesus, J; Stewart, V

1992-01-01

Selection for chlorate resistance yields mol (formerly chl) mutants with defects in molybdenum cofactor synthesis. Complementation and genetic mapping analyses indicated that the Klebsiella pneumoniae mol genes are functionally homologous to those of Escherichia coli and occupy analogous genetic map positions. Hypoxanthine utilization in other organisms requires molybdenum cofactor as a component of xanthine dehydrogenase, and thus most chlorate-resistant mutants cannot use hypoxanthine as a sole source of nitrogen. Surprisingly, the K. pneumoniae mol mutants and the mol+ parent grew equally well with hypoxanthine as the sole nitrogen source, suggesting that K. pneumoniae has a molybdenum cofactor-independent pathway for hypoxanthine utilization. PMID:1400180

High Useful Yield and Isotopic Analysis of Uranium by Resonance Ionization Mass Spectrometry

DOE PAGES

Savina, Michael R.; Isselhardt, Brett H.; Kucher, Andrew; ...

2017-05-09

Useful yields from resonance ionization mass spectrometry can be extremely high compared to other mass spectrometry techniques, but uranium analysis shows strong matrix effects arising from the tendency of uranium to form strongly bound oxide molecules that do not dissociate appreciably on energetic ion bombardment. Here, we demonstrate a useful yield of 24% for metallic uranium. Modeling the laser ionization and ion transmission processes shows that the high useful yield is attributable to a high ion fraction achieved by resonance ionization. We quantify the reduction of uranium oxide surface layers by Ar + and Ga + sputtering. The useful yieldmore » for uranium atoms from a uranium dioxide matrix is 0.4% and rises to 2% when the surface is in sputter equilibrium with the ion beam. The lower useful yield from the oxide is almost entirely due to uranium oxide molecules reducing the neutral atom content of the sputtered flux. We also demonstrate rapid isotopic analysis of solid uranium oxide at a precision of <0.5% relative standard deviation using relatively broadband lasers to mitigate spectroscopic fractionation.« less

High Useful Yield and Isotopic Analysis of Uranium by Resonance Ionization Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Savina, Michael R.; Isselhardt, Brett H.; Kucher, Andrew

Useful yields from resonance ionization mass spectrometry can be extremely high compared to other mass spectrometry techniques, but uranium analysis shows strong matrix effects arising from the tendency of uranium to form strongly bound oxide molecules that do not dissociate appreciably on energetic ion bombardment. Here, we demonstrate a useful yield of 24% for metallic uranium. Modeling the laser ionization and ion transmission processes shows that the high useful yield is attributable to a high ion fraction achieved by resonance ionization. We quantify the reduction of uranium oxide surface layers by Ar + and Ga + sputtering. The useful yieldmore » for uranium atoms from a uranium dioxide matrix is 0.4% and rises to 2% when the surface is in sputter equilibrium with the ion beam. The lower useful yield from the oxide is almost entirely due to uranium oxide molecules reducing the neutral atom content of the sputtered flux. We also demonstrate rapid isotopic analysis of solid uranium oxide at a precision of <0.5% relative standard deviation using relatively broadband lasers to mitigate spectroscopic fractionation.« less

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

PubMed

Kaur, Baljit; Oberoi, H S; Chadha, B S

2014-03-01

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol. Copyright © 2014 Elsevier Ltd. All rights reserved.

High yield neutron generators using the DD reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vainionpaa, J. H.; Harris, J. L.; Piestrup, M. A.

2013-04-19

A product line of high yield neutron generators has been developed at Adelphi technology inc. The generators use the D-D fusion reaction and are driven by an ion beam supplied by a microwave ion source. Yields of up to 5 Multiplication-Sign 10{sup 9} n/s have been achieved, which are comparable to those obtained using the more efficient D-T reaction. The microwave-driven plasma uses the electron cyclotron resonance (ECR) to produce a high plasma density for high current and high atomic ion species. These generators have an actively pumped vacuum system that allows operation at reduced pressure in the target chamber,more » increasing the overall system reliability. Since no radioactive tritium is used, the generators can be easily serviced, and components can be easily replaced, providing essentially an unlimited lifetime. Fast neutron source size can be adjusted by selecting the aperture and target geometries according to customer specifications. Pulsed and continuous operation has been demonstrated. Minimum pulse lengths of 50 {mu}s have been achieved. Since the generators are easily serviceable, they offer a long lifetime neutron generator for laboratories and commercial systems requiring continuous operation. Several of the generators have been enclosed in radiation shielding/moderator structures designed for customer specifications. These generators have been proven to be useful for prompt gamma neutron activation analysis (PGNAA), neutron activation analysis (NAA) and fast neutron radiography. Thus these generators make excellent fast, epithermal and thermal neutron sources for laboratories and industrial applications that require neutrons with safe operation, small footprint, low cost and small regulatory burden.« less

Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

PubMed

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

The diageotropica mutant of tomato lacks high specific activity auxin binding sites

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rayle, D. L.; Lomax, T. L.

1989-01-01

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

Electrorheological fluid with an extraordinarily high yield stress

NASA Astrophysics Data System (ADS)

Zhang, Yuling; Lu, Kunquan; Rao, Guanghui; Tian, Yu; Zhang, Shaohua; Liang, Jingkui

2002-02-01

Surface modified complex strontium titanate microparticles are synthesized by means of a modified sol-gel technique. A suspension composed of these particles immersed in a silicone oil exhibits excellent electrorheological properties attractive to industry and technology applications: a yield stress as high as 27 kPa in an applied electric field of 3 kV/mm, a low leakage current, wide dynamic ranges in temperature and shear rate, and a long-term stability against sedimentation. In addition to the high dielectric constant of strontium titanate, surfactant and water-free character of the particles may be responsible for the dramatic improvement of the electrorheological properties of the suspension.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs).

PubMed

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-12-19

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC 50 : 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC 50 : 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan ™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs)

PubMed Central

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-01-01

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC50: 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC50: 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs. PMID:29340041

PDGFRA-mutant syndrome.

PubMed

Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

2015-07-01

Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

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

PubMed

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

2017-01-18

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

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait

PubMed Central

2010-01-01

Background The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. Results Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. Conclusions We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait.

PubMed

Pham, Anh-Tung; Lee, Jeong-Dong; Shannon, J Grover; Bilyeu, Kristin D

2010-09-09

The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation

Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

PubMed

Briggs, Robert E; Hauglund, Melissa J; Maheswaran, Samuel K; Tatum, Fred M

2013-11-01

A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture. Published by Elsevier Ltd.

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase.

PubMed

Camacho, María; Burgos, Araceli; Chamber-Pérez, Manuel A

2003-04-01

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.

Projecting crop yield in northern high latitude area.

PubMed

Matsumura, Kanichiro

2014-01-01

Changing climatic conditions on seasonal and longer time scales influence agricultural production. Improvement of soil and fertilizer is a strong factor in agricultural production, but agricultural production is influenced by climate conditions even in highly developed countries. It is valuable if fewer predictors make it possible to conduct future projections. Monthly temperature and precipitation, wintertime 500hPa geopotential height, and the previous year's yield are used as predictors to forecast spring wheat yield in advance. Canadian small agricultural divisions (SAD) are used for analysis. Each SAD is composed of a collection of Canadian Agricultural Regions (CAR) of similar weather and growing conditions. Spring wheat yields in each CAR are forecast from the following variables: (a) the previous year's yield, (b) earlier stages of the growing season's climate conditions and, (c) the previous year's wintertime northern hemisphere 500hPa geopotential height field. Arctic outflow events in the Okanagan Valley in Canada are associated with episodes of extremely low temperatures during wintertime. Principal component analysis (PCA) is applied for wintertime northern hemisphere 500hPa geopotential height anomalies. The spatial PCA mode1 is defined as Arctic Oscillation and it influences prevailing westerlies. The prevailing westerlies meanders and influences climatic conditions. The spatial similarity between wintertime top 5 Arctic outflow event year's composites of 500hPa geopotential height anomalies and mode 3's spatial pattern is found. Mode 3's spatial pattern looks like the Pacific/North American (PNA) pattern which describes the variation of atmospheric circulation pattern over the Pacific Ocean and North America. Climate conditions from April to June, May to July, mode 3's time coefficients, and previous year's yield are used for forecasting spring wheat yield in each SAD. Cross-validation procedure which generates eight sets of models for the eight

[The isolation and characteristics of mutants of the Saccharopolyspora erythraea strain resistant to thiostrepton].

PubMed

Nastasiak, I N; Fedorenko, V A; Danilenko, V N

1997-01-01

The formation of thiostreptone resistant spontaneous and nitrosoguanidine-induced mutants in the erythromycin-producing organism Saccharopolyspora erythraea was investigated. The investigated collection of the mutants was heterogeneous by the level of the thiostreptone resistance (2.5 to 20 micrograms/ml). The thiostreptone resistance mutations had a pleiotropic effect: 17 per cent of the mutants was characterized by the growth thermosensitivity and 26 and 5.8 per cent of the mutants were characterized by loss of the ability to form melanine and aerial mycelium respectively. Such phenotypes were most frequent in the mutants resistant to low concentrations of thiostreptone (2 to 5 micrograms/ml). The absolute majority of the isolated thiostreptone resistant mutants was unstable and formed both the antibiotic resistant and the antibiotic sensitive clones. The greatest portion of the strains with high antibiotic activity (20 per cent) was detected among the S. erythraea spontaneous mutants on the medium with 2.5 micrograms/ ml of thiostreptone. It was shown that the instability of the high antibiotic activity in the mutants was associated with loss of the thiostreptone resistance property.

Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

PubMed Central

Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R.; Baquero, Fernando; Martinez, José Luis

2015-01-01

Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRIr) and triclosan-hypersusceptible (TRIhs) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRIr mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRIr mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRIr mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRIr mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRIr mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits. PMID

Detection of Cell Wall Chemical Variation in Zea Mays Mutants Using Near-Infrared Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buyck, N.; Thomas, S.

Corn stover is regarded as the prime candidate feedstock material for commercial biomass conversion in the United States. Variations in chemical composition of Zea mays cell walls can affect biomass conversion process yields and economics. Mutant lines were constructed by activating a Mu transposon system. The cell wall chemical composition of 48 mutant families was characterized using near-infrared (NIR) spectroscopy. NIR data were analyzed using a multivariate statistical analysis technique called Principal Component Analysis (PCA). PCA of the NIR data from 349 maize leaf samples reveals 57 individuals as outliers on one or more of six Principal Components (PCs) atmore » the 95% confidence interval. Of these, 19 individuals from 16 families are outliers on either PC3 (9% of the variation) or PC6 (1% of the variation), the two PCs that contain information about cell wall polymers. Those individuals for which altered cell wall chemistry is confirmed with wet chemical analysis will then be subjected to fermentation analysis to determine whether or not biomass conversion process kinetics, yields and/or economics are significantly affected. Those mutants that provide indications for a decrease in process cost will be pursued further to identify the gene(s) responsible for the observed changes in cell wall composition and associated changes in process economics. These genes will eventually be incorporated into maize breeding programs directed at the development of a truly dual use crop.« less

Ionomic screening of field-grown soybeans identifies mutants with altered seed elemental composition

USDA-ARS?s Scientific Manuscript database

Soybean seeds contain high levels of mineral nutrients essential for human and animal nutrition. High throughput elemental profiling (ionomics) has identified mutants in model plant species grown in controlled environments. Here, we describe a method for identifying potential soybean ionomics mutant...

Comparative analysis on inactivation kinetics of between piezotolerant and piezosensitive mutant strains of Saccharomyces cerevisiae under combinations of high hydrostatic pressure and temperature.

PubMed

Nomura, Kazuki; Kuwabara, Yuki; Kuwabara, Wataru; Takahashi, Hiroyuki; Nakajima, Kanako; Hayashi, Mayumi; Iguchi, Akinori; Shigematsu, Toru

2017-12-01

We previously obtained a pressure-tolerant (piezotolerant) and a pressure sensitive (piezosensitive) mutant strain, under ambient temperature, from Saccharomyces cerevisiae strain KA31a. The inactivation kinetics of these mutants were analyzed at 150 to 250MPa with 4 to 40°C. By a multiple regression analysis, the pressure and temperature dependency of the inactivation rate constants k values of both mutants, as well as the parent strain KA31a, were well approximated with high correlation coefficients (0.92 to 0.95). For both mutants, as well as strain KA31a, the lowest k value was shown at a low pressure levels with around ambient temperature. The k value approximately increased with increase in pressure level, and with increase and decrease in temperature. The piezosensitive mutant strain a924E1 showed piezosensitivity at all pressure and temperature levels, compared with the parent strain KA31a. In contrast, the piezotolerant mutant strain a2568D8 showed piezotolerance at 4 to 20°C, but did not show significant piezotolerance at 40°C. These results of the variable influence of temperature on pressure inactivation of these strains would be important for better understanding of piezosensitive and piezotolerant mechanisms, as well as the pressure inactivation mechanism of S. cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

An In Vivo Quantitative Comparison of Photoprotection in Arabidopsis Xanthophyll Mutants

PubMed Central

Ware, Maxwell A.; Dall’Osto, Luca; Ruban, Alexander V.

2016-01-01

Contribution of different LHCII antenna carotenoids to protective NPQ (pNPQ) were tested using a range of xanthophyll biosynthesis mutants of Arabidopsis: plants were either devoid of lutein (lut2), violaxanthin (npq2), or synthesized a single xanthophyll species, namely violaxanthin (aba4npq1lut2), zeaxanthin (npq2lut2), or lutein (chy1chy2lut5). A novel pulse amplitude modulated (PAM) fluorescence analysis procedure, that used a gradually increasing actinic light intensity, allowed the efficiency of pNPQ to be tested using the photochemical quenching (qP) parameter measured in the dark (qPd). Furthermore, the yield of photosystem II (ΦPSII) was calculated, and the light intensity which induces photoinhibition in 50% of leaves for each mutant was ascertained. Photoprotective capacities of each xanthophyll were quantified, taking into account chlorophyll a/b ratios and excitation pressure. Here, light tolerance, pNPQ capacity, and ΦPSII were highest in wild type plants. Of the carotenoid mutants, lut2 (lutein-deficient) plants had the highest light tolerance, and the joint the highest ΦPSII with violaxanthin only plants. We conclude that all studied mutants possess pNPQ and a more complete composition of xanthophylls in their natural binding sites is the most important factor governing photoprotection, rather than any one specific xanthophyll suggesting a strong structural effect of the molecules upon the LHCII antenna organization and discuss the results significance for future crop development. PMID:27446097

An In Vivo Quantitative Comparison of Photoprotection in Arabidopsis Xanthophyll Mutants.

PubMed

Ware, Maxwell A; Dall'Osto, Luca; Ruban, Alexander V

2016-01-01

Contribution of different LHCII antenna carotenoids to protective NPQ (pNPQ) were tested using a range of xanthophyll biosynthesis mutants of Arabidopsis: plants were either devoid of lutein (lut2), violaxanthin (npq2), or synthesized a single xanthophyll species, namely violaxanthin (aba4npq1lut2), zeaxanthin (npq2lut2), or lutein (chy1chy2lut5). A novel pulse amplitude modulated (PAM) fluorescence analysis procedure, that used a gradually increasing actinic light intensity, allowed the efficiency of pNPQ to be tested using the photochemical quenching (qP) parameter measured in the dark (qPd). Furthermore, the yield of photosystem II (ΦPSII) was calculated, and the light intensity which induces photoinhibition in 50% of leaves for each mutant was ascertained. Photoprotective capacities of each xanthophyll were quantified, taking into account chlorophyll a/b ratios and excitation pressure. Here, light tolerance, pNPQ capacity, and ΦPSII were highest in wild type plants. Of the carotenoid mutants, lut2 (lutein-deficient) plants had the highest light tolerance, and the joint the highest ΦPSII with violaxanthin only plants. We conclude that all studied mutants possess pNPQ and a more complete composition of xanthophylls in their natural binding sites is the most important factor governing photoprotection, rather than any one specific xanthophyll suggesting a strong structural effect of the molecules upon the LHCII antenna organization and discuss the results significance for future crop development.

Design of high-reliability low-cost amorphous silicon modules for high energy yield

NASA Astrophysics Data System (ADS)

Jansen, Kai W.; Varvar, Anthony; Twesme, Edward; Berens, Troy; Dhere, Neelkanth G.

2008-08-01

For PV modules to fulfill their intended purpose, they must generate sufficient economic return over their lifetime to justify their initial cost. Not only must modules be manufactured at a low cost/Wp with a high energy yield (kWh/kWp), they must also be designed to withstand the significant environmental stresses experienced throughout their 25+ year lifetime. Based on field experience, the most common factors affecting the lifetime energy yield of glass-based amorphous silicon (a-Si) modules have been identified; these include: 1) light-induced degradation; 2) moisture ingress and thin film corrosion; 3) transparent conductive oxide (TCO) delamination; and 4) glass breakage. The current approaches to mitigating the effect of these degradation mechanisms are discussed and the accelerated tests designed to simulate some of the field failures are described. In some cases, novel accelerated tests have been created to facilitate the development of improved manufacturing processes, including a unique test to screen for TCO delamination. Modules using the most reliable designs are tested in high voltage arrays at customer and internal test sites, as well as at independent laboratories. Data from tests at the Florida Solar Energy Center has shown that a-Si tandem modules can demonstrate an energy yield exceeding 1200 kWh/kWp/yr in a subtropical climate. In the same study, the test arrays demonstrated low long-term power loss over two years of data collection, after initial stabilization. The absolute power produced by the test arrays varied seasonally by approximately +/-7%, as expected.

Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

PubMed

Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

2016-06-01

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2016-01-01

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants

PubMed Central

2012-01-01

Background Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production. Results Xylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter. Conclusions Spatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by

Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant.

PubMed

Gu, Yajuan; Chang, Xiaodan; Dai, Shan; Song, Qinghua; Zhao, Hongshan; Lei, Pengcheng

2017-09-10

Xeroderma pigmentosum (XP) is a rare, recessive hereditary disease characterized by sunlight hypersensitivity and high incidence of skin cancer with clinical and genetic heterogeneity. We collected two unrelated Chinese patients showing typical symptoms of XPC without neurologic symptoms. Direct sequencing of XPC gene revealed that patient 1 carried IVS1+1G>A and c.958 C>T mutations, and patient 2 carried c.545_546delTA and c.2257_2258insC mutations. All these four mutations introduced premature terminal codons (PTCs) in XPC gene. The nonsense mutation c.958 C>T yielded truncated mutant Q320X, and we studied its function for global genome repair kinetics. Overexpressed Q320X mutant can localize to site of DNA damage, but it is defective in CPD and 6-4PP repair. Readthrough of PTCs is a new approach to treatment of genetic diseases. We found that aminoglycosides could significantly increase the full length protein expression of Q320X mutant, but NER defects were not rescued in vitro. Copyright © 2017 Elsevier B.V. All rights reserved.

Differential gene expression at different stages of mesocarp development in high- and low-yielding oil palm.

PubMed

Wong, Yick Ching; Teh, Huey Fang; Mebus, Katharina; Ooi, Tony Eng Keong; Kwong, Qi Bin; Koo, Ka Loo; Ong, Chuang Kee; Mayes, Sean; Chew, Fook Tim; Appleton, David R; Kulaveerasingam, Harikrishna

2017-06-21

The oil yield trait of oil palm is expected to involve multiple genes, environmental influences and interactions. Many of the underlying mechanisms that contribute to oil yield are still poorly understood. In this study, we used a microarray approach to study the gene expression profiles of mesocarp tissue at different developmental stages, comparing genetically related high- and low- oil yielding palms to identify genes that contributed to the higher oil-yielding palm and might contribute to the wider genetic improvement of oil palm breeding populations. A total of 3412 (2001 annotated) gene candidates were found to be significantly differentially expressed between high- and low-yielding palms at at least one of the different stages of mesocarp development evaluated. Gene Ontologies (GO) enrichment analysis identified 28 significantly enriched GO terms, including regulation of transcription, fatty acid biosynthesis and metabolic processes. These differentially expressed genes comprise several transcription factors, such as, bHLH, Dof zinc finger proteins and MADS box proteins. Several genes involved in glycolysis, TCA, and fatty acid biosynthesis pathways were also found up-regulated in high-yielding oil palm, among them; pyruvate dehydrogenase E1 component Subunit Beta (PDH), ATP-citrate lyase, β- ketoacyl-ACP synthases I (KAS I), β- ketoacyl-ACP synthases III (KAS III) and ketoacyl-ACP reductase (KAR). Sucrose metabolism-related genes such as Invertase, Sucrose Synthase 2 and Sucrose Phosphatase 2 were found to be down-regulated in high-yielding oil palms, compared to the lower yield palms. Our findings indicate that a higher carbon flux (channeled through down-regulation of the Sucrose Synthase 2 pathway) was being utilized by up-regulated genes involved in glycolysis, TCA and fatty acid biosynthesis leading to enhanced oil production in the high-yielding oil palm. These findings are an important stepping stone to understand the processes that lead to

Production and characterization of streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic septicaemia.

PubMed Central

de Alwis, M C; Carter, G R; Chengappa, M M

1980-01-01

A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent. PMID:6778598

Mutant strain of C. acetobutylicum and process for making butanol

DOEpatents

Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

1993-01-01

A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms.

