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3-22 JAEA-Review 2010-065 FACS-based Screening of Yeast Strain Highly Expressing Cellulase K. Ninomiya a) , H. Soda a) , K. Satoh b) , I. Narumi b) and N. Shimizu a) a) Division of Material Engineering, Graduate School of Natural Science and Technology, Kanazawa University, b) Radiation-Applied Biology Division, QuBS, JAEA By using immunocytochemistry and fluorescence-activated cell sorter (FACS), yeast population highly expressing cellulase on the cell surface was enriched from the mutant population prepared by irradiation with carbon ion beams (220 MeV 12 C 5+ , 100 Gy). The cellulase activity of selected yeasts was 5.2 × 10 –3 U/OD unit, whereas cellulase activity of parental strain was 1.6 × 10 -3 U/OD unit. 近年、非食用バイオマスであるセルロースを原料としたエタノール発酵生産への関心が高まっている。セルロース糖化酵素であるセルラーゼを発現する酵母を用いた“セルロースからの直接エタノール発酵生産”では、酵母のセルラーゼ発現量が生産コストを下げるための律速段階の 1 つと言われている。本研究では、セルラーゼ高発現酵母を育種するために、細胞表層に発現したセルラーゼの酵素量を蛍光免疫染色で蛍光強度に置き換えることにより、fluorescence-activated cell sorter(FACS)を用いて、セルラーゼ高発現株を蛍光強度の高い細胞集団として high-throughput に分取することを目的とした。使用菌株:3 種のセルラーゼ endoglucanase II(EGII)、 cellobiohydrase II(CBHII)ならびに β-glucosidase を細胞表層に発現した酵母 Saccharomyces cerevisiae MT8-1/pEG23u31H6/pFCBH2w3/pBG211 株 1) (MT8-1III 株)を用いた。また、MT8-1III 株に炭素イオン(220 MeV 12 C 5+ )を線量 100 Gy で照射し(以下、重イオンビーム照射)、変異株集団を調製した。酵母の培養は SD 培地を用いて 30 °C で行った。蛍光免疫染色および FACS:酵母表層のセルラーゼ分子の量を蛍光量として評価するため、EGII ならびに CBHII のタグとして融合発現させた RGSHis 6 および FLAG に対する一次抗体、それぞれの一次抗体に特異的な Alexa Fluor 488(FITC)および R-phycoerythrin(PE) 標識二次抗体を用いて二重染色を行った。蛍光免疫染色を行った酵母株は、FACS を用いて蛍光強度を測定し、 FITC ならびに PE の蛍光強度の高い細胞集団を目的株として細胞分取を行なった。セルラーゼ活性測定:酵母表層の EGII ならびに CBHII の総括のセルラーゼ活性を、カルボキシメチルセルロース(CMC)を基質として測定した。CMC が加水分解されることにより生じる還元末端の増加速度を 3,5-dinitrosalicylic acid(DNS)法を用いて測定し、セルラーゼ活性として評価した。1 min で 1 μmol の還元末端を生じる酵素活性を 1 U、また、波長 600 nm における濁度 OD 600 = 10 の酵母細胞懸濁液 1 mL を 1 OD unit と定義し、酵母細胞の持つセルラーゼ活性を U/OD unit で表した。 Figure 1(A)に親株である MT8-1III 株の蛍光免疫染色の結果を示す。FITC ならびに PE の蛍光強度(EGII ならびに CBHII の発現量に相当)の相対値が 10 4 以上の領域(P4 領域)に分布する細胞は全体の 1.6%であった。一方、Fig. 1(B)には、親株 MT8-1III 株に対して「重イ - 78 - オンビーム照射による変異導入」と「FACS による P4 領域の細胞集団の選抜」を行なった酵母の蛍光免疫染色の結果を示す。P4 領域に存在する細胞は全体の 6.9% に増加することが分かった。親株 MT8-1III 株と、重イオン変異と P4 領域の選抜を行なった酵母について、セルラーゼ活性を測定した。その結果、親株 MT8-1III 株のセルラーゼ活性が 1.6 × 10 -3 U/OD unit であったのに対し、選抜された酵母集団のセルラーゼ活性は 5.2 ×10 –3 U/OD unit へ向上していることが分かった。 Reference 1) Y. Fujita et al., Appl. Environ. Microbiol. 68 (2002) 5136. PE (Cellobiohydrase II) PE (Cellobiohydrase II) (A) 58.6% 14.0% 21.2% 4.5% (B) 48.3% 15.7% FITC (Endoglucanase II) 24.8% 4.2% FITC (Endoglucanase II) 1.6% 6.9% Fig. 1 Scatter plots of FITC and PE fluorescence corresponding to EGII and CBHII expression on yeast cells, respectively. (A) Parental yeast strain of MT8-1III. (B) Selected yeast from P4 region cells sorted from ion-beam irradiated MT8-1III.
