Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution Ehime University
 
2.Research Area Integrated Fields
 
3.Research Field Production of Novel Useful Substances through Integration of Life Sciences and Chemistry
 
4.Term of Project FY1996〜FY2000
 
5.Project Number 96I00305
 
6.Title of Project Development of A Robust, Highly Efficient Cell-Free Protein Synthesis System

7.Projetct Leader
Name Institution,Department Title of Position
Yaeta Endo Ehime University, Faculty of Engineering Professor

8.Core Members

Names Institution,Department Title of Position
Takuya Ueda The University of Tokyo, Graduate School Professor
Akira Muto Hirosaki University, Faculty of Agriculture and Life Sciences Professor
Tairo Oshima Tokyo University of Pharmacy and Life Science, school of Life Sciences Professor

9.Cooperating Researchers

10.Summary of Research Results

A plant's natural ribotoxins seemed a likely factor in the instability of wheat germ cell-free system, in which contamination with tritin-containing endosperm fragments would be a serious problem. Accordingly, we prepared our cell-free system from extensively washed embryos, and indeed found that the system became far more active. In a batch system we observed continuous translation for 4 hours, and sucrose density gradient analysis showed formation of large polysomes indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for more than 60 hours yielding 1 to 4 mg of enzymatically active proteins, and 0.6 mg of a 126 kDa TMV protein per milliliter reaction.
Our wheat germ extract is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself. In addition to the benefit of a better translation system, these results shed new light on the translational apparatus itself: While it is usually seen as a rather fragile apparatus, it appears instead to be very stable; so stable and important, in fact, that plants chose this machinery as the target for their defensive suicide mechanism, the RIPs. It might also be important to mention here that the observed high stability of the translational apparatus could be an essential requirement for the evolution of life.

11.Key Words

(1)Translation、(2)Wheat germ、(3)Cell-free protein synthesis
(4)Ribosome、(5)ARS、(6)tRNA
(7)mRNA、(8)Translation factors、(9)Production of protein

12.References

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Shoji Ohuchi In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled
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Masatada TAMAKOSHI The organization of the leuC, leuD and leuB genes of the extreme therm ophile Therm us therm ophilus
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Gene 222 1998 125-132

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Nakamura, Y. The major surface antigen of Pneumocystis carinii.
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FEMS Immun. Med. Microbiol. 22 1998 67-74

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J. Med. Mycol. 39 1998 91-98

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Mol. Microbiol. 28 1998 629-640

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Ito, K. The stretch of C-terminal acidic amino acids of translational release factor eRF1 is a primary binding site for eRF3 of fission yeast.
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RNA 4 1998 958-972

Author Title of Article
Ito, K. Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons.
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Proc. Natl. Acad. Sci. USA 95 1998 8165-8169

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Nakamura, Y. How protein reads the stop codon and terminates translation (Review).
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Genes Cells 3 1998 263-278

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Aoki, M. Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalvtic activity and subunit assembly of inorganic
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Biochem. J. 331 1998 143-148

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Arthritis Rheum. 41 1998 1040-1046

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EMBO J. 17 1998 3188-3196

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Soma, A. Cross-species aminoacylation of tRNA with a long variable arm between Escherichia coli and Saccharomyces cerevisiae.
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Nucleic Acids Res. 26 1998 4374-4381

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Ando, K. Specific binding proteins of M. capricolum MCS4 RNA.
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Japanese Journal of Micoplasmology 25 1998 88-90

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Kidou, S. Isolation, characterization and mRNA expression of four cDNAs encoding translation elongation factor 1A from rice (Oryza sativa L. ).
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Plant Mol. Biol. 36 1998 137-148

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Kidou, S. Isolation and characterization of a rice cDNA encoding the γ -subunit of translation elongation factor 1B (eEF1Bγ).
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Biochim. Biophys. Acta, 1442 1998 369-372

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K. Ogata ATP ase associated with ribosomal 30S-5SRNP particles and 40S subunits of rat liver
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J. Biochem. 123 1998 294-304

Author Title of Article
Nitta H. A novel cell-free system for peptide synthesis driven by pyridine,
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Biol. Chem. 379 1998 819-829