PubMed

Yao, Qiu-Mei; Zhou, Jiao; Gale, Robert Peter; Li, Jin-Lan; Li, Ling-Di; Li, Ning; Chen, Shan-Shan; Ruan, Guo-Rui

2015-10-01

Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2(V617F). Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2(V617F) negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

Proline 68 enhances photoisomerization yield in photoactive yellow protein.

PubMed

Rupenyan, Alisa B; Vreede, Jocelyne; van Stokkum, Ivo H M; Hospes, Marijke; Kennis, John T M; Hellingwerf, Klaas J; Groot, Marie Louise

2011-05-26

In proteins and enzymes, the local environment of an active cofactor plays an important role in controlling the outcome of a functional reaction. In photoactive yellow protein (PYP), it ensures photoisomerization of the chromophore, a prerequisite for formation of a signaling state. PYP is the prototype of a PAS domain, and the preferred model system for the studies of molecular mechanisms of biological light sensing. We investigated the effect of replacing proline-68, positioned near but not in direct contact with the chromophore, with other neutral amino acids (alanine, glycine, and valine), using ultrafast spectroscopy probing the visible and the mid-IR spectral regions, and molecular simulation to understand the interactions tuning the efficiency of light signaling. Transient absorption measurements indicate that the quantum yield of isomerization in the mutants is lower than the yield observed for the wild type. Subpicosecond mid-IR spectra and molecular dynamics simulations of the four proteins reveal that the hydrogen bond interactions around the chromophore and the access of water molecules in the active site of the protein determine the efficiency of photoisomerization. The mutants provide additional hydrogen bonds to the chromophore, directly and by allowing more water molecules access to its binding pocket. We conclude that proline-68 in the wild type protein optimizes the yield of photochemistry by maintaining a weak hydrogen bond with the chromophore, at the same time restraining the entrance of water molecules close to the alkylic part of pCa. This study provides a molecular basis for the structural optimization of biological light sensing.

Analagous Population Structures for Two Alphabaculoviruses Highlight a Functional Role for Deletion Mutants

PubMed Central

Serrano, Amaya; Williams, Trevor; Simón, Oihane; López-Ferber, Miguel; Caballero, Primitivo

2013-01-01

A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large-deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origins of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. The homology of nucleotides in the deleted regions was as high as 79%, similar to those of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that independently generates and sustains deletion mutants within each virus population. Functional analyses using different proportions of complete and deletion genotypes were performed with the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete/deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but there were no significant changes in the speed of kill. In contrast, OB yields increased only in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most extensively deleted SeMNPV genotype and contribute toward the elucidation of the role of such mutants in baculovirus populations. PMID:23204420

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant

PubMed Central

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A.; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K.; Altenbach, Christian; Hong, Yuan; Hanson, Susan M.; Palazzo, Maria C.; Chen, Jeannie; Hubbell, Wayne L.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. PMID:24012956

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

PubMed

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K; Altenbach, Christian; Hong, Yuan; Hanson, Susan M; Palazzo, Maria C; Chen, Jeannie; Hubbell, Wayne L; Gurevich, Eugenia V; Gurevich, Vsevolod V

2013-12-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. © 2013.

Characterization of the endosperm starch and the pleiotropic effects of biosynthetic enzymes on their properties in novel mutant rice lines with high resistant starch and amylose content.

PubMed

Itoh, Yuuki; Crofts, Naoko; Abe, Misato; Hosaka, Yuko; Fujita, Naoko

2017-05-01

Resistant starch (RS) is beneficial to human health. In order to reduce the current prevalence of diabetes and obesity, several transgenic and mutant crops containing high RS content are being developed. RS content of steamed rice with starch-branching enzyme (BE)IIb-deficient mutant endosperms is considerably high. To understand the mechanisms of RS synthesis and to increase RS content, we developed novel mutant rice lines by introducing the gene encoding starch synthase (SS)IIa and/or granule-bound starch synthase (GBSS)I from an indica rice cultivar into a japonica rice-based BEIIb-deficient mutant line, be2b. Introduction of SSIIa from an indica rice cultivar produced higher levels of amylopectin chains with degree of polymerization (DP) 11-18 than those in be2b; the extent of the change was slight due to the shortage of donor chains for SSIIa (DP 6-12) owing to BEIIb deficiency. The introduction of GBSSI from an indica rice cultivar significantly increased amylose content (by approximately 10%) in the endosperm starch. RS content of the new mutant lines was the same as or slightly higher than that of the be2b parent line. The relationship linking starch structure, RS content, and starch biosynthetic enzymes in the new mutant lines has also been discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative study of enzymatic activities of new KatG mutants from low- and high-level isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

PubMed

Brossier, Florence; Boudinet, Marlène; Jarlier, Vincent; Petrella, Stéphanie; Sougakoff, Wladimir

2016-09-01

Resistance to isoniazid (INH-R) in Mycobacterium tuberculosis is mainly due to mutations at position 315 (S315T) of the catalase-peroxidase KatG. We identified 16 mutations (including 13 biochemically uncharacterized mutations) in KatG from INH-R clinical isolates of M. tuberculosis showing mutations other than S315T. The KatG enzymatic activities (catalase, peroxidase, free radical production and isonicotinoyl-NAD formation) of wild-type KatG and the 16 mutants were determined and correlated to their spatial location in a KatG model structure. Of all mutations studied, H270R, which conferred a high level of INH-R and results in the disruption of a coordination bond with the heme, caused complete loss of all enzymatic KatG activities. The mutants generally associated with a very high level of INH-R were all characterized by a drastic reduction in catalase activity and a marked decrease in INH activation activities. One mutant, A162E, displayed a behavior similar to S315T, i.e. a moderate decrease in catalase activity and a drastic decrease in the formation of the radical form of INH. Finally, the mutants associated with a low level of INH-R showed a moderate reduction in the four catalytic activities, likely stemming from an overall alteration of the folding and/or stability of the KatG protein. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques

PubMed Central

Chang, David C.; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R.; Ellis, Esther M.; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response. PMID:23935499

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants

PubMed Central

Wifling, D; Löffel, K; Nordemann, U; Strasser, A; Bernhardt, G; Dove, S; Seifert, R; Buschauer, A

2015-01-01

Background and Purpose Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4R) and rat H4 receptor (rH4R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4R-F169V, mH4R-V171F, hH4R-S179A, hH4R-S179M; double mutants: hH4R-F169V+S179A, hH4R-F169V+S179M and mH4R-V171F+M181S. Experimental Approach Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([3H]-histamine), and in functional [35S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. Key Results Constitutive activity decreased from the hH4 receptor via the hH4R-F169V mutant to the hH4R-F169V+S179A and hH4R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. Conclusions and Implications F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

PubMed

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F; Labes, Antje

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

PubMed Central

Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F.; Labes, Antje

2015-01-01

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum. PMID:26460745

Inactivation and deficiency of core proteins of photosystems I and II caused by genetical phylloquinone and plastoquinone deficiency but retained lamellar structure in a T-DNA mutant of Arabidopsis.

PubMed

Shimada, Hiroshi; Ohno, Ryoichi; Shibata, Masaru; Ikegami, Isamu; Onai, Kiyoshi; Ohto, Masa-aki; Takamiya, Ken-ichiro

2005-02-01

Phylloquinone, a substituted 1,4-naphthoquinone with an 18-carbon-saturated phytyl tail, functions as a bound one-electron carrier cofactor at the A1 site of photosystem I (PSI). A Feldmann tag line mutant, no. 2755 (designated as abc4 hereafter), showed pale-green young leaves and white old leaves. The mutated nuclear gene encoded 1,4-dihydroxy-2-naphtoic acid phytyltransferase, an enzyme of phylloquinone biosynthesis, and high-performance liquid chromatography analysis revealed that the abc4 mutant contained no phylloquinone, and only about 3% plastoquinone. Photooxidation of P700 of PSI in the abc4 mutant was not observed, and reduced-versus-oxidized difference spectroscopy indicated that the abc4 mutant had no P700. The maximum quantum yield of photosystem II (PSII) in the abc4 mutant was much decreased, and the electron transfer from PSII to PSI in the abc4 mutant did not occur. For the pale-green leaves of the abc4 mutant plant, the ultrastructure of the chloroplasts was almost the same as that of the wild-type plant. However, the chloroplasts in the albino leaves of the mutant were smaller and had a lot of grana thylakoids and few stroma thylakoids. The amounts of PSI and PSII core subunits in the abc4 mutant were significantly decreased compared with those in the wild type. These results suggested that a deficiency of phylloquinone in PSI caused the abolishment of PSI and a partial defect of PSII due to a significant decrease of plastoquinone, but did not influence the ultrastructure of the chloroplasts in young leaves.

Experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters

PubMed Central

Bastiaans, Eric; Debets, Alfons J. M.; Aanen, Duur K.

2016-01-01

In multicellular organisms, there is a potential risk that cheating mutants gain access to the germline. Development from a single-celled zygote resets relatedness among cells to its maximum value each generation, which should accomplish segregation of cheating mutants from non-cheaters and thereby protect multicellular cooperation. Here we provide the crucial direct comparison between high- and low-relatedness conditions to test this hypothesis. We allow two variants of the fungus Neurospora crassa to evolve, one with and one without the ability to form chimeras with other individuals, thus generating two relatedness levels. While multicellular cooperation remains high in the high-relatedness lines, it significantly decreases in all replicate low-relatedness lines, resulting in an average threefold decrease in spore yield. This reduction is caused by cheating mutants with reduced investment in somatic functions, but increased competitive success when fusing with non-cheaters. Our experiments demonstrate that high genetic relatedness is crucial to sustain multicellular cooperation. PMID:27139112

ketu mutant mice uncover an essential meiotic function for the ancient RNA helicase YTHDC2

PubMed Central

Jain, Devanshi; Puno, M Rhyan; Meydan, Cem; Lailler, Nathalie; Mason, Christopher E; Lima, Christopher D; Anderson, Kathryn V

2018-01-01

Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, ‘ketu’, caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3′→5′ RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis. PMID:29360036

Prediction of dynamic behavior of mutant strains from limited wild-type data.

PubMed

Song, Hyun-Seob; Ramkrishna, Doraiswami

2012-03-01

Metabolic engineering is the field of introducing genetic changes in organisms so as to modify their function towards synthesizing new products of high impact to society. However, engineered cells frequently have impaired growth rates thus seriously limiting the rate at which such products are made. The problem is attributable to inadequate understanding of how a metabolic network functions in a dynamic sense. Predictions of mutant strain behavior in the past have been based on steady state theories such as flux balance analysis (FBA), minimization of metabolic adjustment (MOMA), and regulatory on/off minimization (ROOM). Such predictions are restricted to product yields and cannot address productivity, which is of focal interest to applications. We demonstrate that our framework ( [Song and Ramkrishna, 2010] and [Song and Ramkrishna, 2011]), based on a “cybernetic” view of metabolic systems, makes predictions of the dynamic behavior of mutant strains of Escherichia coli from a limited amount of data obtained from the wild-type. Dynamic frameworks must necessarily address the issue of metabolic regulation, which the cybernetic approach does by postulating that metabolism is an optimal dynamic response of the organism to the environment in driving reactions towards ensuring survival. The predictions made in this paper are without parallel in the literature and lay the foundation for rational metabolic engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

High Titer and Yields Achieved with Novel, Low-Severity Pretreatment Strategy

DOE Office of Scientific and Technical Information (OSTI.GOV)

NREL researchers obtained high concentration sugar syrups in enzymatic hydrolysis that are fermentable to ethanol and other advanced biofuels and intermediate products at high yields. The novel DMR process is simpler and bypasses all severe pretreatment methods, thus reducing the environmental impact. The results are unprecedented. Researchers achieved a high concentration of sugars (230g/L of monomeric sugar and 270 g/L total sugar) and this low toxicity, highly fermentable syrup yielded 86 g/L ethanol (> 90 percent conversion). In addition, the lignin streams from this process can readily be converted to jet or renewable diesel blendstocks through a hydrodeoxygenation step. Themore » NREL-developed, low severity DMR process may potentially replace higher severity chemical pretreatments and associated expensive reactors constructed of exotic alloys with a simpler process, using commercial-scale equipment commonly associated with the pulp and paper industry, to produce high concentration, low toxicity sugar streams and highly reactive lignin streams from non-food renewable biomass for biological and catalytic upgrading to advanced biofuels and chemicals. The simpler DMR process with black liquor recycling could reduce environmental and life-cycle impacts, and repurpose shuttered pulp and paper mills to help revitalize rural economies.« less

Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development.

PubMed

Puckette, Michael; Clark, Benjamin A; Smith, Justin D; Turecek, Traci; Martel, Erica; Gabbert, Lindsay; Pisano, Melia; Hurtle, William; Pacheco, Juan M; Barrera, José; Neilan, John G; Rasmussen, Max

2017-11-15

The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria. IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production. Copyright © 2017 Puckette et al.

Genetic dissimilarity of putative gamma-ray-induced 'Preciosa-AAAB-Pome type' banana (Musa sp) mutants based on multivariate statistical analysis.

PubMed

Pestana, R K N; Amorim, E P; Ferreira, C F; Amorim, V B O; Oliveira, L S; Ledo, C A S; Silva, S O

2011-10-25

Bananas are among the most important fruit crops worldwide, being cultivated in more than 120 countries, mainly by small-scale producers. However, short-stature high-yielding bananas presenting good agronomic characteristics are hard to find. Consequently, wind continues to damage a great number of plantations each year, leading to lodging of plants and bunch loss. Development of new cultivars through conventional genetic breeding methods is hindered by female sterility and the low number of seeds. Mutation induction seems to have great potential for the development of new cultivars. We evaluated genetic dissimilarity among putative 'Preciosa' banana mutants generated by gamma-ray irradiation, using morphoagronomic characteristics and ISSR markers. The genetic distances between the putative 'Preciosa' mutants varied from 0.21 to 0.66, with a cophenetic correlation coefficient of 0.8064. We found good variability after irradiation of 'Preciosa' bananas; this procedure could be useful for banana breeding programs aimed at developing short-stature varieties with good agronomic characteristics.

High-Yield Synthesis and Applications of Anisotropic Gold Nanoparticles

NASA Astrophysics Data System (ADS)

Vigderman, Leonid

This work will describe research directed towards the synthesis of anisotropic gold nanoparticles as well as their functionalization and biological applications. The thesis will begin by describing a new technique for the high-yield synthesis of gold nanorods using hydroquinone as a reducing agent. This addresses important limitations of the traditional nanorod synthesis including low yield of gold ions conversion to metallic form and inability to produce rods with longitudinal surface plasmon peak above 850 nm. The use of hydroquinone was also found to improve the synthesis of gold nanowires via the nanorod-seed mediated procedure developed in our lab. The thesis will next present the synthesis of novel starfruitshaped nanorods, mesorods, and nanowires using a modified nanorod-seed mediated procedure. The starfruit particles displayed increased activity as surfaceenhanced Raman spectroscopy (SERS) substrates as compared to smooth structures. Next, a method for the functionalization of gold nanorods using a cationic thiol, 16-mercaptohexadecyltrimethylammonium bromide (MTAB), will be described. By using this thiol, we were able to demonstrate the complete removal of toxic surfactant from the nanorods and were also able to precisely quantify the grafting density of thiol molecules on the nanorod surface through a combination of several analytical techniques. Finally, this thesis will show that MTABfunctionalized nanorods are nontoxic and can be taken up in extremely high numbers into cancer cells. The thesis will conclude by describing the surprising uptake of larger mesorods and nanowires functionalized with MTAB into cells in high quantities.

Identification of natural high-oleate mutants from the USDA Peanut Germplasm Collection

USDA-ARS?s Scientific Manuscript database

Natural genetic variation may exist in plant germplasm collections. Identifying genetic variation may provide useful materials for breeders to develop new cultivars. After screening 8,846 cultivated peanut germplasm accessions by gas chromatography analysis, we identified three natural mutant lines ...

High Photoluminescence Quantum Yield in Band Gap Tunable Bromide Containing Mixed Halide Perovskites.

PubMed

Sutter-Fella, Carolin M; Li, Yanbo; Amani, Matin; Ager, Joel W; Toma, Francesca M; Yablonovitch, Eli; Sharp, Ian D; Javey, Ali

2016-01-13

Hybrid organic-inorganic halide perovskite based semiconductor materials are attractive for use in a wide range of optoelectronic devices because they combine the advantages of suitable optoelectronic attributes and simultaneously low-cost solution processability. Here, we present a two-step low-pressure vapor-assisted solution process to grow high quality homogeneous CH3NH3PbI3-xBrx perovskite films over the full band gap range of 1.6-2.3 eV. Photoluminescence light-in versus light-out characterization techniques are used to provide new insights into the optoelectronic properties of Br-containing hybrid organic-inorganic perovskites as a function of optical carrier injection by employing pump-powers over a 6 orders of magnitude dynamic range. The internal luminescence quantum yield of wide band gap perovskites reaches impressive values up to 30%. This high quantum yield translates into substantial quasi-Fermi level splitting and high "luminescence or optically implied" open-circuit voltage. Most importantly, both attributes, high internal quantum yield and high optically implied open-circuit voltage, are demonstrated over the entire band gap range (1.6 eV ≤ Eg ≤ 2.3 eV). These results establish the versatility of Br-containing perovskite semiconductors for a variety of applications and especially for the use as high-quality top cell in tandem photovoltaic devices in combination with industry dominant Si bottom cells.

Analysis of a Partial Male-Sterile Mutant of Arabidopsis thaliana Isolated from a Low-Energy Argon Ion Beam Mutagenized Pool

NASA Astrophysics Data System (ADS)

Xu, Min; Bian, Po; Wu, Yuejin; Yu, Zengliang

2008-04-01

A screen for Arabidopsis fertility mutants, mutagenized by low-energy argon ion beam, yielded two partial male-sterile mutants tc243-1 and tc243-2 which have similar phenotypes. tc243-2 was investigated in detail. The segregation ratio of the mutant phenotypes in the M2 pools suggested that mutation behaved as single Mendelian recessive mutations. tc243 showed a series of mutant phenotypes, among which partial male-sterile was its striking mutant characteristic. Phenotype analysis indicates that there are four factors leading to male sterility. a. Floral organs normally develop inside the closed bud, but the anther filaments do not elongate sufficiently to position the locules above the stigma at anthesis. b. The anther locules do not dehisce at the time of flower opening (although limited dehiscence occurs later). c. Pollens of mutant plants develop into several types of pollens at the trinucleated stage, as determined by staining with DAPI (4',6-diamidino-2-phenylindole), which shows a variable size, shape and number of nucleus. d. The viability of pollens is lower than that of the wild type on the germination test in vivo and vitro.

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity

PubMed Central

Sun, Xin; Marque, Leonard O.; Cordner, Zachary; Pruitt, Jennifer L.; Bhat, Manik; Li, Pan P.; Kannan, Geetha; Ladenheim, Ellen E.; Moran, Timothy H.; Margolis, Russell L.; Rudnicki, Dobrila D.

2014-01-01

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient–derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of HdhQ7/Q150 knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT. PMID:25035419

Photosynthesis, light use efficiency, and yield of reduced-chlorophyll soybean mutants in field conditions

USDA-ARS?s Scientific Manuscript database

Reducing chlorophyll (chl) content may improve the conversion efficiency of absorbed radiation into biomass (ec) and therefore yield in dense monoculture crops by improving light penetration and distribution within the canopy. Modeling suggests that reducing chl content may also reduce leaf temperat...

Ultraviolet light-induced mutants of Streptococcus lactis subspecies diacetylactis with enhanced acid- or flavor-producing abilities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuila, R.K.; Ranganathan, B.

1978-04-01

A strain of Streptococcus lactis subspecies diacetylactis S/sub 1/ isolated from fresh milk was exposed to 7200 ergs/mm/sup 2/ of ultraviolet radiation. Over 8100 colonies surviving from 7.4 x 10/sup 6/ cells exposed to radiation were screened on citrate agar for detection and isolation of mutants with increased flavor and/or acid production. Of the survivors, 960 were type-I mutants that exhibited clear zone on citrate agar after 18 h (presumed to be high diacetyl producers), and 288 were type-II mutants which did not exhibit clear zones on citrate agar for up to 72 h (high acid producers). Type-II mutants producedmore » an average .93 percent titratable acidity which was 34 percent more than the .69 percent of the parent. Reduction in titratable acidity (56 percent less) was considerable in type-I mutants, compared with the parent culture. Diacetyl + acetoin production by type-I mutants was 137.9 ppM which has 4.5 times more than that of the parental strain. Acetaldehyde production in the mutants varied from 1.5 to 34.5 ppM (parent culture 3.0 ppM). The mutants with increased acid and high acetoin plus diacetyl production were stable after 50 subcultures in milk.« less

Kasugamycin-dependent mutants of Escherichia coli.

PubMed Central

Dabbs, E R

1978-01-01

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

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

PubMed Central

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

2012-01-01

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

Lack of chemically induced mutation in repair-deficient mutants of yeast.

PubMed

Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

A genome-scale Escherichia coli kinetic metabolic model k-ecoli457 satisfying flux data for multiple mutant strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khodayari, Ali; Maranas, Costas D.