3-23 Molecular Analysis of Carbon Ion Induced Mutations in Yeast Saccharomyces cerevisiae Cells K. Shimizu a) , Y. Matuo a, b) , Y. Izumi b) , Y. Hase c) , S. Nozawa c) , A. N. Sakamoto c) and I. Narumi c) a) Radioisotope Research Center, Osaka University, b) Research Institute of Nuclear Engineering, University of Fukui, c) Radiation-Applied Biology Division, QuBS, JAEA 1. Introduction This study is intended to elucidate the molecular mechanism of the mutagenesis caused by ion beam irradiation. Ion beam irradiation is expected to increase mutation frequency and spectrum, since it has a high linear energy transfer (LET). However, the detailed molecular mechanism of its action has not been proven. Recently, we reported that the main mutations induced by high-LET carbon-ion irradiation were GC to TA transversions 1) . DNA adducts such as 8-oxodG, which are produced by interactions of reactive oxygen species (ROS) with these molecules, can create various pairing formations in DNA molecules, for example, pairing with dA in the syn conformation to produce a GC to TA mutation 2) . In this study, we used the yeast mutant strains ogg1 and msh2, which are deficient in mismatch repair mechanisms. Mutations involving 8-oxodGs were caused by ion beam irradiation in the ogg1 and msh2 mutant strains. 2. Materials and methods 2.1. Yeast strains S. cerevisiae haploid strains used in this study were S288c (MATα, RAD), rad52 (MATα, G160/2b), ogg1 (MATα, OGG1) and msh2 (MATα, msh2). 2.2. Irradiation methods To determine the biological effectiveness of high LET ion beam irradiation, yeast cells were exposed to heavy ions accelerated at the AVF cyclotron (TIARA; JAEA Takasaki, Takasaki, Japan). As for the physical properties of the carbon ions, the JAEA-Review 2010-065 mean LET in the yeast was estimated to be 107 keV/μm water equivalent. This LET value was calculated by means of the ELOSS code program, which uses the elementary composition and the density of the target to calculate the energy loss of ion particles. 3. Results and Discussion One of the most common consequences of ROS exposure is the incorporation of oxidized nucleotides such as 8-oxodG into the genome. Mismatch repair has been shown to repair oxidative damage by lowering the level of 8-oxodG that is incorporated into the genome, presumably by recognizing mismatches different from those recognized by base excision repair. The distribution of the mutations induced by carbon ion beam irradiation is shown in Fig. 1. In the ogg1 mutant strain, there were minor hot spots at positions 330, 515, 602, and 745 (Fig. 1 (C)). The mutations in msh2 mutant were distributed evenly for base substitution, except for a minor hot spot at position 345. These results suggest that the incorporation of damaged nucleotides was not uniform. In comparison with the surrounding sequence context of mutational base sites, the C residues in the 5’- (A/T) C (A/T) -3’ sequence were found to be easily mutated (data not shown). These observations are in agreement with results obtained in wild type cells. References 1) Y. Matuo et al., Mutat. Res. 602 (2006) 7. 2) M. Inoue et al., J. Biol. Chem. 273 (1998) 11069. Fig. 1 The mutation spectra induced by carbon ion beam irradiation of yeast. White triangles: base substitute mutations, inverted black triangles: deletion/insertions. (A) nucleotide position number of URA3 gene; (B) mutation spectrum of wild type; (C) ogg1; (D) msh2. - 79 -
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JAEA-Review 2010-065 JAEA Takasaki
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JAEA-Review 2010-065 PREFACE This r
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JAEA-Review 2010-065 and pulsed hea
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JAEA-Review 2010-065 1-23 Studies o
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JAEA-Review 2010-065 3-24 Lethal Ef
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JAEA-Review 2010-065 4-07 Fabricati
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JAEA-Review 2010-065 5. Status of I
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JAEA-Review 2010-065 1-13 Alpha-rad
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1-01 Radiation Resistance of InGaP/
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1-03 Evaluation of Soft Error Rates
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1-05 Heavy-ion Induced Current in S
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Total Ionizing Dose Tolerance of Si
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1-09 Evaluation of Single Event Eff
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Hydrogen Diffusion in a-Si:H Thin F
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1-13 Alpha-radiolysis of Organic Ex
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Study on Stability of Cs·Sr Solven
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Irradiation Effect of Gamma-Rays on
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1-19 Development of Radiation Resis
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1-21 Alpha-Ray Irradiation Damage o
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Page 45 and 46: 1-25 Effect of Groundwater Radiolys
Page 47 and 48: 1-27 Simulation of Neutron Damage M