Kinetic models of metabolism at a genome scale that faithfully recapitulate the effect of multiple genetic interventions would be transformative in our ability to reliably design novel overproducing microbial strains. Here, we introduce k-ecoli457, a genome-scale kinetic model of Escherichia coli metabolism that satisfies fluxomic data for wild-type and 25 mutant strains under different substrates and growth conditions. The k-ecoli457 model contains 457 model reactions, 337 metabolites and 295 substrate-level regulatory interactions. Parameterization is carried out using a genetic algorithm by simultaneously imposing all available fluxomic data (about 30 measured fluxes per mutant). Furthermore, the Pearson correlation coefficient between experimentalmore » data and predicted product yields for 320 engineered strains spanning 24 product metabolites is 0.84. This is substantially higher than that using flux balance analysis, minimization of metabolic adjustment or maximization of product yield exhibiting systematic errors with correlation coefficients of, respectively, 0.18, 0.37 and 0.47.« less

A genome-scale Escherichia coli kinetic metabolic model k-ecoli457 satisfying flux data for multiple mutant strains

DOE PAGES

Khodayari, Ali; Maranas, Costas D.

2016-12-20

Kinetic models of metabolism at a genome scale that faithfully recapitulate the effect of multiple genetic interventions would be transformative in our ability to reliably design novel overproducing microbial strains. Here, we introduce k-ecoli457, a genome-scale kinetic model of Escherichia coli metabolism that satisfies fluxomic data for wild-type and 25 mutant strains under different substrates and growth conditions. The k-ecoli457 model contains 457 model reactions, 337 metabolites and 295 substrate-level regulatory interactions. Parameterization is carried out using a genetic algorithm by simultaneously imposing all available fluxomic data (about 30 measured fluxes per mutant). Furthermore, the Pearson correlation coefficient between experimentalmore » data and predicted product yields for 320 engineered strains spanning 24 product metabolites is 0.84. This is substantially higher than that using flux balance analysis, minimization of metabolic adjustment or maximization of product yield exhibiting systematic errors with correlation coefficients of, respectively, 0.18, 0.37 and 0.47.« less

Rational design of an enzyme mutant for anti-cocaine therapeutics

NASA Astrophysics Data System (ADS)

Zheng, Fang; Zhan, Chang-Guo

2008-09-01

(-)-Cocaine is a widely abused drug and there is no available anti-cocaine therapeutic. The disastrous medical and social consequences of cocaine addiction have made the development of an effective pharmacological treatment a high priority. An ideal anti-cocaine medication would be to accelerate (-)-cocaine metabolism producing biologically inactive metabolites. The main metabolic pathway of cocaine in body is the hydrolysis at its benzoyl ester group. Reviewed in this article is the state-of-the-art computational design of high-activity mutants of human butyrylcholinesterase (BChE) against (-)-cocaine. The computational design of BChE mutants have been based on not only the structure of the enzyme, but also the detailed catalytic mechanisms for BChE-catalyzed hydrolysis of (-)-cocaine and (+)-cocaine. Computational studies of the detailed catalytic mechanisms and the structure-and-mechanism-based computational design have been carried out through the combined use of a variety of state-of-the-art techniques of molecular modeling. By using the computational insights into the catalytic mechanisms, a recently developed unique computational design strategy based on the simulation of the rate-determining transition state has been employed to design high-activity mutants of human BChE for hydrolysis of (-)-cocaine, leading to the exciting discovery of BChE mutants with a considerably improved catalytic efficiency against (-)-cocaine. One of the discovered BChE mutants (i.e., A199S/S287G/A328W/Y332G) has a ˜456-fold improved catalytic efficiency against (-)-cocaine. The encouraging outcome of the computational design and discovery effort demonstrates that the unique computational design approach based on the transition-state simulation is promising for rational enzyme redesign and drug discovery.

Polymorphic variation in susceptibility and metabolism of triclosan-resistant mutants of Escherichia coli and Klebsiella pneumoniae clinical strains obtained after exposure to biocides and antibiotics.

PubMed

Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R; Baquero, Fernando; Martinez, José Luis; Coque, Teresa M

2015-01-01

Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRI(r)) and triclosan-hypersusceptible (TRI(hs)) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI(r) mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRI(r) mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRI(r) mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRI(r) mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI(r) mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive

The tomato mutant ars1 (altered response to salt stress 1) identifies an R1-type MYB transcription factor involved in stomatal closure under salt acclimation.

PubMed

Campos, Juan F; Cara, Beatriz; Pérez-Martín, Fernando; Pineda, Benito; Egea, Isabel; Flores, Francisco B; Fernandez-Garcia, Nieves; Capel, Juan; Moreno, Vicente; Angosto, Trinidad; Lozano, Rafael; Bolarin, Maria C

2016-06-01

A screening under salt stress conditions of a T-DNA mutant collection of tomato (Solanum lycopersicum L.) led to the identification of the altered response to salt stress 1 (ars1) mutant, which showed a salt-sensitive phenotype. Genetic analysis of the ars1 mutation revealed that a single T-DNA insertion in the ARS1 gene was responsible of the mutant phenotype. ARS1 coded for an R1-MYB type transcription factor and its expression was induced by salinity in leaves. The mutant reduced fruit yield under salt acclimation while in the absence of stress the disruption of ARS1 did not affect this agronomic trait. The stomatal behaviour of ars1 mutant leaves induced higher Na(+) accumulation via the transpiration stream, as the decreases of stomatal conductance and transpiration rate induced by salt stress were markedly lower in the mutant plants. Moreover, the mutation affected stomatal closure in a response mediated by abscisic acid (ABA). The characterization of tomato transgenic lines silencing and overexpressing ARS1 corroborates the role of the gene in regulating the water loss via transpiration under salinity. Together, our results show that ARS1 tomato gene contributes to reduce transpirational water loss under salt stress. Finally, this gene could be interesting for tomato molecular breeding, because its manipulation could lead to improved stress tolerance without yield penalty under optimal culture conditions. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

In vitro capability of faropenem to select for resistant mutants of Streptococcus pneumoniae and Haemophilus influenzae.

PubMed

Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C

2008-02-01

When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones.

Functional characterization of mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking short domains within the large, lumen-exposed loop of the chlorophyll protein CP47 in photosystem II.

PubMed

Gleiter, H M; Haag, E; Shen, J R; Eaton-Rye, J J; Inoue, Y; Vermaas, W F; Renger, G

1994-10-11

Several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions or a single-site mutation within the large, lumen-exposed loop (loop E) of the chlorophyll a-binding photosystem II core protein, CP47, are analyzed for their functional properties by measuring the flash-induced pattern of thermoluminescence, oxygen yield, and fluorescence quantum yield. A physiological and biochemical characterization of these mutant strains has been given in two previous reports [Eaton-Rye, J.J., & Vermaas, W.F.J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J.J., Renger, G., & Vermaas, S. F.J. (1993) Biochemistry 32, 4444-4454]. The results of the present study show that deletion of charged and conserved amino acids in a region roughly located between residues 370 and 390 decreases the binding affinity of the extrinsic PS II-O protein to photosystem II. Marked differences with PSII-O deletion mutants are observed with respect to Ca2+ requirement and the flash-induced pattern of oxygen evolution. Under conditions where a sufficient light activation is provided, the psbB mutants assayed in this study reveal normal S-state parameters and lifetimes. The results bear two basic implications: (i) the manganese involved in water oxidation can still be bound in a functionally normal or only slightly distorted manner, and (ii) the binding of the extrinsic PS II-O protein to photosystem II is impaired in mutants carrying a deletion in the domain between residues 370 and 390, but the presence of the PS II-O protein is still of functional relevance for the PS II complex, e.g., for maintenance of a high-affinity binding site for Ca2+ and/or involvement during the process of photoactivation.

Identification of Proteus mirabilis Mutants with Increased Sensitivity to Antimicrobial Peptides

PubMed Central

McCoy, Andrea J.; Liu, Hongjian; Falla, Timothy J.; Gunn, John S.

2001-01-01

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility. PMID:11408219

Secondary electron emission yield from high aspect ratio carbon velvet surfaces

NASA Astrophysics Data System (ADS)

Jin, Chenggang; Ottaviano, Angelica; Raitses, Yevgeny

2017-11-01

The plasma electrons bombarding a plasma-facing wall surface can induce secondary electron emission (SEE) from the wall. A strong SEE can enhance the power losses by reducing the wall sheath potential and thereby increasing the electron flux from the plasma to the wall. The use of the materials with surface roughness and the engineered materials with surface architecture is known to reduce the effective SEE by trapping the secondary electrons. In this work, we demonstrate a 65% reduction of SEE yield using a velvet material consisting of high aspect ratio carbon fibers. The measurements of SEE yield for different velvet samples using the electron beam in vacuum demonstrate the dependence of the SEE yield on the fiber length and the packing density, which is strongly affected by the alignment of long velvet fibers with respect to the electron beam impinging on the velvet sample. The results of SEE measurements support the previous observations of the reduced SEE measured in Hall thrusters.

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Donnini, Claudia

2005-05-01

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Development of High Yield Feedstocks and Biomass Conversion Technology for Renewable Energy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Andrew G.; Crow, Susan; DeBeryshe, Barbara

2015-04-09

This project had two main goals. The first goal was to evaluate several high yielding tropical perennial grasses as feedstock for biofuel production, and to characterize the feedstock for compatible biofuel production systems. The second goal was to assess the integration of renewable energy systems for Hawaii. The project focused on high-yield grasses (napiergrass, energycane, sweet sorghum, and sugarcane). Field plots were established to evaluate the effects of elevation (30, 300 and 900 meters above sea level) and irrigation (50%, 75% and 100% of sugarcane plantation practice) on energy crop yields and input. The test plots were extensive monitored including:more » hydrologic studies to measure crop water use and losses through seepage and evapotranspiration; changes in soil carbon stock; greenhouse gas flux (CO 2, CH 4, and N 2O) from the soil surface; and root morphology, biomass, and turnover. Results showed significant effects of environment on crop yields. In general, crop yields decrease as the elevation increased, being more pronounced for sweet sorghum and energycane than napiergrass. Also energy crop yields were higher with increased irrigation levels, being most pronounced with energycane and less so with sweet sorghum. Daylight length greatly affected sweet sorghum growth and yields. One of the energy crops (napiergrass) was harvested at different ages (2, 4, 6, and 8 months) to assess the changes in feedstock characteristics with age and potential to generate co-products. Although there was greater potential for co-products from younger feedstock, the increased production was not sufficient to offset the additional cost of harvesting multiple times per year. The feedstocks were also characterized to assess their compatibility with biochemical and thermochemical conversion processes. The project objectives are being continued through additional support from the Office of Naval Research, and the Biomass Research and Development Initiative. Renewable

Impaired phloem loading in zmsweet13a,b,c sucrose transporter triple knock-out mutants in Zea mays.

PubMed

Bezrutczyk, Margaret; Hartwig, Thomas; Horschman, Marc; Char, Si Nian; Yang, Jinliang; Yang, Bing; Frommer, Wolf B; Sosso, Davide

2018-04-01

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H + symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. Here we analysed the contribution of SWEETs to phloem loading in maize. We identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. ZmSWEET13 paralogues (a, b, c) are among the most highly expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants were severely stunted. Photosynthesis of mutants was impaired and leaves accumulated high levels of soluble sugars and starch. RNA-seq revealed profound transcriptional deregulation of genes associated with photosynthesis and carbohydrate metabolism. Genome-wide association study (GWAS) analyses may indicate that variability in ZmSWEET13s correlates with agronomical traits, especifically flowering time and leaf angle. This work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Z. mays. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

High Photoluminescence Quantum Yield in Band Gap Tunable Bromide Containing Mixed Halide Perovskites

DOE PAGES

Sutter-Fella, Carolin M.; Li, Yanbo; Amani, Matin; ...

2015-12-21

Hybrid organic-inorganic halide perovskite based semiconductor materials are attractive for use in a wide range of optoelectronic devices because they combine the advantages of suitable optoelectronic attributes and simultaneously low-cost solution processability. Here, we present a two-step low-pressure vapor-assisted solution process to grow high quality homogeneous CH 3NH 3PbI 3-xBr x perovskite films over the full band gap range of 1.6-2.3 eV. Photoluminescence light-in versus light-out characterization techniques are used to provide new insights into the optoelectronic properties of Br-containing hybrid organic-inorganic perovskites as a function of optical carrier injection by employing pump-powers over a 6 orders of magnitude dynamicmore » range. The internal luminescence quantum yield of wide band gap perovskites reaches impressive values up to 30%. This high quantum yield translates into substantial quasi-Fermi level splitting and high "luminescence or optically implied" open-circuit voltage. Most importantly, both attributes, high internal quantum yield and high optically implied open-circuit voltage, are demonstrated over the entire band gap range (1.6 eV ≤ E g ≤ 2.3 eV). These results establish the versatility of Br-containing perovskite semiconductors for a variety of applications and especially for the use as high-quality top cell in tandem photovoltaic devices in combination with industry dominant Si bottom cells. (Figure Presented).« less

Trade-offs between high yields and greenhouse gas emissions in irrigation wheat cropland in China

NASA Astrophysics Data System (ADS)

Cui, Z. L.; Wu, L.; Ye, Y. L.; Ma, W. Q.; Chen, X. P.; Zhang, F. S.

2014-04-01

Although the concept of producing higher yields with reduced greenhouse gas (GHG) emissions is a goal that attracts increasing public and scientific attention, the trade-off between high yields and GHG emissions in intensive agricultural production is not well understood. Here, we hypothesize that there exists a mechanistic relationship between wheat grain yield and GHG emission, and that could be transformed into better agronomic management. A total 33 sites of on-farm experiments were investigated to evaluate the relationship between grain yield and GHG emissions using two systems (conventional practice, CP; high-yielding systems, HY) of intensive winter wheat (Triticum aestivum L.) in China. Furthermore, we discussed the potential to produce higher yields with lower GHG emissions based on a survey of 2938 farmers. Compared to the CP system, grain yield was 39% (2352 kg ha-1) higher in the HY system, while GHG emissions increased by only 10%, and GHG emission intensity was reduced by 21%. The current intensive winter wheat system with farmers' practice had a median yield and maximum GHG emission rate of 6050 kg ha-1 and 4783 kg CO2 eq ha-1, respectively; however, this system can be transformed to maintain yields while reducing GHG emissions by 26% (6077 kg ha-1, and 3555 kg CO2 eq ha-1). Further, the HY system was found to increase grain yield by 39% with a simultaneous reduction in GHG emissions by 18% (8429 kg ha-1, and 3905 kg CO2 eq ha-1, respectively). In the future, we suggest moving the trade-off relationships and calculations from grain yield and GHG emissions to new measures of productivity and environmental protection using innovative management technologies.

EMMA—mouse mutant resources for the international scientific community

PubMed Central

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

Variable sensitivity of US maize yield to high temperatures across developmental stages

NASA Astrophysics Data System (ADS)

Butler, E. E.; Huybers, P. J.

2013-12-01

The sensitivity of maize to high temperatures has been widely demonstrated. Furthermore, field work has indicated that reproductive development stages are particularly sensitive to stress, but this relationship has not been quantified across a wide geographic region. Here, the relationship between maize yield and temperature variations is examined as a function of developmental stage. US state-level data from the National Agriculture Statistics Service provide dates for six growing stages: planting, silking, doughing, dented, mature, and harvested. Temperatures that correspond to each developmental stage are then inferred from a network of weather station observations interpolated to the county level, and a multiple linear regression technique is employed to estimate the sensitivity of county yield outcomes to variations in growing-degree days and an analogous measure of high temperatures referred to as killing-degree days. Uncertainties in the transition times between county-level growth stages are accounted for. Results indicate that the silking and dented stages are generally the most sensitive to killing degree days, with silking the most sensitive stage in the US South and dented the most sensitive in the US North. These variable patterns of sensitivity aid in interpreting which weather events are of greatest significance to maize yields and provide some insight into how shifts in planting time or changes in developmental timing would influence the risks associated with exposure to high temperatures.

Facile high-yield synthesis of polyaniline nanosticks with intrinsic stability and electrical conductivity.

PubMed

Li, Xin-Gui; Li, Ang; Huang, Mei-Rong

2008-01-01

Chemical oxidative polymerization at 15 degrees C was used for the simple and productive synthesis of polyaniline (PAN) nanosticks. The effect of polymerization media on the yield, size, stability, and electrical conductivity of the PAN nanosticks was studied by changing the concentration and nature of the acid medium and oxidant and by introducing organic solvent. Molecular and supramolecular structure, size, and size distribution of the PAN nanosticks were characterized by UV/Vis and IR spectroscopy, X-ray diffraction, laser particle-size analysis, and transmission electron microscopy. Introduction of organic solvent is advantageous for enhancing the yield of PAN nanosticks but disadvantageous for formation of PAN nanosticks with small size and high conductivity. The concentration and nature of the acid medium have a major influence on the polymerization yield and conductivity of the nanosized PAN. The average diameter and length of PAN nanosticks produced with 2 M HNO(3) and 0.5 M H(2)SO(4) as acid media are about 40 and 300 nm, respectively. The PAN nanosticks obtained in an optimal medium (i.e., 2 M HNO(3)) exhibit the highest conductivity of 2.23 S cm(-1) and the highest yield of 80.7 %. A mechanism of formation of nanosticks instead of nanoparticles is proposed. Nanocomposite films of the PAN nanosticks with poly(vinyl alcohol) show a low percolation threshold of 0.2 wt %, at which the film retains almost the same transparency and strength as pure poly(vinyl alcohol) but 262 000 times the conductivity of pure poly(vinyl alcohol) film. The present synthesis of PAN nanosticks requires no external stabilizer and provides a facile and direct route for fabrication of PAN nanosticks with high yield, controllable size, intrinsic self-stability, strong redispersibility, high purity, and optimizable conductivity.

The Mysterious Rescue of adg1-1/tpt-2 – an Arabidopsis thaliana Double Mutant Impaired in Acclimation to High Light – by Exogenously Supplied Sugars

PubMed Central

Heinrichs, Luisa; Schmitz, Jessica; Flügge, Ulf-Ingo; Häusler, Rainer E.

2012-01-01

An Arabidopsis thaliana double mutant (adg1-1/tpt-2) defective in the day- and night-path of photoassimilate export from the chloroplast due to a knockout in the triose phosphate/phosphate translocator (TPT; tpt-2) and a lack of starch [mutation in ADP glucose pyrophosphorylase (AGPase); adg1-1] exhibits severe growth retardation, a decrease in the photosynthetic capacity, and a high chlorophyll fluorescence (HCF) phenotype under high light conditions. These phenotypes could be rescued when the plants were grown on sucrose (Suc) or glucose (Glc). Here we address the question whether Glc-sensing hexokinase1 (HXK1) defective in the Glc insensitive 2 (gin2-1) mutant is involved in the sugar-dependent rescue of adg1-1/tpt-2. Triple mutants defective in the TPT, AGPase, and HXK1 (adg1-1/tpt-2/gin2-1) were established as homozygous lines and grown together with Col-0 and Landsberg erecta (Ler) wild-type plants, gin2-1, the adg1-1/tpt-2 double mutant, and the adg1-1/tpt-2/gpt2-1 triple mutant [additionally defective in the glucose 6-phosphate/phosphate translocator 2 (GPT2)] on agar in the presence or absence of 50 mM of each Glc, Suc, or fructose (Fru). The growth phenotype of the double mutant and both triple mutants could be rescued to a similar extent only by Glc and Suc, but not by Fru. All three sugars were capable of rescuing the HCF and photosynthesis phenotype, irrespectively of the presence or absence of HXK1. Quantitative RT-PCR analyses of sugar-responsive genes revealed that plastidial HXK (pHXK) was up-regulated in adg1-1/tpt-2 plants grown on sugars, but showed no response in adg1-1/tpt-2/gin2-1. It appears likely that soluble sugars are directly taken up by the chloroplasts and enter further metabolism, which consumes ATP and NADPH from the photosynthetic light reaction and thereby rescues the photosynthesis phenotype of the double mutant. The implication of sugar turnover and probably signaling inside the chloroplasts for the concept of retrograde

Phenotypic mutant library: potential for gene discovery

USDA-ARS?s Scientific Manuscript database

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

Alterations in Topoisomerase IV and DNA Gyrase in Quinolone-Resistant Mutants of Mycoplasma hominis Obtained In Vitro

PubMed Central

Bébéar, Cécile M.; Renaudin, Hélène; Charron, Alain; Bové, Joseph M.; Bébéar, Christiane; Renaudin, Joel

1998-01-01

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426→Asn substitution in ParE. GyrA changes (Ser83→Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83→Leu or Ser84→Trp) were detected in the first-step mutants prior to ParC changes (Glu84→Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParC (Ser80→Ile), or ParC (Ser80→Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin. PMID:9736554

High-Yield Production of Levulinic Acid from Pretreated Cow Dung in Dilute Acid Aqueous Solution.

PubMed

Su, Jialei; Shen, Feng; Qiu, Mo; Qi, Xinhua

2017-02-14

Agricultural waste cow dung was used as feedstock for the production of a high value-added chemical levulinic acid (LA) in dilute acid aqueous solutions. A high LA yield of 338.9 g/kg was obtained from the pretreated cow dung, which was much higher than that obtained from the crude cow dung (135 g/kg), mainly attributed to the breakage of the lignin fraction in the lignocellulose structure of the cow dung by potassium hydroxide (KOH) pretreatment, and thus enhanced the accessibility of cow dung to the acid sites in the catalytic reaction. Meanwhile, another value-added chemical formic acid could be obtained with a yield of ca. 160 g/kg in the process, implying a total production of ca. 500 g/kg yield for LA and formic acid from the pretreated cow dung with the proposed process. The developed process was shown to be tolerant to high initial substrate loading with a satisfied LA yield. This work provides a promising strategy for the value-increment utilization of liglocellulosic agricultural residues.

Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

PubMed

Suzuki, Sho W; Onodera, Jun; Ohsumi, Yoshinori

2011-02-25

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase

PubMed Central

Franke, Kamila B.; Bukau, Bernd; Mogk, Axel

2017-01-01

The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings. PMID:28275610

Wafer scale fabrication of carbon nanotube thin film transistors with high yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Boyuan; Liang, Xuelei, E-mail: liangxl@pku.edu.cn, E-mail: ssxie@iphy.ac.cn; Yan, Qiuping

Carbon nanotube thin film transistors (CNT-TFTs) are promising candidates for future high performance and low cost macro-electronics. However, most of the reported CNT-TFTs are fabricated in small quantities on a relatively small size substrate. The yield of large scale fabrication and the performance uniformity of devices on large size substrates should be improved before the CNT-TFTs reach real products. In this paper, 25 200 devices, with various geometries (channel width and channel length), were fabricated on 4-in. size ridged and flexible substrates. Almost 100% device yield were obtained on a rigid substrate with high out-put current (>8 μA/μm), high on/off current ratiomore » (>10{sup 5}), and high mobility (>30 cm{sup 2}/V·s). More importantly, uniform performance in 4-in. area was achieved, and the fabrication process can be scaled up. The results give us more confidence for the real application of the CNT-TFT technology in the near future.« less

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities

PubMed Central

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

2016-01-01

Summary The RAS/MAPK-signalling pathway is frequently deregulated in non-small cell lung cancer (NSCLC), often through KRAS activating mutations1-3. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations4-7. We recently showed that advanced lung tumours from KrasG12D/+;p53-null mice frequently exhibit KrasG12D allelic enrichment (KrasG12D/Kraswild-type>1)7, implying that mutant Kras copy gains are positively selected during progression. Through a comprehensive analysis of mutant Kras homozygous and heterozygous MEFs and lung cancer cells we now show that these genotypes are phenotypically distinct. In particular, KrasG12D/G12D cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the TCA cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous NSCLC cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of KrasG12D copy gain), but not in the corresponding early tumours (KrasG12D heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprised of two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated based on their relative mutant allelic content. We also provide the first in vivo evidence of metabolic rewiring during lung cancer malignant progression. PMID:26909577

Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy

PubMed Central

Dai, Xin; Jiang, Ying; Tan, Chalet

2015-01-01

KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors. PMID:25946136

Mutant fatty acid desaturase

DOEpatents

Shanklin, John; Cahoon, Edgar B.

2004-02-03

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

Characterization and Complementation of a Chlorophyll-Less Dominant Mutant GL1 in Lagerstroemia indica.

PubMed

Wang, Shu'an; Wang, Peng; Gao, Lulu; Yang, Rutong; Li, Linfang; Zhang, Enliang; Wang, Qing; Li, Ya; Yin, Zengfang

2017-05-01

Crape myrtle (Lagerstroemia indica) is a woody ornamental plant popularly grown because of its long-lasting, midsummer blooms and beautiful colors. The GL1 dominant mutant is the first chlorophyll-less mutant identified in crape myrtle. It was obtained from a natural yellow leaf bud mutation. We previously revealed that leaf color of the GL1 mutant is affected by light intensity. However, the mechanism of the GL1 mutant on light response remained unclear. The acclimation response of mutant and wild-type (WT) plants was assessed in a time series after transferring from low light (LL) to high light (HL) by analyzing chlorophyll synthesis precursor content, photosynthetic performance, and gene expression. In LL conditions, coproporphyrinogen III (Coprogen III) content had the greatest amount of accumulation in the mutant compared with WT, increasing by 100%. This suggested that the yellow leaf phenotype of the GL1 dominant mutant might be caused by disruption of coproporphyrinogen III oxidase (CPO) biosynthesis. Furthermore, the candidate gene, oxygen-independent CPO (HEMN), might only affect expression of upstream genes involved in chlorophyll metabolism in the mutant. Moreover, two genes, photosystem II (PSII) 10 kDa protein (psbR) and chlorophyll a/b binding protein gene (CAB1), had decreased mRNA levels in the GL1 mutant within the first 96 h following LL/HL transfer compared with the WT. Hierarchical clustering revealed that these two genes shared a similar expression trend as the oxygen-dependent CPO (HEMF). These findings provide evidence that GL1 is highly coordinated with PSII stability and chloroplast biogenesis.

Chaperone-mediated autophagy degrades mutant p53

PubMed Central

Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-guang; Iwanicki, Marcin P.; Ofengeim, Dimitry; Coloff, Jonathan L.; Pan, Lifeng; Ince, Tan A.; Kroemer, Guido; Brugge, Joan S.; Yuan, Junying

2013-01-01

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924

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

PubMed

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

2016-10-01

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

Effect of high oleic acid soybean on seed oil, protein concentration, and yield

USDA-ARS?s Scientific Manuscript database

Soybeans with high oleic acid content are desired by oil processors because of their improved oxidative stability for broader use in food, fuel and other products. However, non-GMO high-oleic soybeans have tended to have low seed yield. The objective of this study was to test non-GMO, high-oleic s...

High-yield production of herbicidal thaxtomins and analogs in a nonpathogenic Streptomyces strain.

PubMed

Jiang, Guangde; Zhang, Yucheng; Powell, Magan M; Zhang, Peilan; Zuo, Ran; Zhang, Yi; Kallifidas, Dimitrios; Tieu, Albert M; Luesch, Hendrik; Loria, Rosemary; Ding, Yousong

2018-03-30

Thaxtomins are virulence factors of most plant pathogenic Streptomyces strains. Due to their potent herbicidal activity, attractive environmental compatibility and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the United States Environmental Protection Agency. However, the low yield of thaxtomins in native Streptomyces producers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster from S. scabiei 87.22 was cloned and expressed in S. albus J1074 after chromosomal integration. The production of thaxtomins and nitro-tryptophan analogs were observed using LC-MS analysis. When culturing the engineered S. albus J1074 in the minimal medium TMDc, the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than S. scabiei 87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/L. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system in S. albus J1074 was capitalized to produce one unnatural fluorinated analog 5-F-thaxtomin A, whose structure was elucidated by a combination of MS and 1D and 2D NMR analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated that S. albus J1074 has the potential to produce thaxtomins and thereof with high yield, fostering their agricultural applications. IMPORTANCE Thaxtomins are agriculturally valuable herbicidal natural products but the productivity of native producers is limiting. Heterologous expression of thaxtomin gene cluster in S. albus J1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap

Characterization of Novel Materials with Very Low Secondary Electron Emission Yield for Use in High-Power Microwave Devices

NASA Astrophysics Data System (ADS)

Svimonishvili, Tengiz; Zameroski, Nathan; Gilmore, Mark; Schamiloglu, Edl; Gaudet, John; Yan, Lincan

2004-11-01

Secondary Electron Emission (SEE) results from bombarding materials with electrons, atoms, or ions. The amount of secondary emission depends on factors such as bulk and surface properties of materials, energy of incident particles, and their angle of incidence. Total secondary electron emission yield, defined as the number of secondary electrons ejected per primary electron, is an important material parameter. Materials with high yield find use, for instance, in photomultiplier tubes, whereas materials with low yield, such as graphite, are used for SEE suppression in high-power microwave devices. The lower the SEE yield, the better the performance of high-power microwave devices (for example, gyrotrons). Employing a low-energy electron gun (energy range from 5 eV to 2000 eV), our work aims at characterizing and eventually identifying novel materials (with the lowest possible SEE yield) that will enhance operation and efficiency of high-power microwave devices.

Microbial electrolysis cells for high yield hydrogen gas production from organic matter.

PubMed

Logan, Bruce E; Call, Douglas; Cheng, Shaoan; Hamelers, Hubertus V M; Sleutels, Tom H J A; Jeremiasse, Adriaan W; Rozendal, René A

2008-12-01

The use of electrochemically active bacteria to break down organic matter, combined with the addition of a small voltage (> 0.2 V in practice) in specially designed microbial electrolysis cells (MECs), can result in a high yield of hydrogen gas. While microbial electrolysis was invented only a few years ago, rapid developments have led to hydrogen yields approaching 100%, energy yields based on electrical energy input many times greater than that possible by water electrolysis, and increased gas production rates. MECs used to make hydrogen gas are similar in design to microbial fuel cells (MFCs) that produce electricity, but there are important differences in architecture and analytical methods used to evaluate performance. We review here the materials, architectures, performance, and energy efficiencies of these MEC systems that show promise as a method for renewable and sustainable energy production, and wastewater treatment.

Mutant E. coli strain with increased succinic acid production

DOEpatents

Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

1998-01-01

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Mutant E. coli strain with increased succinic acid production

DOEpatents

Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

2001-09-25

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Mutant E. coli strain with increased succinic acid production

DOEpatents

Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

2002-01-01

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans

PubMed Central

2012-01-01

Background Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid. A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. Results Polar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. Conclusions In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial

In Vitro Capability of Faropenem To Select for Resistant Mutants of Streptococcus pneumoniae and Haemophilus influenzae▿ †

PubMed Central

Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C.

2008-01-01

When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones. PMID:18086853

Sputtering yields of carbon based materials under high particle flux with low energy

NASA Astrophysics Data System (ADS)

Nakamura, K.; Nagase, A.; Dairaku, M.; Akiba, M.; Araki, M.; Okumura, Y.

1995-04-01

A new ion source which can produce high particle flux beams at low energies has been developed. This paper presents preliminary results on the sputtering yield of the carbon fiber reinforced composites (CFCs) measured with the new ion source. The sputtering yields of 1D and 2D CFCs, which are candidate materials for the divertor armour tiles, have been measured by the weight loss method under the hydrogen and deuterium particle fluxes of 2 ˜ 7 × 10 20/m 2 s at 50 ˜ 150 eV. Preferential sputtering of the matrix was observed on CFCs which included the matrix of 40 ˜ 60 w%. The energy dependence of the sputtering yields was weak. The sputtering yields of CFCs normally irradiated with deuterium beam were from 0.073 to 0.095, and were around three times larger than those with hydrogen beam.

Improved Properties of Baker's Yeast Mutants Resistant to 2-Deoxy-d-Glucose

PubMed Central

Rincón, Ana M.; Codón, Antonio C.; Castrejón, Francisco; Benítez, Tahía

2001-01-01

We isolated spontaneous mutants from Saccharomyces cerevisiae (baker's yeast V1) that were resistant to 2-deoxy-d-glucose and had improved fermentative capacity on sweet doughs. Three mutants could grow at the same rate as the wild type in minimal SD medium (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) and had stable elevated levels of maltase and/or invertase under repression conditions but lower levels in maltose-supplemented media. Two of the mutants also had high levels of phosphatase active on 2-deoxy-d-glucose-6-phosphate. Dough fermentation (CO2 liberation) by two of the mutants was faster and/or produced higher final volumes than that by the wild type, both under laboratory and industrial conditions, when the doughs were supplemented with glucose or sucrose. However, the three mutants were slower when fermenting plain doughs. Fermented sweet bakery products obtained with these mutants were of better quality than those produced by the wild type, with regard to their texture and their organoleptic properties. PMID:11526034

Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

PubMed

van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

2015-03-01

We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

Problem-Solving Test: Tryptophan Operon Mutants

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

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

Secondary electron emission yield from high aspect ratio carbon velvet surfaces

DOE PAGES

Jin, Chenggang; Ottaviano, Angelica; Raitses, Yevgeny

2017-11-01

The plasma electrons bombarding a plasma-facing wall surface can induce secondary electron emission (SEE) from the wall. A strong SEE can enhance the power losses by reducing the wall sheath potential and thereby increasing the electron flux from the plasma to the wall. The use of the materials with surface roughness and the engineered materials with surface architecture is known to reduce the effective SEE by trapping the secondary electrons. In this work, we demonstrate a 65% reduction of SEE yield using a velvet material consisting of high aspect ratio carbon fibers. The measurements of SEE yield for different velvetmore » samples using the electron beam in vacuum demonstrate the dependence of the SEE yield on the fiber length and the packing density, which is strongly affected by the alignment of long velvet fibers with respect to the electron beam impinging on the velvet sample. Furthermore, the results of SEE measurements support the previous observations of the reduced SEE measured in Hall thrusters.« less

A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

PubMed Central

Tang, Xiaohu; Seyb, Kathleen I.; Huang, Mickey; Schuman, Eli R.; Shi, Ping; Zhu, Haining; Glicksman, Marcie A.

2013-01-01

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells. PMID:22140121

Use of a highly transparent zebrafish mutant for investigations in the development of the vertebrate auditory system (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wisniowiecki, Anna M.; Mattison, Scott P.; Kim, Sangmin; Riley, Bruce; Applegate, Brian E.

2016-03-01

Zebrafish, an auditory specialist among fish, offer analogous auditory structures to vertebrates and is a model for hearing and deafness in vertebrates, including humans. Nevertheless, many questions remain on the basic mechanics of the auditory pathway. Phase-sensitive Optical Coherence Tomography has been proven as valuable technique for functional vibrometric measurements in the murine ear. Such measurements are key to building a complete understanding of auditory mechanics. The application of such techniques in the zebrafish is impeded by the high level of pigmentation, which develops superior to the transverse plane and envelops the auditory system superficially. A zebrafish double mutant for nacre and roy (mitfa-/- ;roya-/- [casper]), which exhibits defects for neural-crest derived melanocytes and iridophores, at all stages of development, is pursued to improve image quality and sensitivity for functional imaging. So far our investigations with the casper mutants have enabled the identification of the specialized hearing organs, fluid-filled canal connecting the ears, and sub-structures of the semicircular canals. In our previous work with wild-type zebrafish, we were only able to identify and observe stimulated vibration of the largest structures, specifically the anterior swim bladder and tripus ossicle, even among small, larval specimen, with fully developed inner ears. In conclusion, this genetic mutant will enable the study of the dynamics of the zebrafish ear from the early larval stages all the way into adulthood.

Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment.

PubMed

Yu, Wei; Yang, Fan; Yang, Jinghui; Ma, Lei; Cun, Yina; Song, Shaohui; Liao, Guoyang

2016-11-01

Usage of influenza vaccine is the best choice measure for preventing and conclusion of influenza virus infection. Although it has been used of chicken embryo to produce influenza vaccine, following with WHO recommended vaccine strain, there were uncontrollable factors and its deficiencies, specially, during an influenza pandemic in the world. The Vero cells are used for vaccine production of a few strains including influenza virus, because of its homology with human, recommended by WHO. However, as known most of the influenza viruses strains could not culture by Vero cells. It was used two high-yield influenza viruses adapted in Vero cells as donor viruses, such as A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B), to construct high-yield wild influenza virus in Vero cells under antibody selection pressure. After reassortment and passages, it obtained the new Vaccine strains with A/Tianjin/15/2009Va (H1N1), A/Fujian/196/2009Va (H3N2) and B/Chongqing/1384/2010Va (B), which was not only completely keeping their original antigenic (HA and NA), but also grown well in Vero cells with high-yield. All results of gene analysis and HA, HI shown that this reassortment method could be used to find new direction to product the influenza vaccine. J. Med. Virol. 88:1914-1921, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Effect of space flight on yield of Monascus purpureus].

PubMed

Yin, Hong; Xie, Shen-yi; Zhang, Guang-ming; Xie, Shen-meng

2003-10-01

To select high Lovastatin-producing microbial breed by space flight. Monascus purpureus species was carried into space by the recoverable spaceship, "Shenzhou 3". After flight, the strain was rejuvenized, segregated and selected. The content of Lovastatin produced in the solid fermentation was examined. Mutants with high productivity of Lovastatin were obtained. A series of tests showed that the acquired character of the mutants was stable. Space flight is an effective method for the selection of fine strains.

Novel functional Renilla luciferase mutant provides long-term serum stability and high luminescence activity.

PubMed

Song, Woo Chul; Sung, Hye-Jin; Park, Kyung Soo; Choi, Jeong-Woo; Cho, Je-Yeol; Um, Soong Ho

2013-10-01

Fluorescent and luminescent chemical probes are essential in recent medical diagnostics. However, the use of these probes in vivo has raised concerns due to their low sensitivity, background signal interference, and non-biocompatibility. Therefore, biological chromophores have received much attention as new alternatives. In particular, luciferase, a class of oxidative enzyme with bioluminescence, has emerged as a promising fluorophore due to its improved biocompatibility. However, the enzyme usually possesses weaker luminescence and stability relative to its chemically-based competitors. Here, we report a novel functional mutant luciferase with both enhanced luminescence and long-term serum stability. For the preparation of the modified Renilla luciferase, a new bacterial subcloning design was established. The luciferase coding DNA sequence was redesigned so that mutant luciferase could be easily expressed in an Escherichia coli system. The mutant Renilla luciferase, which we called "m-Rluc," demonstrated characteristic enzymatic functions and showed a 5.6-fold increase in luminescence activity. In addition, the enzyme's physiological stability remained >80% for more than 5days, in contrast to conventional luciferase, termed "hrluc," which disappeared within a few hours. We suggest that this novel biological luciferase probe may be a great tool for both in vitro and in vivo medical diagnostics. Copyright © 2013 Elsevier Inc. All rights reserved.

Limitations to photosynthesis under light and heat stress in three high-yielding wheat genotypes.

PubMed

Monneveux, Philippe; Pastenes, Claudio; Reynolds, Matthew P

2003-06-01

Three high-yielding wheat genotypes (T. aestivum L., c.v. Siete Cerros, Seri and Bacanora, released in 1966, 1982 and 1988, respectively) were grown under irrigation in two high radiation, low relative humidity environments (Tlaltizapan and Ciudad Obregon CIMMYT experimental stations, Mexico). Gas exchange and fluorescence parameters were assessed on the flag leaf during the day. Carbon isotope discrimination (delta) was analysed in flag leaf at anthesis and in grain at maturity. In both environments, gas exchange and fluorescence parameters varied markedly with irradiance and temperature. Analysis of their respective variation indicated the occurrence of photo-respiration and photo-inhibition, particularly in Tlaltizapan, the warmest environment, and in Siete Cerros. In Ciudad Obregon (high-yielding environment) lower Ci (internal CO2 concentration) and delta La (carbon isotope discrimination of the leaf) suggested a higher intrinsic photosynthetic capacity in the variety Bacanora. Higher yield of this genotype was also associated with higher Fv'/Fo' (ratio of photochemical and non photochemical rate constants in the light) and Fm'/Fm (ratio of the non photochemical rate constants in the dark and light adapted state).

Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

PubMed

Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly

2014-01-01

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

Simulations of super-ellipse hohlraum targets as a path to high neutron yields

NASA Astrophysics Data System (ADS)

Milovich, Jose; Amendt, Peter; Storm, Erik; Robey, Harry; Haan, Steve; Landen, Otto; Meezan, Nathan; Lindl, John

2017-10-01

Recently neutron yields in excess of 1016 have been achieved at the National Ignition Facility (NIF) using a low-density gas fill hohlraum and a subscale high-density-carbon capsule. The laser power used was near the current maximum level allowed on the inner cones of the NIF laser. While more energy can be extracted from the laser to provide additional improvement on the neutron yield, a more efficient design is desired. A new effort has begun to investigate alternatives to the current cylinder-shaped hohlraum for driving larger capsules (1.1 mm outer radius). If these new hohlraums can preserve the implosion symmetry, the additional absorbed energy is expected to provide a path to high neutron yield and potential ignition. Super-ellipse hohlraums, a generalization of an earlier rugby hohlraum design, have the advantage of a larger waist diameter and reduced parasitic energy losses from the corners of cylindrical hohlraums while still being able to produce the required capsule drive at the current energy and power limits available at the NIF. We will present plausible designs of these hohlraums based on the Lamé mathematical construction, and discuss their prospects to reach high neutron gains. Prepared by LLNL under Contract DE-AC52-07NA27344.

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

PubMed

Wilke, Sonja; Krausze, Joern; Gossen, Manfred; Groebe, Lothar; Jäger, Volker; Gherardi, Ermanno; van den Heuvel, Joop; Büssow, Konrad

2010-06-01

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

Regioselective alkane hydroxylation with a mutant AlkB enzyme

DOEpatents

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.

Mutant E. coli strain with increased succinic acid production

DOEpatents

Donnelly, M.; Millard, C.S.; Stols, L.

1998-06-23

A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

Towards a "Golden Standard" for computing globin stability: Stability and structure sensitivity of myoglobin mutants.

PubMed

Kepp, Kasper P

2015-10-01

Fast and accurate computation of protein stability is increasingly important for e.g. protein engineering and protein misfolding diseases, but no consensus methods exist for important proteins such as globins, and performance may depend on the type of structural input given. This paper reports benchmarking of six protein stability calculators (POPMUSIC 2.1, I-Mutant 2.0, I-Mutant 3.0, CUPSAT, SDM, and mCSM) against 134 experimental stability changes for mutations of sperm-whale myoglobin. Six different high-resolution structures were used to test structure sensitivity that may impair protein 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 SDMMutant 2.0Mutant 3.0Mutant 2.0Mutant 3.0Mutant 3.0 (0.05)Mutant 2.0 (0.09)Mutant 2.0 is proficient for this purpose, as further validated against a data set of related cytochrome c like proteins. The results also emphasize the importance of high-quality crystal structures and reveal structure-dependent effects even in the near-atomic resolution limit. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly oil-producing microalgae selected through directed-evolution on a microfludic chip

NASA Astrophysics Data System (ADS)

Mestler, Troy; Estevez-Torres, Andre; Lambert, Guillaume; Austin, Robert H.