Page 49 and 50: 1-29 Irradiation Hardening in Ion-i
Page 51 and 52: 1-31 Preparation of Anion-Exchange
Page 53 and 54: 1-33 Radiation-Induced Graft Polyme
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Page 58 and 59: 2-02 Development of Zwitterionic Mo
Page 60 and 61: Modification of Hydroxypropyl Cellu
Page 62 and 63: The Recovery of Precious Metals Usi
Page 64 and 65: 2-08 ESR Study on Carboxymethyl Chi
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Page 69 and 70: JAEA-Review 2010-065 3-28 Electron
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Page 74 and 75: 3-02 Mutagenic effects of He ion pa
Page 76 and 77: 3-04 Functional Analysis of Flavono
Page 78 and 79: 3-06 Generation New Ornamental Plan
Page 80 and 81: 3-08 Stability of Flower-colour Mut
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Page 84 and 85: 3-12 Producing New Gene Resources i
Page 86 and 87: 3-14 Production of Soybean Mutants
Page 88 and 89: Assessment of Irradiation Treatment
Page 90 and 91: 3-18 Effect of Different LET Radiat
Page 92 and 93: 3-20 JAEA-Review 2010-065 Fungicide
Page 96 and 97: 3-24 Lethal Effects of Different LE
Page 98 and 99: 3-26 JAEA-Review 2010-065 Ion Beam
Page 100 and 101: 3-28 Electron Spin Relaxation Behav
Page 102 and 103: 3-30 Target Irradiation of Individu
Page 104 and 105: 3-32 Carbon-ion Microbeam Induces B
Page 106 and 107: 3-34 Biological Effects of Carbon I
Page 108 and 109: 3-36 Difference in Bystander Lethal
Page 110 and 111: 3-38 Analysis of Lethal Effect Medi
Page 112 and 113: 3-40 Irradiation with Carbon Ion Be
Page 114 and 115: 3-42 Expression of Two Gelsolins in
Page 116 and 117: 3-44 Analysis of Bystander Cell Sig
Page 118 and 119: 3-46 Quantitative Evaluation of Ric
Page 120 and 121: 3-48 Visualization of 107 Cd Accumu
Page 122 and 123: 3-50 Uniformity Measurement of Newl
Page 124 and 125: 3-52 Imaging and Biodistribution of
Page 126 and 127: 3-54 Improvement of Spatial Resolut
Page 128 and 129: 3-56 The Optimum Conditions in the
Page 130 and 131: 3-58 Evaluation of Cisplatin Concen
Page 132 and 133: 3-60 JAEA-Review 2010-065 Analysis
Page 134 and 135: 3-62 JAEA-Review 2010-065 Sensitivi
Page 136 and 137: JAEA-Review 2010-065 4-14 The Effec
Page 138 and 139: JAEA-Review 2010-065 4-39 Relations
Page 141 and 142: 4-01 Hydrogen Gasochromism of WO3 F
Page 143 and 144: 4-03 Synthesis of Single-Crystallin
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4-05 Synergy Effects in Electron/Io
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Fabrication of Diluted Magnetic Sem
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4-09 Gas Permeation Characteristics
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4-11 Control of Radial Size of Poly
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4-13 Behavior of N Atoms in Nitridi
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4-15 JAEA-Review 2010-065 Annealing
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Low Temperature Ion Channeling of F
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4-19 Radiation-Induced Electrical D
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4-21 Study on Cu Precipitation in E
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4-23 Evaluation of Fluorescence Mat
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4-25 Evaluation of the ZrC Layer fo
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4-27 Structure Analysis of K/Si(111
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4-29 LET Effect on the Radiation In
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4-31 Stabilization of Measurement S
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Systematic Measurement of Neutron a
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4-35 Measurement of Neutron Fluence
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4-37 Evaluation of the Response Cha
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4-39 Relationship between Internucl
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4-41 Analysis of Radiation Damage a
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4-43 Positive Secondary Ion Emissio
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4-45 JAEA-Review 2010-065 Ion Induc
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4-47 Fabrication of Dielectrophoret
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4-49 Fast Single-Ion Hit System for
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4-51 Development of Beam Generation
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JAEA-Review 2010-065 5. Status of I
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Operation of the AVF Cyclotron T. N
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Operation of Electron Accelerator a
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5-06 FACILITY USE PROGRAM in Takasa
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5-08 Radioactive Waste Management i
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Appendix 2 List of Related Patents
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Appendix 4 Type of Research Collabo
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表1.SI 基本単位基本量 SI
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