2009-03-01

Some species of photosynthetic microalgae produce signi?cant amounts of oil which can be easily converted to diesel fuel. However, as it stands today, biodiesel is signi?cantly more expensive than fossil fuels. We wish to improve the oil yield and production rate of a single species of microalgae through directed evolution. We propose to utilize our microfabication technology to create microhabitats to control the nutrient environment of the species, monitor oil production through Raman Spectroscopy, and punish colonies of algae which have low oil yield. We believe this process will produce a mutant species with a high oil yield.

Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

PubMed

Rhaese, H J; Hoch, J A; Groscurth, R

1977-03-01

To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

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

PubMed Central

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

2013-01-01

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

Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species.

PubMed

Yao, Shuo; Brandt, Anders; Egsgaard, Helge; Gjermansen, Claes

2012-12-01

Triacylglycerols, an energy storage compound in microalgae, are known to be accumulated after nitrogen starvation of microalgae cells. Microalgae could be of importance for future biodiesel production due to their fast growth rate and high oil content. In collections of temperature sensitive mutants of Chlamydomonas reinhardtii and Chlorella vulgaris, nine out of fourty-one mutants in C. reinhardtii and eleven out of fifty-three mutants in C. vulgaris contained increased amounts of neutral lipids, predominantly as triacylglycerols. Upon temperature induced cell-cycle arrest, these mutants showed enlarged cellular volume compared with the wild type. The C. reinhardtii mutants were analyzed further and one type of mutants displayed a shift in lipid composition from polar membrane lipids to neutral lipids after a temperature up-shift, while the second type of mutants accumulated more total lipid per cell, predominantly as neutral lipids as compared with the wild type. Three C. reinhardtii mutants were analyzed further and found to be arrested after DNA synthesis but prior to cell division in the cell cycle. These mutants will be useful in order to further understand neutral lipid accumulation in microalgae and suggest possibilities for biodiesel production by specific induction of lipid accumulation in miroalgal cultures by cell-cycle inhibition. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Misfolded rhodopsin mutants display variable aggregation properties.

PubMed

Gragg, Megan; Park, Paul S-H

2018-06-08

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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

2005-01-28

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

Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.

PubMed

Xu, Li; Liu, Xiaohong; Yin, Zhenhao; Liu, Qian; Lu, Lili; Xiao, Min

2016-12-01

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp 252 , Asp 257 , Asp 264 , Glu 530 , Arg 548 , His 553 , and Trp 555 ) and may form the hydrophobic pocket in stabilizing donor (Trp 261 , Tyr 302 , Tyr 316 , and Trp 369 ) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp 257 . Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

Very high CO2 reduces photosynthesis, dark respiration and yield in wheat

NASA Technical Reports Server (NTRS)

Reuveni, J.; Bugbee, B.

1997-01-01

Although terrestrial CO2 concentrations, [CO2] are not expected to reach 1000 micromoles mol-1 for many decades, CO2 levels in closed systems such as growth chambers and glasshouses, can easily exceed this concentration. CO2 levels in life support systems in space can exceed 10000 micromoles mol-1 (1%). Here we studied the effect of six CO2 concentrations, from ambient up to 10000 micromoles mol-1, on seed yield, growth and gas exchange of two wheat cultivars (USU-Apogee and Veery-l0). Elevating [CO2] from 350 to 1000 micromoles mol-1 increased seed yield (by 33%), vegetative biomass (by 25%) and number of heads m-2 (by 34%) of wheat plants. Elevation of [CO2] from 1000 to 10000 micromoles mol-1 decreased seed yield (by 37%), harvest index (by 14%), mass per seed (by 9%) and number of seeds per head (by 29%). This very high [CO2] had a negligible, non-significant effect on vegetative biomass, number of heads m-2 and seed mass per head. A sharp decrease in seed yield, harvest index and seeds per head occurred by elevating [CO2] from 1000 to 2600 micromoles mol-1. Further elevation of [CO2] from 2600 to 10000 micromoles mol-1 caused a further but smaller decrease. The effect of CO2 on both wheat cultivars was similar for all growth parameters. Similarly there were no differences in the response to high [CO2] between wheat grown hydroponically in growth chambers under fluorescent lights and those grown in soilless media in a glasshouse under sunlight and high pressure sodium lamps. There was no correlation between high [CO2] and ethylene production by flag leaves or by wheat heads. Therefore, the reduction in seed set in wheat plants is not mediated by ethylene. The photosynthetic rate of whole wheat plants was 8% lower and dark respiration of the wheat heads 25% lower when exposed to 2600 micromoles mol-1 CO2 compared to ambient [CO2]. It is concluded that the reduction in the seed set can be mainly explained by the reduction in the dark respiration in wheat heads

Diversity pattern in Sesamum mutants selected for a semi-arid cropping system.

PubMed

Murty, B R; Oropeza, F

1989-02-01

Due to the complex requirements of moisture stress, substantial genetic diversity with a wide array of character combinations and effective simultaneous selection for several variables is necessary for improving the productivity and adaptation of a component crop in order for it to fit into a cropping system under semi-arid tropical conditions. Sesamum indicum L. is grown in Venezuela after rice/sorghum/or maize under such conditions. A mutation breeding program was undertaken using six locally adapted varieties to develop genotypes suitable for the above system. The diversity pattern for nine variables was assessed by multivariate analysis in 301 M4 progenies. Analysis of the characteristic roots and principal components in three methods of selection, i.e., M2 bulks (A), individual plant selection throughout (B), and selection in M3 for single variable (C), revealed differences in the pattern of variation between varieties, selection methods, and varieties x methods interactions. Method B was superior to the others and gave 17 of the 21 best M5 progenies. 'Piritu' and 'CF' varieties yielded the most productive progenies in M5 and M6. Diversity was large and selection was effective for such developmental traits as earliness and synchrony, combined with multiple disease resistance, which could be related to their importance by multivariate analyses. Considerable differences in the variety of character combinations among the high yielding. M5 progenies of 'CF' and 'Piritu' suggested possible further yield improvement. The superior response of 'Piritu' and 'CF' over other varieties in yield and adaptation was due to major changes in plant type and character associations. Multilocation testing of M5 generations revealed that the mutant progenies had a 40%-100% yield superiority over the parents; this was combined with earliness, synchrony, and multiple disease resistance, and was confirmed in the M6 generation grown on a commercial scale. This study showed that

Supportability of a High-Yield-Stress Slurry in a New Stereolithography-Based Ceramic Fabrication Process

NASA Astrophysics Data System (ADS)

He, Li; Song, Xuan

2018-03-01

In recent years, ceramic fabrication using stereolithography (SLA) has gained in popularity because of its high accuracy and density that can be achieved in the final part of production. One of the key challenges in ceramic SLA is that support structures are required for building overhanging features, whereas removing these support structures without damaging the components is difficult. In this research, a suspension-enclosing projection-stereolithography process is developed to overcome this challenge. This process uses a high-yield-stress ceramic slurry as the feedstock material and exploits the elastic force of the material to support overhanging features without the need for building additional support structures. Ceramic slurries with different solid loadings are studied to identify the rheological properties most suitable for supporting overhanging features. An analytical model of a double doctor-blade module is established to obtain uniform and thin recoating layers from a high-yield-stress slurry. Several test cases highlight the feasibility of using a high-yield-stress slurry to support overhanging features in SLA.

Crop Yield Predictions - High Resolution Statistical Model for Intra-season Forecasts Applied to Corn in the US

NASA Astrophysics Data System (ADS)

Cai, Y.

2017-12-01

Accurately forecasting crop yields has broad implications for economic trading, food production monitoring, and global food security. However, the variation of environmental variables presents challenges to model yields accurately, especially when the lack of highly accurate measurements creates difficulties in creating models that can succeed across space and time. In 2016, we developed a sequence of machine-learning based models forecasting end-of-season corn yields for the US at both the county and national levels. We combined machine learning algorithms in a hierarchical way, and used an understanding of physiological processes in temporal feature selection, to achieve high precision in our intra-season forecasts, including in very anomalous seasons. During the live run, we predicted the national corn yield within 1.40% of the final USDA number as early as August. In the backtesting of the 2000-2015 period, our model predicts national yield within 2.69% of the actual yield on average already by mid-August. At the county level, our model predicts 77% of the variation in final yield using data through the beginning of August and improves to 80% by the beginning of October, with the percentage of counties predicted within 10% of the average yield increasing from 68% to 73%. Further, the lowest errors are in the most significant producing regions, resulting in very high precision national-level forecasts. In addition, we identify the changes of important variables throughout the season, specifically early-season land surface temperature, and mid-season land surface temperature and vegetation index. For the 2017 season, we feed 2016 data to the training set, together with additional geospatial data sources, aiming to make the current model even more precise. We will show how our 2017 US corn yield forecasts converges in time, which factors affect the yield the most, as well as present our plans for 2018 model adjustments.

High yield production of extracellular recombinant levansucrase by Bacillus megaterium.

PubMed

Korneli, Claudia; Biedendieck, Rebekka; David, Florian; Jahn, Dieter; Wittmann, Christoph

2013-04-01

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L(-1)) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL(-1) on fructose and 17.2 U mL(-1) on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL(-1), reflecting a levansucrase concentration of 0.52 g L(-1). This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

High-yield production of a stable Vero cell-based vaccine candidate against the highly pathogenic avian influenza virus H5N1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Fangye; Zhou, Jian; Ma, Lei

Highlights: Black-Right-Pointing-Pointer Vero cell-based HPAI H5N1 vaccine with stable high yield. Black-Right-Pointing-Pointer Stable high yield derived from the YNVa H3N2 backbone. Black-Right-Pointing-Pointer H5N1/YNVa has a similar safety and immunogenicity to H5N1delta. -- Abstract: Highly pathogenic avian influenza (HPAI) viruses pose a global pandemic threat, for which rapid large-scale vaccine production technology is critical for prevention and control. Because chickens are highly susceptible to HPAI viruses, the supply of chicken embryos for vaccine production might be depleted during a virus outbreak. Therefore, developing HPAI virus vaccines using other technologies is critical. Meeting vaccine demand using the Vero cell-based fermentation process hasmore » been hindered by low stability and yield. In this study, a Vero cell-based HPAI H5N1 vaccine candidate (H5N1/YNVa) with stable high yield was achieved by reassortment of the Vero-adapted (Va) high growth A/Yunnan/1/2005(H3N2) (YNVa) virus with the A/Anhui/1/2005(H5N1) attenuated influenza vaccine strain (H5N1delta) using the 6/2 method. The reassorted H5N1/YNVa vaccine maintained a high hemagglutination (HA) titer of 1024. Furthermore, H5N1/YNVa displayed low pathogenicity and uniform immunogenicity compared to that of the parent virus.« less

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis.

PubMed

Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J; Xiang, Cheng-Bin

2010-07-01

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a 'sulphur-free' solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance.

Processable high-carbon-yielding polymer for micro- and nanofabrication

NASA Astrophysics Data System (ADS)

Perpall, Mark W.; Zengin, Huseyin; Perera, K. Prasanna U.; Zhou, Wensheng; Shah, Hiren; Wu, Xinyu; Creager, Stephen E.; Smith, Dennis W., Jr.; Foulger, Stephen H.; Ballato, John M.

2003-01-01

Bis-ortho-Diynyl Arene (BODA) monomers polymerize to network polynapthalene by the thermally-driven Bergman cyclization and subsequent radical polymerization via oligomeric intermediates that can be melt or solution processed. Further heating of the network to 1000 °C affords a high-yield glassy carbon structure that retains the approximate size and dimensions of the polymer precursor. The higher carbon-yield for BODA networks (75- 80 % by mass) is significantly greater than that of traditional phenol-formaldehyde resins and other carbon precursor polymers leading to its greater dimensional stability. Phenyl terminated BODA derived polymers were fabricated using microprocessing such as the micromolding in capillaries (MIMIC) technique, direct microtransfer molding, and molding in quartz capillary tubes. Nano-scale fabrication using closed packed silica spheres as templates was demonstrated with an hydroxy-terminated monomer which exhibits greatly enhanced compatibility for silica surfaces. After pyrolysis to glassy carbon, the silica is chemically etched leaving an inverse carbon opal photonic crystal which is electrically conductive. The wavelength of light diffracted is a function of the average refractive index of the carbon/ filler composite, which can be modified for use as sensitive detector elements.

Metaphase to Anaphase (mat) Transition–Defective Mutants inCaenorhabditis elegans

PubMed Central

Golden, Andy; Sadler, Penny L.; Wallenfang, Matthew R.; Schumacher, Jill M.; Hamill, Danielle R.; Bates, Gayle; Bowerman, Bruce; Seydoux, Geraldine; Shakes, Diane C.

2000-01-01

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants “mat” for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog. PMID:11134076

High-Yield Synthesis and Optical Properties of Carbon Nanotube Porins

DOE PAGES

Tunuguntla, Ramya H.; Chen, Xi; Belliveau, Allison; ...

2017-01-18

Carbon nanotube porins (CNTPs) are a convenient membrane-based model system for studying nanofluidic transport that replicates a number of key structural features of biological membrane channels. We present a generalized approach for CNTP synthesis using sonochemistry-assisted segmenting of carbon nanotubes. Prolonged tip sonication in the presence of lipid molecules debundles and fragments long carbon nanotube aggregates into stable and water-soluble individual CNTPs with lengths in the range 5–20 nm. We discuss the main parameters that determine the efficiency and the yield of this process, describe the optimized conditions for high-yield CNTP synthesis, and demonstrate that this methodology can be adaptedmore » for synthesis of CNTPs of different diameters. We also present the optical properties of CNTPs and show that a combination of Raman and UV–vis–NIR spectroscopy can be used to monitor the quality of the CNTP synthesis. Altogether, CNTPs represent a versatile nanopore building block for creating higher-order functional biomimetic materials.« less

High-wafer-yield, high-performance vertical cavity surface-emitting lasers

NASA Astrophysics Data System (ADS)

Li, Gabriel S.; Yuen, Wupen; Lim, Sui F.; Chang-Hasnain, Constance J.

1996-04-01

Vertical cavity surface emitting lasers (VCSELs) with very low threshold current and voltage of 340 (mu) A and 1.5 V is achieved. The molecular beam epitaxially grown wafers are grown with a highly accurate, low cost and versatile pre-growth calibration technique. One- hundred percent VCSEL wafer yield is obtained. Low threshold current is achieved with a native oxide confined structure with excellent current confinement. Single transverse mode with stable, predetermined polarization direction up to 18 times threshold is also achieved, due to stable index guiding provided by the structure. This is the highest value reported to data for VCSELs. We have established that p-contact annealing in these devices is crucial for low voltage operation, contrary to the general belief. Uniform doping in the mirrors also appears not to be inferior to complicated doping engineering. With these design rules, very low threshold voltage VCSELs are achieved with very simple growth and fabrication steps.

Probing the role of highly conserved residues in triosephosphate isomerase--analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme.

PubMed

Bandyopadhyay, Debarati; Murthy, Mathur R N; Balaram, Hemalatha; Balaram, Padmanabhan

2015-10-01

Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodium falciparum triosephosphate isomerase (PfTIM) enzyme (EC 5.3.1.1) as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of Kd values for dimer dissociation (Q64N = 73.7 ± 9.2 nm and Q64E = 44.6 ± 8.4 nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 °C and 45 °C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity. Structural data have been deposited in the Protein Data Bank under accession numbers 4ZZ9, 5BMW, 5BMX, 5BNK and 5BRB. © 2015 FEBS.

A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies

PubMed Central

Oria, Maria P.; Hamaker, Bruce R.; Axtell, John D.; Huang, Chia-Ping

2000-01-01

The endosperm of a sorghum mutant cultivar, with high in vitro uncooked and cooked protein digestibilities, was examined by transmission electron microscopy and α-, β-, and γ-kafirins (storage proteins) were localized within its protein bodies. Transmission electron microscopy micrographs revealed that these protein bodies had a unique microstructure related to high protein digestibility. They were irregular in shape and had numerous invaginations, often reaching to the central area of the protein body. Protein bodies from normal cultivars, such as P721N studied here, with much lower uncooked and cooked digestibilities are spherical and contain no invaginations. Immunocytochemistry results showed that the relative location of α- and β-kafirins within the protein bodies of the highly digestible genotype were similar to the normal cultivar, P721N. γ-Kafirin, however, was concentrated in dark-staining regions at the base of the folds instead of at the protein body periphery, as is typical of normal cultivars. The resulting easy accessibility of digestive enzymes to α-kafirin, the major storage protein, in addition to the increased surface area of the protein bodies of the highly digestible cultivar appear to account for its high in vitro protein digestibility. PMID:10792028

Suppression of mutants aberrant in light intensity responses of complementary chromatic adaptation.

PubMed Central

Casey, E S; Kehoe, D M; Grossman, A R

1997-01-01

Complementary chromatic adaptation is a process in which cyanobacteria alter the pigment protein (phycocyanin and phycoerythrin) composition of their light-harvesting complexes, the phycobilisomes, to help optimize the absorbance of prevalent wavelengths of light in the environment. Several classes of mutants that display aberrant complementary chromatic adaptation have been isolated. One of the mutant classes, designated "blue" or FdB, accumulates high levels of the blue chromoprotein phycocyanin in low-intensity green light, a condition that normally suppresses phycocyanin synthesis. We demonstrate here that the synthesis of the phycocyanin protein and mRNA in the FdB mutants can be suppressed by increasing the intensity of green light. Hence, these mutants have a decreased sensitivity to green light with respect to suppression of phycocyanin synthesis. Although we were unable to complement the blue mutants, we did isolate genes that could suppress the mutant phenotype. These genes, which have been identified previously, encode a histidine kinase sensor and response regulator protein that play key roles in controlling complementary chromatic adaptation. These findings are discussed with respect to the mechanism by which light quality and quantity control the biosynthesis of the phycobilisome. PMID:9226271

Creating high yield water soluble luminescent graphene quantum dots via exfoliating and disintegrating carbon nanotubes and graphite flakes.

PubMed

Lin, Liangxu; Zhang, Shaowei

2012-10-21

We have developed an effective method to exfoliate and disintegrate multi-walled carbon nanotubes and graphite flakes. With this technique, high yield production of luminescent graphene quantum dots with high quantum yield and low oxidization can be achieved.

Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis.

PubMed

Falbel, T G; Meehl, J B; Staehelin, L A

1996-10-01

Analyses of a series of allelic chlorina mutants of wheat (Triticum aestivum L.), which have partial blocks in chlorophyll (Chl) synthesis and, therefore, a limited Chl supply, reinforce the principle that Chl is required for the stable accumulation of Chl-binding proteins and that only reaction centers accumulate when the supply of Chl is severely limited. Depending on the rate of Chl accumulation (determined by the severity of the mutation) and on the rate of turnover of Chl and its precursors (determined by the environment in which the plant is grown), the mutants each reach an equilibrium of Chl synthesis and degradation. Together these mutants generate a spectrum of phenotypes. Under the harshest conditions (high illumination), plants with moderate blocks in Chl synthesis have membranes with very little Chl and Chl-proteins and membrane stacks resembling the thylakoids of the lethal xantha mutants of barely grown at low to medium light intensities (which have more severe blocks). In contrast, when grown under low-light conditions the same plants with moderate blocks have thylakoids resembling those of the wild type. The wide range of phenotypes of Chl b-deficient mutants has historically produced more confusion than enlightenment, but incomparable growth conditions can now explain the discrepancies reported in the literature.

Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis.

PubMed Central

Falbel, T G; Meehl, J B; Staehelin, L A

1996-01-01

Analyses of a series of allelic chlorina mutants of wheat (Triticum aestivum L.), which have partial blocks in chlorophyll (Chl) synthesis and, therefore, a limited Chl supply, reinforce the principle that Chl is required for the stable accumulation of Chl-binding proteins and that only reaction centers accumulate when the supply of Chl is severely limited. Depending on the rate of Chl accumulation (determined by the severity of the mutation) and on the rate of turnover of Chl and its precursors (determined by the environment in which the plant is grown), the mutants each reach an equilibrium of Chl synthesis and degradation. Together these mutants generate a spectrum of phenotypes. Under the harshest conditions (high illumination), plants with moderate blocks in Chl synthesis have membranes with very little Chl and Chl-proteins and membrane stacks resembling the thylakoids of the lethal xantha mutants of barely grown at low to medium light intensities (which have more severe blocks). In contrast, when grown under low-light conditions the same plants with moderate blocks have thylakoids resembling those of the wild type. The wide range of phenotypes of Chl b-deficient mutants has historically produced more confusion than enlightenment, but incomparable growth conditions can now explain the discrepancies reported in the literature. PMID:8883392

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

PubMed

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

2012-11-30

This paper describes the isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel. Following UV mutagenesis, 2000 Chlorella and 2800 Scenedesmus colonies were screened with a method based on Nile Red fluorescence. Several mutants with high Nile Red fluorescence were selected by this high-throughput method in both species. Growth and biomass parameters of the strongest mutants were analyzed in detail. All of the four Chlorella mutants showed no significant changes in growth rate, cell weight, cell size, protein and chlorophyll contents on a per cell basis. Whereas all contained elevated total lipid and TAG content per unit of dry weight, two of them were also affected for starch metabolism, suggesting a change in biomass/storage carbohydrate composition. Two Scenedesmus mutants showed a 1.5 and 2-fold increased cell weight and larger cells compared to the wild type, which led to a general increase of biomass including total lipid and TAG content on a per cell basis. Such mutants could subsequently be used as commercial oleaginous algae and serve as an alternative to conventional petrol. Copyright © 2012 Elsevier B.V. All rights reserved.

Polarity-defective mutants of Aspergillus nidulans.

PubMed

Osherov, N; Mathew, J; May, G S

2000-12-01

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the alpha subunit of yeast mitochondrial phenylalanyl--tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed. Copyright 2000 Academic Press.

Patients with Long-Term Oral Carriage Harbor High-Persister Mutants of Candida albicans▿

PubMed Central

LaFleur, Michael D.; Qi, Qingguo; Lewis, Kim

2010-01-01

Fungal biofilms produce a small number of persister cells which can tolerate high concentrations of fungicidal agents. Persisters form upon attachment to a surface, an important step in the pathogenesis of Candida strains. The periodic application of antimicrobial agents may select for strains with increased levels of persister cells. In order to test this possibility, 150 isolates of Candida albicans and C. glabrata were obtained from cancer patients who were at high risk for the development of oral candidiasis and who had been treated with topical chlorhexidine once a day. Persister levels were measured by exposing biofilms growing in the wells of microtiter plates to high concentrations of amphotericin B and plating for survivors. The persister levels of the isolates varied from 0.2 to 9%, and strains isolated from patients with long-term carriage had high levels of persisters. High-persister strains were isolated from every patient with Candida carriage of more than 8 consecutive weeks but from no patients with transient carriage. All of the high-persister isolates had an amphotericin B MIC that was the same as that for the wild type, indicating that these strains were drug-tolerant rather than drug-resistant mutants. Biofilms of the majority of high-persister strains also showed an increased tolerance to chlorhexidine and had the same MIC for this antimicrobial as the wild type. This study suggests that persister cells are clinically relevant, and antimicrobial therapy selects for high-persister strains in vivo. The drug tolerance of persisters may be a critical but overlooked component responsible for antimicrobial drug failure and relapsing infections. PMID:19841146

Brassinosteroid-Insensitive Dwarf Mutants of Arabidopsis Accumulate Brassinosteroids1

PubMed Central

Noguchi, Takahiro; Fujioka, Shozo; Choe, Sunghwa; Takatsuto, Suguru; Yoshida, Shigeo; Yuan, Heng; Feldmann, Kenneth A.; Tax, Frans E.

1999-01-01

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels. PMID:10557222

The Tennessee Mouse Genome Consortium: Identification of ocular mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jablonski, Monica M.; Wang, Xiaofei; Lu, Lu

2005-06-01

The Tennessee Mouse Genome Consortium (TMGC) is in its fifth year of a ethylnitrosourea (ENU)-based mutagenesis screen to detect recessive mutations that affect the eye and brain. Each pedigree is tested by various phenotyping domains including the eye, neurohistology, behavior, aging, ethanol, drug, social behavior, auditory, and epilepsy domains. The utilization of a highly efficient breeding protocol and coordination of various universities across Tennessee makes it possible for mice with ENU-induced mutations to be evaluated by nine distinct phenotyping domains within this large-scale project known as the TMGC. Our goal is to create mutant lines that model human diseases andmore » disease syndromes and to make the mutant mice available to the scientific research community. Within the eye domain, mice are screened for anterior and posterior segment abnormalities using slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, eye weight, histology, and immunohistochemistry. As of January 2005, we have screened 958 pedigrees and 4800 mice, excluding those used in mapping studies. We have thus far identified seven pedigrees with primary ocular abnormalities. Six of the mutant pedigrees have retinal or subretinal aberrations, while the remaining pedigree presents with an abnormal eye size. Continued characterization of these mutant mice should in most cases lead to the identification of the mutated gene, as well as provide insight into the function of each gene. Mice from each of these pedigrees of mutant mice are available for distribution to researchers for independent study.« less

High-throughput, image-based screening of pooled genetic variant libraries

PubMed Central

Emanuel, George; Moffitt, Jeffrey R.; Zhuang, Xiaowei

2018-01-01

Image-based, high-throughput screening of genetic perturbations will advance both biology and biotechnology. We report a high-throughput screening method that allows diverse genotypes and corresponding phenotypes to be imaged in numerous individual cells. We achieve genotyping by introducing barcoded genetic variants into cells and using massively multiplexed FISH to measure the barcodes. We demonstrated this method by screening mutants of the fluorescent protein YFAST, yielding brighter and more photostable YFAST variants. PMID:29083401

31 CFR 356.21 - How are awards at the high yield or discount rate calculated?

Code of Federal Regulations, 2010 CFR

2010-07-01

... discount rate calculated? 356.21 Section 356.21 Money and Finance: Treasury Regulations Relating to Money... high yield or discount rate calculated? (a) Awards to submitters. We generally prorate bids at the highest accepted yield or discount rate under § 356.20(a)(2) of this part. For example, if 80.15% is the...

Different mutational function of low- and high-linear energy transfer heavy-ion irradiation demonstrated by whole-genome resequencing of Arabidopsis mutants.

PubMed

Kazama, Yusuke; Ishii, Kotaro; Hirano, Tomonari; Wakana, Taeko; Yamada, Mieko; Ohbu, Sumie; Abe, Tomoko

2017-12-01

Heavy-ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Several studies have indicated that the value of LET affects DNA lesion formation in several ways, including the efficiency and the density of double-stranded break induction along the particle path. We assumed that the mutation type can be altered by selecting an appropriate LET value. Here, we quantitatively demonstrate differences in the mutation type induced by irradiation with two representative ions, Ar ions (LET: 290 keV μm -1 ) and C ions (LET: 30.0 keV μm -1 ), by whole-genome resequencing of the Arabidopsis mutants produced by these irradiations. Ar ions caused chromosomal rearrangements or large deletions (≥100 bp) more frequently than C ions, with 10.2 and 2.3 per mutant genome under Ar- and C-ion irradiation, respectively. Conversely, C ions induced more single-base substitutions and small indels (<100 bp) than Ar ions, with 28.1 and 56.9 per mutant genome under Ar- and C-ion irradiation, respectively. Moreover, the rearrangements induced by Ar-ion irradiation were more complex than those induced by C-ion irradiation, and tended to accompany single base substitutions or small indels located close by. In conjunction with the detection of causative genes through high-throughput sequencing, selective irradiation by beams with different effects will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Enhanced electron yield from laser-driven wakefield acceleration in high-Z gas jets.

PubMed

Mirzaie, Mohammad; Hafz, Nasr A M; Li, Song; Liu, Feng; He, Fei; Cheng, Ya; Zhang, Jie

2015-10-01

An investigation of the electron beam yield (charge) form helium, nitrogen, and neon gas jet plasmas in a typical laser-plasma wakefield acceleration experiment is carried out. The charge measurement is made by imaging the electron beam intensity profile on a fluorescent screen into a charge coupled device which was cross-calibrated with an integrated current transformer. The dependence of electron beam charge on the laser and plasma conditions for the aforementioned gases are studied. We found that laser-driven wakefield acceleration in low Z-gas jet targets usually generates high-quality and well-collimated electron beams with modest yields at the level of 10-100 pC. On the other hand, filamentary electron beams which are observed from high-Z gases at higher densities reached much higher yields. Evidences for cluster formation were clearly observed in the nitrogen gas jet target, where we received the highest electron beam charge of ∼1.7 nC. Those intense electron beams will be beneficial for the applications on the generation of bright X-rays, gamma rays radiations, and energetic positrons via the bremsstrahlung or inverse-scattering processes.

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

NASA Astrophysics Data System (ADS)

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.

User Guide for the LORE1 Insertion Mutant Resource.

PubMed

Mun, Terry; Małolepszy, Anna; Sandal, Niels; Stougaard, Jens; Andersen, Stig U

2017-01-01

Lotus japonicus is a model legume used in the study of plant-microbe interactions, especially in the field of biological nitrogen fixation due to its ability to enter into a symbiotic relationship with a soil bacterium, Mesorhizobium loti. The LORE1 mutant population is a valuable resource for reverse genetics in L. japonicus due to its non-transgenic nature, high tagging efficiency, and low copy count. Here, we outline a workflow for identifying, ordering, and establishing homozygous LORE1 mutant lines for a gene of interest, LjFls2, including protocols for growth and genotyping of a segregating LORE1 population.

Genes and Alcohol Consumption: Studies with Mutant Mice

PubMed Central

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

High quantum-yield phosphors via quantum splitting and upconversion

NASA Astrophysics Data System (ADS)

Jeong, Joayoung

The Gd3+ ion has been used to induce quantum splitting in luminescent materials by using cross-relaxation energy transfer (CRET). In Nd:LiGdF4, quantum splitting results from a two-step CRET between Gd3+ and Nd3+, first involving a transition 6G→6I on Gd3+ and an excitation within the 4f3 configuration of Nd3+ followed by a second CRET that brings Gd3+ to 6P7/2. The excited Nd3+ ion rapidly relaxes nonradiatively to the emitting 4F3/2. The excited Gd3+ ion then transfers its energy back to Nd3+, which gives rise to the second photon. The result is a quantum yield of 1.05 +/- 0.35 with emission in the NIR following excitation at 175 nm. GdF3:Pr3+, Eu 3+ also exhibits quantum splitting, but only at very low concentration of Pr3+ (0.3%) and Eu3+ (0.2%), resulting in a quantum yield of approximately 20% under 160-nm excitation. Host intrinsic emission via a self-trapped exciton (STE) was also examined as a means to sensitize Gd3+ emission. The material ScPO4:Gd 3+ exhibits a high absolute quantum yield of 0.9 +/- 0.2 under 170-nm excitation, demonstrating a potentially new and efficient pathway for exciting quantum splitting phosphors. Single crystals of the material GdZrF7 were grown, and its structure was established via single-crystal X-ray diffraction methods. Doped samples of GdZrF7:Yb3+, Er3+ exhibit bright up-conversion luminescence with light output that is up to twice that of a commercial material based on the host Gd2O2S. When doped with Eu3+, the fluoride also emits a nearly white color under vacuum ultraviolet excitation with an absolute quantum yield near 0.9. The new compound Gd4.67(SiO4)3S was synthesized and studied. The structure was established via single-crystal X-ray methods, and the luminescence of Tb3+ samples was investigated.

The Anti-Methicillin-Resistant Staphylococcus aureus Quinolone WCK 771 Has Potent Activity against Sequentially Selected Mutants, Has a Narrow Mutant Selection Window against Quinolone-Resistant Staphylococcus aureus, and Preferentially Targets DNA Gyrase▿ †

PubMed Central

Bhagwat, Sachin S.; Mundkur, Lakshmi A.; Gupte, Shrikant V.; Patel, Mahesh V.; Khorakiwala, Habil F.

2006-01-01

WCK 771 is a broad-spectrum fluoroquinolone with enhanced activity against quinolone-resistant staphylococci. To understand the impact of the target-level interactions of WCK 771 on its antistaphylococcal pharmacodynamic properties, we determined the MICs for genetically defined mutants and studied the mutant prevention concentrations (MPCs), the frequency of mutation, and the cidality against the wild type and double mutants. There was a twofold increase in the MICs of WCK 771 for single gyrA mutants, indicating that DNA gyrase is its primary target. All first- and second-step mutants selected by WCK 771 revealed gyrA and grlA mutations, respectively. The MICs of WCK 771 and clinafloxacin were found to be superior to those of other quinolones against strains with double and triple mutations. WCK 771 was also cidal for high-density double mutants at low concentrations. WCK 771 and clinafloxacin showed narrow mutant selection windows compared to those of the other quinolones. Against a panel of 50 high-level quinolone-resistant clinical isolates of staphylococci (ciprofloxacin MIC ≥ 16 μg/ml), the WCK 771 MPCs were ≤2 μg/ml for 68% of the strains and ≤4 μg/ml for 28% of the strains. Our results demonstrate that gyrA is the primary target of WCK 771 and that it has pharmacodynamic properties remarkably different from those of quinolones with dual targets (garenoxacin and moxifloxacin) and topoisomerase IV-specific quinolones (trovafloxacin). WCK 771 displayed an activity profile comparable to that of clinafloxacin, a dual-acting quinolone with a high affinity to DNA gyrase. Overall, the findings signify the key role of DNA gyrase in determining the optimal antistaphylococcal features of quinolones. PMID:16940059

Flow “Fine” Synthesis: High Yielding and Selective Organic Synthesis by Flow Methods

PubMed Central

2015-01-01

Abstract The concept of flow “fine” synthesis, that is, high yielding and selective organic synthesis by flow methods, is described. Some examples of flow “fine” synthesis of natural products and APIs are discussed. Flow methods have several advantages over batch methods in terms of environmental compatibility, efficiency, and safety. However, synthesis by flow methods is more difficult than synthesis by batch methods. Indeed, it has been considered that synthesis by flow methods can be applicable for the production of simple gasses but that it is difficult to apply to the synthesis of complex molecules such as natural products and APIs. Therefore, organic synthesis of such complex molecules has been conducted by batch methods. On the other hand, syntheses and reactions that attain high yields and high selectivities by flow methods are increasingly reported. Flow methods are leading candidates for the next generation of manufacturing methods that can mitigate environmental concerns toward sustainable society. PMID:26337828

Isolation and characterization of low-sulphur-tolerant mutants of Arabidopsis

PubMed Central

Wu, Yu; Zhao, Qing; Gao, Lei; Yu, Xiao-Min; Fang, Ping; Oliver, David J.; Xiang, Cheng-Bin

2010-01-01

Sulphur is an essential element for plant growth and development as well as for defence against biotic and abiotic stresses. Increasing sulphate utilization efficiency (SUE) is an important issue for crop improvement. Little is known about the genetic determinants of sulphate utilization efficiency. No gain-of-function mutants with improved SUE have been reported to date. Here the isolation and characterization of two low-sulphur-tolerant mutants, sue3 and sue4 are reported using a high-throughput genetic screen where a ‘sulphur-free’ solid medium was devised to give the selection pressure necessary to suppress the growth of the wild-type seedlings. Both mutants showed improved tolerance to low sulphur conditions and well-developed root systems. The mutant phenotype of both sue3 and sue4 was specific to sulphate deficiency and the mutants displayed enhanced tolerance to heavy metal and oxidative stress. Genetic analysis revealed that sue3 was caused by a single recessive nuclear mutation while sue4 was caused by a single dominant nuclear mutation. The recessive locus in sue3 is the previously identified VirE2-interacting Protein 1. The dominant locus in sue4 is a function-unknown locus activated by the four enhancers on the T-DNA. The function of SUE3 and SUE4 in low sulphur tolerance was confirmed either by multiple mutant alleles or by recapitulation analysis. Taken together, our results demonstrate that this genetic screen is a reasonable approach to isolate Arabidopsis mutants with improved low sulphur tolerance and potentially with enhanced sulphate utilization efficiency. The two loci identified in sue3 and sue4 should assist in understanding the molecular mechanisms of low sulphur tolerance. PMID:20547563

Extracting DNA from 'jaws': high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material.

PubMed

Nielsen, E E; Morgan, J A T; Maher, S L; Edson, J; Gauthier, M; Pepperell, J; Holmes, B J; Bennett, M B; Ovenden, J R

2017-05-01

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield. © 2016 John Wiley & Sons Ltd.

Identification of a novel Lymantria dispar nucleopolyhedrovirus mutant that exhibits abnormal polyhedron formation and virion occlusion.

PubMed

Slavicek, J M; Mercer, M J; Pohlman, D; Kelly, M E; Bischoff, D S

1998-07-01

In previous studies on the formation of Lymantria dispar nuclear polyhedrosis virus (LdMNPV) few polyhedra (FP) mutants, several polyhedron formation mutants (PFM) were identified that appeared to be unique. These viral mutants are being characterized to investigate the processes of polyhedron formation and virion occlusion. LdMNPV isolate PFM-1 is one of these mutants, and is described in this report. Genetic techniques were used to determine if isolate PFM-1 contained a mutation in the polyhedrin or 25K FP gene. Wild-type viruses were recovered after coinfection of Ld652Y cells with isolate PFM-1 and a FP mutant, and with isolates PFM-1 and PFM-C (isolate PFM-C contains a mutation in the polyhedrin gene). These viruses were analyzed by genomic restriction endonuclease digestion and found to be chimeras of the original PFMs used in the coinfections. Marker rescue studies mapped the mutation in isolate PFM-1 to a genomic region that does not include the polyhedrin or 25K FP genes. Isolate PFM-1 produced approximately 14-fold fewer polyhedra than LdMNPV isolate A21-MPV, an isolate that produces wild-type levels of polyhedra, and approximately 2-fold more polyhedra compared to the FP isolate 122-2. Polyhedra generated by isolate PFM-1 were normal in size and shape but contained very few viral nucleocapsids. The same amount of budded virus (BV) was released from cells infected with isolates PFM-1 and A21-MPV. In contrast, isolate 122-2 yielded significantly more BV than isolates PFM-1 and A21-MPV.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Do UV-A radiation and blue light during growth prime leaves to cope with acute high-light in photoreceptor mutants of Arabidopsis thaliana?

PubMed

Brelsford, Craig C; Morales, Luis O; Nezval, Jakub; Kotilainen, Titta K; Hartikainen, Saara M; Aphalo, Pedro J; Robson, T Matthew

2018-04-28

We studied how plants acclimated to growing conditions that included combinations of blue light and ultraviolet-A (UV-A) radiation, and whether their growing environment affected their photosynthetic capacity during and after a brief period of acute high light (as might happen during an under-canopy sunfleck). Arabidopsis thaliana Landsberg erecta wild-type were compared with mutants lacking functional blue-light-and-UV photoreceptors: phototropin 1PHOT1, cryptochromes (CRY1 and CRY2) and UV RESISTANT LOCUS 8 (uvr8). This was achieved using LED lamps in a controlled environment to create treatments with or without blue light, in a split-plot design with or without UV-A radiation. We compared the accumulation of phenolic compounds under growth conditions and after exposure to 30 minutes of high light at the end of the experiment (46 days), and likewise measured the operational efficiency of photosystem II (φPSII a proxy for photosynthetic performance) and dark-adapted maximum quantum yield (F v /F m to assess PSII damage). Our results indicate that cryptochromes are the main photoreceptors regulating phenolic-compound accumulation in response to blue light and UV-A radiation, and a lack of functional cryptochromes impairs photosynthetic performance under high light. Our findings also reveal a role for UVR8 in accumulating flavonoids in response to a low UV-A dose. Interestingly, phototropin 1 partially-mediated constitutive accumulation of phenolic compounds in the absence of blue light. Low irradiance blue light and UV-A did not improve φPSII and F v /F m upon our acute high light treatment, however CRYs played an important role in ameliorating high-light stress. This article is protected by copyright. All rights reserved.

Imaginal Disc Abnormalities in Lethal Mutants of Drosophila

PubMed Central

Shearn, Allen; Rice, Thomas; Garen, Alan; Gehring, Walter

1971-01-01

Late lethal mutants of Drosophila melanogaster, dying after the larval stage of development, were isolated. The homozygous mutant larvae were examined for abnormal imaginal disc morphology, and the discs were injected into normal larval hosts to test their capacities to differentiate into adult structures. In about half of the mutants analyzed, disc abnormalities were found. Included among the abnormalities were missing discs, small discs incapable of differentiating, morphologically normal discs with limited capacities for differentiation, and discs with homeotic transformations. In some mutants all discs were affected, and in others only certain discs. The most extreme abnormal phenotype is a class of “discless” mutants. The viability of these mutant larvae indicates that the discs are essential only for the development of an adult and not of a larva. The late lethals are therefore a major source of mutants for studying the genetic control of disc formation. Images PMID:5002822

High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction.

PubMed

Lee, Seon-Hwa; Hong, Seung-Hye; Kim, Kyoung-Rok; Oh, Deok-Kun

2017-08-01

To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. Fructose at 1 M (180 g l -1 ) was converted to 0.8 M (144 g l -1 ) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l -1 h -1 . No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

Neutron temporal diagnostic for high-yield deuterium-tritium cryogenic implosions on OMEGA

DOE PAGES

Stoeckl, C.; Boni, R.; Ehrne, F.; ...

2016-05-10

A next-generation neutron temporal diagnostic (NTD) capable of recording high-quality data for the highest anticipated yield cryogenic DT implosion experiments was recently installed at the Omega Laser Facility. A high-quality measurement of the neutron production width is required to determine the hot-spot pressure achieved in inertial confinement fusion experiments—a key metric in assessing the quality of these implosions. The design of this NTD is based on a fast-rise-time plastic scintillator, which converts the neutron kinetic energy to 350- to 450-nm-wavelength light. The light from the scintillator inside the nose-cone assembly is relayed ~16 m to a streak camera in amore » well-shielded location. An ~200× reduction in neutron background was observed during the first high-yield DT cryogenic implosions compared to the current NTD installation on OMEGA. An impulse response of ~40±10 ps was measured in a dedicated experiment using hard x rays from a planar target irradiated with a 10-ps short pulse from the OMEGA EP laser. Furthermore, the measured instrument response includes contributions from the scintillator rise time, optical relay, and streak camera.« less

Neutron temporal diagnostic for high-yield deuterium–tritium cryogenic implosions on OMEGA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoeckl, C.; Boni, R.; Ehrne, F.

A next-generation neutron temporal diagnostic (NTD) capable of recording high-quality data for the highest anticipated yield cryogenic deuterium–tritium (DT) implosion experiments was recently installed at the Omega Laser Facility. A high-quality measurement of the neutron production width is required to determine the hot-spot pressure achieved in inertial confinement fusion experiments—a key metric in assessing the quality of these implosions. The design of this NTD is based on a fast-rise-time plastic scintillator, which converts the neutron kinetic energy to 350- to 450-nm-wavelength light. The light from the scintillator inside the nose-cone assembly is relayed ∼16 m to a streak camera inmore » a well-shielded location. An ∼200× reduction in neutron background was observed during the first high-yield DT cryogenic implosions compared to the current NTD installation on OMEGA. An impulse response of ∼40 ± 10 ps was measured in a dedicated experiment using hard x-rays from a planar target irradiated with a 10-ps short pulse from the OMEGA EP laser. The measured instrument response includes contributions from the scintillator rise time, optical relay, and streak camera.« less

Neutron temporal diagnostic for high-yield deuterium-tritium cryogenic implosions on OMEGA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoeckl, C.; Boni, R.; Ehrne, F.

A next-generation neutron temporal diagnostic (NTD) capable of recording high-quality data for the highest anticipated yield cryogenic DT implosion experiments was recently installed at the Omega Laser Facility. A high-quality measurement of the neutron production width is required to determine the hot-spot pressure achieved in inertial confinement fusion experiments—a key metric in assessing the quality of these implosions. The design of this NTD is based on a fast-rise-time plastic scintillator, which converts the neutron kinetic energy to 350- to 450-nm-wavelength light. The light from the scintillator inside the nose-cone assembly is relayed ~16 m to a streak camera in amore » well-shielded location. An ~200× reduction in neutron background was observed during the first high-yield DT cryogenic implosions compared to the current NTD installation on OMEGA. An impulse response of ~40±10 ps was measured in a dedicated experiment using hard x rays from a planar target irradiated with a 10-ps short pulse from the OMEGA EP laser. Furthermore, the measured instrument response includes contributions from the scintillator rise time, optical relay, and streak camera.« less

Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants.

PubMed

Nicolson, T; Rüsch, A; Friedrich, R W; Granato, M; Ruppersberg, J P; Nüsslein-Volhard, C

1998-02-01

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

Field-based high throughput phenotyping rapidly identifies genomic regions controlling yield components in rice

PubMed Central

Tanger, Paul; Klassen, Stephen; Mojica, Julius P.; Lovell, John T.; Moyers, Brook T.; Baraoidan, Marietta; Naredo, Maria Elizabeth B.; McNally, Kenneth L.; Poland, Jesse; Bush, Daniel R.; Leung, Hei; Leach, Jan E.; McKay, John K.

2017-01-01

To ensure food security in the face of population growth, decreasing water and land for agriculture, and increasing climate variability, crop yields must increase faster than the current rates. Increased yields will require implementing novel approaches in genetic discovery and breeding. Here we demonstrate the potential of field-based high throughput phenotyping (HTP) on a large recombinant population of rice to identify genetic variation underlying important traits. We find that detecting quantitative trait loci (QTL) with HTP phenotyping is as accurate and effective as traditional labor-intensive measures of flowering time, height, biomass, grain yield, and harvest index. Genetic mapping in this population, derived from a cross of an modern cultivar (IR64) with a landrace (Aswina), identified four alleles with negative effect on grain yield that are fixed in IR64, demonstrating the potential for HTP of large populations as a strategy for the second green revolution. PMID:28220807

One-step synthesis of high-yield biodiesel from waste cooking oils by a novel and highly methanol-tolerant immobilized lipase.

PubMed

Wang, Xiumei; Qin, Xiaoli; Li, Daoming; Yang, Bo; Wang, Yonghua

2017-07-01

This study reported a novel immobilized MAS1 lipase from marine Streptomyces sp. strain W007 for synthesizing high-yield biodiesel from waste cooking oils (WCO) with one-step addition of methanol in a solvent-free system. Immobilized MAS1 lipase was selected for the transesterification reactions with one-step addition of methanol due to its much more higher biodiesel yield (89.50%) when compared with the other three commercial immobilized lipases (<10%). The highest biodiesel yield (95.45%) was acquired with one-step addition of methanol under the optimized conditions. Moreover, it was observed that immobilized MAS1 lipase retained approximately 70% of its initial activity after being used for four batch cycles. Finally, the obtained biodiesel was further characterized using FT-IR, 1 H and 13 C NMR spectroscopy. These findings indicated that immobilized MAS1 lipase is a promising catalyst for biodiesel production from WCO with one-step addition of methanol under high methanol concentration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, Susan I.

mutation. Characterization of mutant and wild-type plants has revealed that sugars inhibit breakdown of seed storage lipids. In addition, high concentrations of exogenous sugars largely eliminate the development of mature chloroplasts by developing seedlings. Affymetrix GeneChip experiments have revealed that expression of many plant genes is partially regulated by sugar levels, with approximately two percent of genes exhibiting alterations in steady-state mRNA levels in response to changing sugar concentrations. Ultimately, a better understanding of plant sugar responses may allow improvements in rates of carbon fixation and manipulation of carbon partitioning. These improvements will be needed to help make production of energy from biomass more economically attractive.« less

Astaxanthin synthesis by Xanthophyllomyces dendrorhous DSM 5626 and its astaxanthin overproducing mutants on xylose media under different illumination.

PubMed

Stachowiak, Barbara

2014-01-01

Astaxanthin is the most important and expensive carotenoid pigment used in aquaculture. Its commercial attractiveness is also related with its antioxidant potential. Xanthophyllomyces dendrorhous yeast is considered to be promising for commercial production of astaxanthin. The aim of this study was to investigate the possibility of the growth and astaxanthin production by X. dendrorhous strains 011 media containing xylose under different illumination. A', dendrorhous DSM 5626 and its mutants: 10BE and 26UV were used in this study. The cultures were carried out 011 hydrolysed rye stillage (HS) and YM medium with xylose (YM-K). Cell concentration, total carotenoid and astaxanthin yields were assessed in 5-day cultures. The effect of illumination in the range of 0-5.000 lx 011 growth and on astaxanthin production of yeasts in cultures run 011 YM-K medium was also examined. For the tested yeast strains better growth parameters and astaxanthin yields were obtained on the YM-K medium. 011 which for all strains the highest pigment yields were recorded at 600-1.000 lx. The highest concentration of astaxanthin in cells was recorded for 26UV in a culture at 1.000 lx (0.51 g∙kg-1 DCW). The volume yield of the pigment regardless of strain was highest in cultures at 600 lx. In this case 10BE was found to be the best astaxanthin producer with a yield of 2.15 mg dm-3. Astaxanthin synthesis in X. dendrorhous DSM 5626 and its mutants was better 011 YM-K medium comparing to hydrolysed rye stillage. Moreover, carotenogenesis in the studied yeast strains was subjected to marked photoregulation. Illumination within the range of 600-1.000 lx promotes carotenogenesis and astaxanthin production, while exceeding a certain light capacity results in microbial cell death.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 parentalmore » 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.« less

Nicotinamide Ribosyl Uptake Mutants in Haemophilus influenzae

PubMed Central

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

2003-01-01

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. PMID:12933892

Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

PubMed

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.

Isolation and Identification of Pathogenicity Mutant of Curvularia lunata via Restriction Enzyme-Mediated Integration.

PubMed

Wang, Y J; Liu, T; Hou, J M; Zuo, Y H

2013-09-01

In this report, 156 hygromycin-resistant mutants were generated via restriction enzyme-mediated insertional (REMI) mutagenesis. All mutants were subjected to a bioassay on detached leaves. Five mutants (T4, T39, T71, T91, and T135) showed reduced symptom development, whereas one mutant (T120) did not exhibit any symptoms on the leaves compared with the wild type. The pathogenicity of these mutants was further assayed through the spray inoculation of whole seedlings. The results demonstrated that the pathogenicity of the T4, T39, T71, T91, and T135 mutants was reduced, whereas the T120 mutant lost its pathogenicity. Southern blot analysis revealed that the plasmids were inserted at different sites in the genome with different copy numbers. Flanking sequences approximately 550, 860, and 150 bp were obtained from T7, T91, and T120, respectively through plasmids rescue. Sequence analysis of the flanking sequences from T7 and T91 showed no homology to any known sequences in GenBank. The flanking sequence from the T120 mutant was highly homologous to MAPKK kinases, which regulates sexual/asexual development, melanization, pathogenicity from Cochliobolus heterostrophus. These results indicate that REMI and plasmids rescue have great potential for finding pathogenicity genes.

High char yield epoxy curing agents

NASA Technical Reports Server (NTRS)

Delvigs, P.; Serafini, T. T.; Vanucci, R. D.

1981-01-01

Class of imide-amine curing agents preserves structural integrity, prevents fiber release, and is fully compatible with conventional epoxy resins; agents do not detract from composite properties while greatly reducing char yield. Materials utilizing curing are used in aerospace, automotive, and other structural components where deterioration must be minimized and fiber release avoided in event of fire.

Characterization and classification of zebrafish brain morphology mutants

PubMed Central

Lowery, Laura Anne; De Rienzo, Gianluca; Gutzman, Jennifer H.; Sive, Hazel

2010-01-01

The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several time-points, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, while others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, class 1 mutants show midline separation defects, corresponding to epithelial junction defects; class 2 mutants show reduced brain ventricle size; class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. PMID:19051268

Climate Change Impacts on Sediment Yield in Headwaters of a High-latitude Region in China

NASA Astrophysics Data System (ADS)

Zhou, Y.; Xu, Y. J.; Wang, J., , Dr; Weihua, X.; Huang, Y.

2017-12-01

Climate change is expected to have strongest effects in higher latitude regions. Despite intensive research on possible hydrological responses to global warming in these regions, our knowledge of climate change on surface erosion and sediment yield in high-latitude headwaters is limited. In this study, we used the Soil and Water Assessment Tool (SWAT) to predict future runoff and sediment yield from the headwaters of a high-latitude river basin in China's far northeast. The SWAT model was first calibrated with historical discharge records and the model parameterization achieved satisfactory validation. The calibrated model was then applied to two greenhouse gas concentration trajectories, RCP4.5 and RCP8.5, for the period from 2020 to 2050 to estimate future runoff. Sediment yields for this period were predicted using a discharge-sediment load rating curve developed from field measurements in the past nine years. Our preliminary results show an increasing trend of sediment yield under both climate change scenarios, and that the increase is more pronounced in the summer and autumn months. Changes in precipitation and temperature seem to exert variable impacts on runoff and sediment yield at interannual and seasonal scales in these headwaters. These findings imply that the current river basin management in the region needs to be reviewed and improved in order to be effective under a changing climate.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

Genetic transformation of Neurospora tetrasperma, demonstration of repeat-induced point mutation (RIP) in self-crosses and a screen for recessive RIP-defective mutants.

PubMed Central

Bhat, Ashwin; Tamuli, Ranjan; Kasbekar, Durgadas P

2004-01-01

The pseudohomothallic fungus Neurospora tetrasperma is naturally resistant to the antibiotic hygromycin. We discovered that mutation of its erg-3 (sterol C-14 reductase) gene confers a hygromycin-sensitive phenotype that can be used to select transformants on hygromycin medium by complementation with the N. crassa erg-3+ and bacterial hph genes. Cotransformation of hph with PCR-amplified DNA of other genes enabled us to construct strains duplicated for the amplified DNA. Using transformation we constructed self-fertile strains that were homoallelic for an ectopic erg-3+ transgene and a mutant erg-3 allele at the endogenous locus. Self-crosses of these strains yielded erg-3 mutant ascospores that produced colonies with the characteristic morphology on Vogel's sorbose agar described previously for erg-3 mutants of N. crassa. The mutants were generated by repeat-induced point mutation (RIP), a genome defense process that causes numerous G:C to A:T mutations in duplicated DNA sequences. Homozygosity for novel recessive RIP-deficient mutations was signaled by self-crosses of erg-3-duplication strains that fail to produce erg-3 mutant progeny. Using this assay we isolated a UV-induced mutant with a putative partial RIP defect. RIP-induced mutants were isolated in rid-1 and sad-1, which are essential genes, respectively, for RIP and another genome defense mechanism called meiotic silencing by unpaired DNA. PMID:15280231

Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

PubMed Central

Zhu, Y Q; Remington, K M; North, T W

1996-01-01

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis. PMID:8878567

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

PubMed

Jones, Stuart; Ahmet, Jonathan; Ayton, Kelly; Ball, Matthew; Cockerill, Mark; Fairweather, Emma; Hamilton, Nicola; Harper, Paul; Hitchin, James; Jordan, Allan; Levy, Colin; Lopez, Ruth; McKenzie, Eddie; Packer, Martin; Plant, Darren; Simpson, Iain; Simpson, Peter; Sinclair, Ian; Somervaille, Tim C P; Small, Helen; Spencer, Gary J; Thomson, Graeme; Tonge, Michael; Waddell, Ian; Walsh, Jarrod; Waszkowycz, Bohdan; Wigglesworth, Mark; Wiseman, Daniel H; Ogilvie, Donald

2016-12-22

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Creating a High-Touch Recruitment Event: Utilizing Faculty to Recruit and Yield Students

ERIC Educational Resources Information Center

Freed, Lindsey R.; Howell, Leanne L.

2018-01-01

The following article describes the planning and implementation of a university student recruitment event that produced a high (new) student yield. Detailed descriptions of how staff and faculty worked together to plan and implement this event are described.

Studies of fission fragment yields via high-resolution γ-ray spectroscopy

NASA Astrophysics Data System (ADS)

Wilson, J. N.; Lebois, M.; Qi, L.; Amador-Celdran, P.; Bleuel, D.; Briz, J. A.; Carroll, R.; Catford, W.; Witte, H. De; Doherty, D. T.; Eloirdi, R.; Georgiev, G.; Gottardo, A.; Goasduff, A.; Hadyñska-Klek, K.; Hauschild, K.; Hess, H.; Ingeberg, V.; Konstantinopoulos, T.; Ljungvall, J.; Lopez-Martens, A.; Lorusso, G.; Lozeva, R.; Lutter, R.; Marini, P.; Matea, I.; Materna, T.; Mathieu, L.; Oberstedt, A.; Oberstedt, S.; Panebianco, S.; Podolyak, Zs.; Porta, A.; Regan, P. H.; Reiter, P.; Rezynkina, K.; Rose, S. J.; Sahin, E.; Seidlitz, M.; Serot, O.; Shearman, R.; Siebeck, B.; Siem, S.; Smith, A. G.; Tveten, G. M.; Verney, D.; Warr, N.; Zeiser, F.; Zielinska, M.

2018-03-01

Precise spectroscopic information on the fast neutron induced fission of the 238U(n,f) reaction was recently gained using a new technique which involved coupling of the Miniball high resolution y-ray spectrometer and the LICORNE directional neutron source. The experiment allowed measurement of the isotopic fission yields for around 40 even-even nuclei at an incident neutron energy of around 2 MeV where yield data are very sparse. In addition spectroscopic information on very neutron-rich fission products was obtained. Results were compared to models, both the JEFF-3.1.1 data base and the GEF code, and large discrepancies for the S1 fission mode in the Sn/Mo isotope pair were discovered. This suggests that current models are overestimating the role played by spherical shell effects in fast neutron induced fission. In late 2017 and 2018 the nu-ball hybrid spectrometer will be constructed at the IPN Orsay to perform further experimental investigations with directional neutrons coupled to a powerful hybrid Ge/LaBr3 detector array. This will open up new possibilities for measurements of fission yields for fast-neutron-induced fission using the spectroscopic technique and will be complimentary to other methods being developed.

Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gandhi, Prafull S.; Page, Michael J.; Chen, Zhiwei

2009-09-15

The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215-217 {beta}-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve themore » design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* {yields} E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.« less

Targeting Autophagy Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib

PubMed Central

Wang, Weibin; Kang, Helen; Zhao, Yinu; Min, Irene; Wyrwas, Brian; Moore, Maureen; Teng, Lisong; Zarnegar, Rasa; Jiang, Xuejun

2017-01-01

Context: The RAF inhibitor vemurafenib has provided a major advance for the treatment of patients with BRAF-mutant metastatic melanoma. However, BRAF-mutant thyroid cancer is relatively resistant to vemurafenib, and the reason for this disparity remains unclear. Anticancer therapy–induced autophagy can trigger adaptive drug resistance in a variety of cancer types and treatments. To date, role of autophagy during BRAF inhibition in thyroid cancer remains unknown. Objective: In this study, we investigate if autophagy is activated in vemurafenib-treated BRAF-mutant thyroid cancer cells, and whether autophagy inhibition improves or impairs the treatment efficacy of vemurafenib. Design: Autophagy level was determined by western blot assay and transmission electron microscopy. The combined effects of autophagy inhibitor and vemurafenib were assessed in terms of cell viability in vitro and tumor growth rate in vivo. Whether the endoplasmic reticulum (ER) stress was in response to vemurafenib-induced autophagy was also analyzed. Results: Vemurafenib induced a high level of autophagy in BRAF-mutant thyroid cancer cells. Inhibition of autophagy by either a pharmacological inhibitor or interfering RNA knockdown of essential autophagy genes augmented vemurafenib-induced cell death. Vemurafenib-induced autophagy was independent of MAPK signaling pathway and was mediated through the ER stress response. Finally, administration of vemurafenib with the autophagy inhibitor hydroxychloroquine promoted more pronounced tumor suppression in vivo. Conclusions: Our data demonstrate that vemurafenib induces ER stress response–mediated autophagy in thyroid cancer and autophagy inhibition may be a beneficial strategy to sensitize BRAF-mutant thyroid cancer to vemurafenib. PMID:27754804

Targeting Autophagy Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

PubMed

Wang, Weibin; Kang, Helen; Zhao, Yinu; Min, Irene; Wyrwas, Brian; Moore, Maureen; Teng, Lisong; Zarnegar, Rasa; Jiang, Xuejun; Fahey, Thomas J

2017-02-01

The RAF inhibitor vemurafenib has provided a major advance for the treatment of patients with BRAF-mutant metastatic melanoma. However, BRAF-mutant thyroid cancer is relatively resistant to vemurafenib, and the reason for this disparity remains unclear. Anticancer therapy-induced autophagy can trigger adaptive drug resistance in a variety of cancer types and treatments. To date, role of autophagy during BRAF inhibition in thyroid cancer remains unknown. In this study, we investigate if autophagy is activated in vemurafenib-treated BRAF-mutant thyroid cancer cells, and whether autophagy inhibition improves or impairs the treatment efficacy of vemurafenib. Autophagy level was determined by western blot assay and transmission electron microscopy. The combined effects of autophagy inhibitor and vemurafenib were assessed in terms of cell viability in vitro and tumor growth rate in vivo. Whether the endoplasmic reticulum (ER) stress was in response to vemurafenib-induced autophagy was also analyzed. Vemurafenib induced a high level of autophagy in BRAF-mutant thyroid cancer cells. Inhibition of autophagy by either a pharmacological inhibitor or interfering RNA knockdown of essential autophagy genes augmented vemurafenib-induced cell death. Vemurafenib-induced autophagy was independent of MAPK signaling pathway and was mediated through the ER stress response. Finally, administration of vemurafenib with the autophagy inhibitor hydroxychloroquine promoted more pronounced tumor suppression in vivo. Our data demonstrate that vemurafenib induces ER stress response-mediated autophagy in thyroid cancer and autophagy inhibition may be a beneficial strategy to sensitize BRAF-mutant thyroid cancer to vemurafenib. Copyright © 2017 by the Endocrine Society

Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

PubMed

Pratter, S M; Eixelsberger, T; Nidetzky, B

2015-12-01

A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular characterization of baculovirus Bombyx mori nucleopolyhedrovirus polyhedron mutants.

PubMed

Katsuma, S; Noguchi, Y; Shimada, T; Nagata, M; Kobayashi, M; Maeda, S

1999-01-01

Four newly isolated and two previously isolated polyhedron mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were studied. Two polyhedron deficient mutants, #126 and #136, produced small uncrystallized particles of polyhedrin in the nuclei and cytoplasm of infected cells. Mutant #211 produced a large number of variably sized polyhedra in the nucleus and #220 produced a few large cuboidal polyhedra in the nucleus. Mutant #24 and #128 were previously isolated BmNPV mutants. Mutant #24 could not produce polyhedrin mRNA and polyhedra produced by mutant #128 lacked oral infectivity. Nucleotide sequence analysis indicated that five mutants (#126, #136, #211, #220 and #128) had amino acid substitutions in polyhedrin and mutant #24 had a point mutation only in the promoter region of the polyhedrin gene. Cotransfection experiments showed that the altered phenotypes were due to the mutations found in the polyhedrin gene regions. In mutants #126 and #136, amino acid sequences of the nuclear localization signal of polyhedrin were identical to those of wild-type BmNPV, suggesting that this sequence was necessary but not sufficient for nuclear localization of polyhedrin. Electron microscopic observation revealed that fewer occluded virions were contained in polyhedra of #128 and #220.

Computational Design of a Thermostable Mutant of Cocaine Esterase via Molecular Dynamics Simulations

PubMed Central

Huang, Xiaoqin; Gao, Daquan; Zhan, Chang-Guo

2015-01-01

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ~11 min at physiological temperature (37°C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the RMSD and RMSF of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modeling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37°C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability may be considered as a valuable tool for computational design of thermostable mutants of an enzyme. PMID:21373712

Computational design of a thermostable mutant of cocaine esterase via molecular dynamics simulations.

PubMed

Huang, Xiaoqin; Gao, Daquan; Zhan, Chang-Guo

2011-06-07

Cocaine esterase (CocE) has been known as the most efficient native enzyme for metabolizing naturally occurring cocaine. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only ∼11 min at physiological temperature (37 °C). It is highly desirable to develop a thermostable mutant of CocE for therapeutic treatment of cocaine overdose and addiction. To establish a structure-thermostability relationship, we carried out molecular dynamics (MD) simulations at 400 K on wild-type CocE and previously known thermostable mutants, demonstrating that the thermostability of the active form of the enzyme correlates with the fluctuation (characterized as the root-mean square deviation and root-mean square fluctuation of atomic positions) of the catalytic residues (Y44, S117, Y118, H287, and D259) in the simulated enzyme. In light of the structure-thermostability correlation, further computational modelling including MD simulations at 400 K predicted that the active site structure of the L169K mutant should be more thermostable. The prediction has been confirmed by wet experimental tests showing that the active form of the L169K mutant had a half-life of 570 min at 37 °C, which is significantly longer than those of the wild-type and previously known thermostable mutants. The encouraging outcome suggests that the high-temperature MD simulations and the structure-thermostability relationship may be considered as a valuable tool for the computational design of thermostable mutants of an enzyme.

High-Yield Secretion of Multiple Client Proteins in Aspergillus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segato, F.; Damasio, A. R. L.; Goncalves, T. A.

2012-07-15

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degradingmore » enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.« less

Purkinje cells from RyR2 mutant mice are highly arrhythmogenic but responsive to targeted therapy.

PubMed

Kang, Guoxin; Giovannone, Steven F; Liu, Nian; Liu, Fang-Yu; Zhang, Jie; Priori, Silvia G; Fishman, Glenn I

2010-08-20

The Purkinje fiber network has been proposed as the source of arrhythmogenic Ca(2+) release events in catecholaminergic polymorphic ventricular tachycardia (CPVT), yet evidence supporting this mechanism at the cellular level is lacking. We sought to determine the frequency and severity of spontaneous Ca(2+) release events and the response to the antiarrhythmic agent flecainide in Purkinje cells and ventricular myocytes from RyR2(R4496C/+) CPVT mutant mice and littermate controls. We crossed RyR2(R4496C/+) knock-in mice with the newly described Cntn2-EGFP BAC transgenic mice, which express a fluorescent reporter gene in cells of the cardiac conduction system, including the distal Purkinje fiber network. Isolated ventricular myocytes (EGFP(-)) and Purkinje cells (EGFP(+)) from wild-type hearts and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorimetry. Both wild-type and RyR2(R4496C/+) mutant Purkinje cells displayed significantly slower kinetics of activation and relaxation compared to ventricular myocytes of the same genotype, and tau(decay) in the mutant Purkinje cells was significantly slower than that observed in wild-type Purkinje cells. Of the 4 groups studied, RyR2(R4496C/+) mutant Purkinje cells were also most likely to develop spontaneous Ca(2+) release events, and the number of events per cell was also significantly greater. Furthermore, with isoproterenol treatment, although all 4 groups showed increases in the frequency of arrhythmogenic Ca(2+(i)) events, the RyR2(R4496C/+) Purkinje cells responded with the most profound abnormalities in intracellular Ca(2+) handling, including a significant increase in the frequency of unstimulated Ca(2+(i)) events and the development of alternans, as well as isolated and sustained runs of triggered beats. Both Purkinje cells and ventricular myocytes from wild-type mice showed suppression of spontaneous Ca(2+) release events with flecainide, whereas in RyR2(R

Method for reproducibly preparing a low-melting high-carbon yield precursor

DOEpatents

Smith, Wesley E.; Napier, Jr., Bradley

1978-01-01

The present invention is directed to a method for preparing a reproducible synthetic carbon precursor by the autoclave polymerization of indene (C.sub.9 H.sub.8) at a temperature in the range of 470.degree.-485.degree. C, and at a pressure in the range of about 1000 to about 4300 psi. Volatiles in the resulting liquid indene polymer are removed by vacuum outgassing to form a solid carbon precursor characterized by having a relatively low melting temperature, high-carbon yield, and high reproducibility which provide for the fabrication of carbon and graphite composites having strict requirements for reproducible properties.

Field-based high throughput phenotyping rapidly identifies genomic regions controlling yield components in rice

DOE PAGES

Tanger, Paul; Klassen, Stephen; Mojica, Julius P.; ...

2017-02-21

In order to ensure food security in the face of population growth, decreasing water and land for agriculture, and increasing climate variability, crop yields must increase faster than the current rates. Increased yields will require implementing novel approaches in genetic discovery and breeding. We demonstrate the potential of field-based high throughput phenotyping (HTP) on a large recombinant population of rice to identify genetic variation underlying important traits. We find that detecting quantitative trait loci (QTL) with HTP phenotyping is as accurate and effective as traditional labor- intensive measures of flowering time, height, biomass, grain yield, and harvest index. Furthermore, geneticmore » mapping in this population, derived from a cross of an modern cultivar (IR64) with a landrace (Aswina), identified four alleles with negative effect on grain yield that are fixed in IR64, demonstrating the potential for HTP of large populations as a strategy for the second green revolution.« less

Field-based high throughput phenotyping rapidly identifies genomic regions controlling yield components in rice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanger, Paul; Klassen, Stephen; Mojica, Julius P.

In order to ensure food security in the face of population growth, decreasing water and land for agriculture, and increasing climate variability, crop yields must increase faster than the current rates. Increased yields will require implementing novel approaches in genetic discovery and breeding. We demonstrate the potential of field-based high throughput phenotyping (HTP) on a large recombinant population of rice to identify genetic variation underlying important traits. We find that detecting quantitative trait loci (QTL) with HTP phenotyping is as accurate and effective as traditional labor- intensive measures of flowering time, height, biomass, grain yield, and harvest index. Furthermore, geneticmore » mapping in this population, derived from a cross of an modern cultivar (IR64) with a landrace (Aswina), identified four alleles with negative effect on grain yield that are fixed in IR64, demonstrating the potential for HTP of large populations as a strategy for the second green revolution.« less

High Throughput Screening of Esterases, Lipases and Phospholipases in Mutant and Metagenomic Libraries: A Review.

PubMed

Peña-García, Carlina; Martínez-Martínez, Mónica; Reyes-Duarte, Dolores; Ferrer, Manuel

2016-01-01

Nowadays, enzymes can be efficiently identified and screened from metagenomic resources or mutant libraries. A set of a few hundred new enzymes can be found using a simple substrate within few months. Hence, the establishment of collections of enzymes is no longer a big hurdle. However, a key problem is the relatively low rate of positive hits and that a timeline of several years from the identification of a gene to the development of a process is the reality rather than the exception. Major problems are related to the time-consuming and cost-intensive screening process that only very few enzymes finally pass. Accessing to the highest possible enzyme and mutant diversity by different, but complementary approaches is increasingly important. The aim of this review is to deliver state-of-art status of traditional and novel screening protocols for targeting lipases, esterases and phospholipases of industrial relevance, and that can be applied at high throughput scale (HTS) for at least 200 distinct substrates, at a speed of more than 105 - 108 clones/day. We also review fine-tuning sequence analysis pipelines and in silico tools, which can further improve enzyme selection by an unprecedent speed (up to 1030 enzymes). If the hit rate in an enzyme collection could be increased by HTS approaches, it can be expected that also the very further expensive and time-consuming enzyme optimization phase could be significantly shortened, as the processes of enzyme-candidate selection by such methods can be adapted to conditions most likely similar to the ones needed at industrial scale.

Exogenously Applied Plant Growth Regulators Enhance the Morpho-Physiological Growth and Yield of Rice under High Temperature.

PubMed

Fahad, Shah; Hussain, Saddam; Saud, Shah; Hassan, Shah; Ihsan, Zahid; Shah, Adnan N; Wu, Chao; Yousaf, Muhammad; Nasim, Wajid; Alharby, Hesham; Alghabari, Fahad; Huang, Jianliang

2016-01-01

A 2-year experiment was conducted to ascertain the effects of exogenously applied plant growth regulators (PGR) on rice growth and yield attributes under high day (HDT) and high night temperature (HNT). Two rice cultivars (IR-64 and Huanghuazhan) were subjected to temperature treatments in controlled growth chambers and four different combinations of ascorbic acid (Vc), alpha-tocopherol (Ve), brassinosteroids (Br), methyl jasmonates (MeJA), and triazoles (Tr) were applied. High temperature severely affected rice morphology, and also reduced leaf area, above-, and below-ground biomass, photosynthesis, and water use efficiency, while increased the leaf water potential of both rice cultivars. Grain yield and its related attributes except number of panicles, were reduced under high temperature. The HDT posed more negative effects on rice physiological attributes, while HNT was more detrimental for grain formation and yield. The Huanghuazhan performed better than IR-64 under high temperature stress with better growth and higher grain yield. Exogenous application of PGRs was helpful in alleviating the adverse effects of high temperature. Among PGR combinations, the Vc+Ve+MejA+Br was the most effective treatment for both cultivars under high temperature stress. The highest grain production by Vc+Ve+MejA+Br treated plants was due to enhanced photosynthesis, spikelet fertility and grain filling, which compensated the adversities of high temperature stress. Taken together, these results will be of worth for further understanding the adaptation and survival mechanisms of rice to high temperature and will assist in developing heat-resistant rice germplasm in future.

Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum

DOE PAGES

Papanek, Beth A.; Biswas, Ranjita; Rydzak, Thomas; ...

2015-09-12

Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, we study a mutant strain of C. thermocellum that was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulosemore » Avicel, effectively eliminating formate, acetate, and lactate production and reducing H 2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4 mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO 2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1 mM ethanol on 10 g/L Avicel. In conclusion, with the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.« less

Dictyostelium discoideum mutants with conditional defects in phagocytosis

PubMed Central

1994-01-01

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s). PMID:7519624

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

PubMed Central

Ducasse, D A; Mushegian, A R; Shepherd, R J

1995-01-01

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants. PMID:7543587

Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalayanamitr, A.; Bhumiratana, A.; Flegel, T.W.

A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.

Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

PubMed Central

Rossouw, D.; Heyns, E. H.; Setati, M. E.; Bosch, S.

2013-01-01

The ability of Saccharomyces cerevisiae to efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1 and ACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ and tdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, the TPS1 gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of the TPS1 gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines. PMID:23793638

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves

PubMed Central

Vanhercke, Thomas; El Tahchy, Anna; Liu, Qing; Zhou, Xue-Rong; Shrestha, Pushkar; Divi, Uday K; Ral, Jean-Philippe; Mansour, Maged P; Nichols, Peter D; James, Christopher N; Horn, Patrick J; Chapman, Kent D; Beaudoin, Frederic; Ruiz-López, Noemi; Larkin, Philip J; de Feyter, Robert C; Singh, Surinder P; Petrie, James R

2014-01-01

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications. PMID:24151938

Purification and characterization of an alginate lyase from marine Bacterium Vibrio sp. mutant strain 510-64.

PubMed

Hu, Xiaoke; Jiang, Xiaolu; Hwang, Huey-Min

2006-08-01

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.

Engineering a vitamin B12 high-throughput screening system by riboswitch sensor in Sinorhizobium meliloti.

PubMed

Cai, Yingying; Xia, Miaomiao; Dong, Huina; Qian, Yuan; Zhang, Tongcun; Zhu, Beiwei; Wu, Jinchuan; Zhang, Dawei

2018-05-11

As a very important coenzyme in the cell metabolism, Vitamin B 12 (cobalamin, VB 12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB 12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB 12 production. High-yield VB 12 -producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. By the help of engineered strains with varied capacities of VB 12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device. A flow cytometry high-throughput screening system was developed based on the btuB riboswitch with high efficiency to identify positive mutants. Mutation of Sinorhizobium meliloti (S. meliloti) was optimized using the novel mutation technique of atmospheric and room temperature plasma (ARTP). Finally, the mutant S. meliloti MC5-2 was obtained and considered as a candidate for industrial applications. After 7 d's cultivation on a rotary shaker at 30 °C, the VB 12 titer of S. meliloti MC5-2 reached 156 ± 4.2 mg/L, which was 21.9% higher than that of the wild type strain S. meliloti 320 (128 ± 3.2 mg/L). The genome of S. meliloti MC5-2 was sequenced, and gene mutations were identified and analyzed. To our knowledge, it is the first time that a riboswitch element was used in S. meliloti. The flow cytometry high-throughput screening system was successfully developed and a high-yield VB 12 producing strain was obtained. The identified and analyzed gene mutations gave useful information for developing high-yield strains by metabolic engineering. Overall, this work provides a useful high-throughput screening method for developing high VB 12 -yield strains.

An annotated database of Arabidopsis mutants of acyl lipid metabolism

DOE PAGES

McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

2014-12-10

Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism ( fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypesmore » of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

Determining the Critical Dose Threshold of Electron-Induced Electron Yield for Minimally Charged Highly Insulating Materials

NASA Astrophysics Data System (ADS)

Hoffmann, Ryan; Dennison, J. R.; Abbott, Jonathan

2006-03-01

When incident energetic electrons interact with a material, they excite electrons within the material to escape energies. The electron emission is quantified as the ratio of emitted electrons to incident particle flux, termed electron yield. Measuring the electron yield of insulators is difficult due to dynamic surface charge accumulation which directly affects landing energies and the potential barrier that emitted electrons must overcome. Our recent measurements of highly insulating materials have demonstrated significant changes in total yield curves and yield decay curves for very small electron doses equivalent to a trapped charge density of <10^10 electrons /cm^3. The Chung-Everhart theory provides a basic model for the behavior of the electron emission spectra which we relate to yield decay curves as charge is allowed to accumulate. Yield measurements as a function of dose for polyimide (Kapton^TM) and microcrystalline SiO2 will be presented. We use our data and model to address the question of whether there is a minimal dose threshold at which the accumulated charge no longer affects the yield.

Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum Fenna-Matthews-Olson (FMO) Complex: High Resolution Spectroscopic and Modeling Studies.

PubMed

Khmelnitskiy, Anton; Saer, Rafael G; Blankenship, Robert E; Jankowiak, Ryszard

2018-04-12

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub 821 and Sub 817 ) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd

High-yield exfoliation of tungsten disulphide nanosheets by rational mixing of low-boiling-point solvents

NASA Astrophysics Data System (ADS)

Sajedi-Moghaddam, Ali; Saievar-Iranizad, Esmaiel

2018-01-01

Developing high-throughput, reliable, and facile approaches for producing atomically thin sheets of transition metal dichalcogenides is of great importance to pave the way for their use in real applications. Here, we report a highly promising route for exfoliating two-dimensional tungsten disulphide sheets by using binary combination of low-boiling-point solvents. Experimental results show significant dependence of exfoliation yield on the type of solvents as well as relative volume fraction of each solvent. The highest yield was found for appropriate combination of isopropanol/water (20 vol% isopropanol and 80 vol% water) which is approximately 7 times higher than that in pure isopropanol and 4 times higher than that in pure water. The dramatic increase in exfoliation yield can be attributed to perfect match between the surface tension of tungsten disulphide and binary solvent system. Furthermore, solvent molecular size also has a profound impact on the exfoliation efficiency, due to the steric repulsion.

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

PubMed Central

Prakash, Louise; Prakash, Satya

1977-01-01

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev ) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups. PMID:195865

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis.

PubMed

Pencík, Aleš; Casanova-Sáez, Rubén; Pilarová, Veronika; Žukauskaite, Asta; Pinto, Rui; Micol, José Luis; Ljung, Karin; Novák, Ondrej

2018-04-27

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism.

PubMed

Zheng, Xuelian; Yang, Shixin; Zhang, Dengwei; Zhong, Zhaohui; Tang, Xu; Deng, Kejun; Zhou, Jianping; Qi, Yiping; Zhang, Yong

2016-07-01

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

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

PubMed

Brokaw, C J; Luck, D J

1985-01-01

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

High-Throughput Sequencing of Campylobacter jejuni Insertion Mutant Libraries Reveals mapA as a Fitness Factor for Chicken Colonization

PubMed Central

Johnson, Jeremiah G.; Livny, Jonathan

2014-01-01

Campylobacter jejuni is a leading cause of gastrointestinal infections worldwide, due primarily to its ability to asymptomatically colonize the gastrointestinal tracts of agriculturally relevant animals, including chickens. Infection often occurs following consumption of meat that was contaminated by C. jejuni during harvest. Because of this, much interest lies in understanding the mechanisms that allow C. jejuni to colonize the chicken gastrointestinal tract. To address this, we generated a C. jejuni transposon mutant library that is amenable to insertion sequencing and introduced this mutant pool into day-of-hatch chicks. Following deep sequencing of C. jejuni mutants in the cecal outputs, several novel factors required for efficient colonization of the chicken gastrointestinal tract were identified, including the predicted outer membrane protein MapA. A mutant strain lacking mapA was constructed and found to be significantly reduced for chicken colonization in both competitive infections and monoinfections. Further, we found that mapA is required for in vitro competition with wild-type C. jejuni but is dispensable for growth in monoculture. PMID:24633877

High-throughput sequencing of Campylobacter jejuni insertion mutant libraries reveals mapA as a fitness factor for chicken colonization.

PubMed

Johnson, Jeremiah G; Livny, Jonathan; Dirita, Victor J

2014-06-01

Campylobacter jejuni is a leading cause of gastrointestinal infections worldwide, due primarily to its ability to asymptomatically colonize the gastrointestinal tracts of agriculturally relevant animals, including chickens. Infection often occurs following consumption of meat that was contaminated by C. jejuni during harvest. Because of this, much interest lies in understanding the mechanisms that allow C. jejuni to colonize the chicken gastrointestinal tract. To address this, we generated a C. jejuni transposon mutant library that is amenable to insertion sequencing and introduced this mutant pool into day-of-hatch chicks. Following deep sequencing of C. jejuni mutants in the cecal outputs, several novel factors required for efficient colonization of the chicken gastrointestinal tract were identified, including the predicted outer membrane protein MapA. A mutant strain lacking mapA was constructed and found to be significantly reduced for chicken colonization in both competitive infections and monoinfections. Further, we found that mapA is required for in vitro competition with wild-type C. jejuni but is dispensable for growth in monoculture.

Characterization of a Weak Allele of Zebrafish cloche Mutant

PubMed Central

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

2011-01-01

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

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

PubMed

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

Electron-induced electron yields of uncharged insulating materials

NASA Astrophysics Data System (ADS)

Hoffmann, Ryan Carl

Presented here are electron-induced electron yield measurements from high-resistivity, high-yield materials to support a model for the yield of uncharged insulators. These measurements are made using a low-fluence, pulsed electron beam and charge neutralization to minimize charge accumulation. They show charging induced changes in the total yield, as much as 75%, even for incident electron fluences of <3 fC/mm2, when compared to an uncharged yield. The evolution of the yield as charge accumulates in the material is described in terms of electron recapture, based on the extended Chung and Everhart model of the electron emission spectrum and the dual dynamic layer model for internal charge distribution. This model is used to explain charge-induced total yield modification measured in high-yield ceramics, and to provide a method for determining electron yield of uncharged, highly insulating, high-yield materials. A sequence of materials with progressively greater charge susceptibility is presented. This series starts with low-yield Kapton derivative called CP1, then considers a moderate-yield material, Kapton HN, and ends with a high-yield ceramic, polycrystalline aluminum oxide. Applicability of conductivity (both radiation induced conductivity (RIC) and dark current conductivity) to the yield is addressed. Relevance of these results to spacecraft charging is also discussed.

Epidemiology and impact of Fasciola hepatica exposure in high-yielding dairy herds

PubMed Central

Howell, Alison; Baylis, Matthew; Smith, Rob; Pinchbeck, Gina; Williams, Diana

2015-01-01

The liver fluke Fasciola hepatica is a trematode parasite with a worldwide distribution and is the cause of important production losses in the dairy industry. The aim of this observational study was to assess the prevalence of exposure to F. hepatica in a group of high yielding dairy herds, to determine the risk factors and investigate their associations with production and fertility parameters. Bulk milk tank samples from 606 herds that supply a single retailer with liquid milk were tested with an antibody ELISA for F. hepatica. Multivariable linear regression was used to investigate the effect of farm management and environmental risk factors on F. hepatica exposure. Higher rainfall, grazing boggy pasture, presence of beef cattle on farm, access to a stream or pond and smaller herd size were associated with an increased risk of exposure. Univariable regression was used to look for associations between fluke exposure and production-related variables including milk yield, composition, somatic cell count and calving index. Although causation cannot be assumed, a significant (p < 0.001) negative association was seen between F. hepatica exposure and estimated milk yield at the herd level, representing a 15% decrease in yield for an increase in F. hepatica exposure from the 25th to the 75th percentile. This remained significant when fertility, farm management and environmental factors were controlled for. No associations were found between F. hepatica exposure and any of the other production, disease or fertility variables. PMID:26093971

Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

PubMed

Govorun, R D; Koshlan, I V; Koshlan, N A; Krasavin, E A; Shmakova, N L

2002-01-01

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET. c2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

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

PubMed

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

2014-02-01

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

The pleiotropic ABNORMAL FLOWER AND DWARF1 affects plant height, floral development and grain yield in rice.