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Y. Endo Mechanisms of Action of Ribotoxins
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J. Toxicol. 17 1998 427-439

Author Title of Article
Takemoto, C. Nucleotide sequences of animal mitochondrial tRNAs(Met) possibly recognizing both AUG and AUA codons.
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Nucleic Acids Symposium Series. 42 1999 77-78

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Chordata, Urochordata) Complete DNA Sequence of the Mitochondrial Genome of the Ascidian Halocvnthia roretzi
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Genetics 153, 1851-1862   1999  

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Tomita, K. Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences:a unique wobble rule In animal mitochondria
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Nucleic Acids Research 27, 4291-4297,   1999  

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Suzuki, T. High Sensitive Analysis of Modified Nucleosides by LC/MS Using ESI/Iontrap Mass Spectrometry
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J. Mass Spectrom. Soc. Jpn. 47 1999 168-176

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Sasuga, J. Gene Contents and Organization of a Mitochondrial DNA Segment of the Squid Loligo bleekeri
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J. Mol. Evol. 48 1999 692-702

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Kondow, A. An extra tRNAGly(U*CU)found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as glycine
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Nucleic Acids Research 27 1999 2554-2559

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Ogawa, T. A cytotoxic ribonuclease targeting specific transfer rna anticodons
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Science 283 1999 2097-2100

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Tomita, T. The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as
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Nucleic Acids Research 27 1999 1683-1689

Author Title of Article
Yasukawa, T. Modification Defect at Anticodon Wobble Nucleotide of Mitochondrial tRNAsLeu(UUR) with Pathogenic Mutations of Mitochondrial
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J. Biol. Chem. 275 1999 4251-4257

Author Title of Article
Hohsaka, T. Efficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems
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J. Am. Chem. Soc. 121 1999 34-40

Author Title of Article
Hohsaka, T. Incorporation of Two Different Nonnatural Amino Acids Independently into a Single Protein through Extension of the Genetic Code
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J. Am. Chem. Soc. 121 1999 12194-12195

Author Title of Article
宍戸昌彦 “人工光機能蛋白質 光電子移動 DNA光分解 抗体光制御"
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光化学 30 1999 58-63

Author Title of Article
Sisido, M. Extension of Protein Functions by the Incorporation of Nonnatural Amino Acids
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Bull. Chem. Soc. Jpn. 72 1999 1409-1425

Author Title of Article
Hideo Nakano Efficient coupled transcription/translation for PCR template by a hollow fiber membrane bioreactor.
Journal Volume Year Pages Concerned
Biotechnology and Bioengineering 64 1999 194-199

Author Title of Article
Junhao Yang Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system.
Journal Volume Year Pages Concerned
Journal of Bioscience and Bioengineering 88 1999 605-609

Author Title of Article
Kigawa, T. Cell-free production and stable-isotope labeling of milligram quantities of proteins
Journal Volume Year Pages Concerned
FEBS Lett. 442 1999 15-19

Author Title of Article
木川隆則 “無細胞タンパク質合成系による大量調製法"
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蛋白質核酸酵素 44 1999 598-605

Author Title of Article
M. Esaki, Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cvtochorme b2
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Proc. Natl. Acad. Sci. USA 96 1999 11770-11775

Author Title of Article
Y. Kubo, Two distinct mechanisms operate in reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdi1p/Yge1p chaperone svstem
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J. Mol. Biol. 286 1999 447-464

Author Title of Article
T. Kanamori, Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer
Journal Volume Year Pages Concerned
Proc. Natl. Acad. Sci. USA 96 1999 3634-3639

Author Title of Article
T. Asai, Sec-dependent pathway and DpH-depemdent pathway do not share a common translocation pore in thvlakoidal protein transport
Journal Volume Year Pages Concerned
J. Biol. Chem. 274 1999 20075-20078

Author Title of Article
Masatada TAMAKOSHI An efficient gene replacemant and deletion system for an extreme thermophile, Thermus thermophilus.
Journal Volume Year Pages Concerned
FEMS Microbiol. Letters 173 1999 431-437

Author Title of Article
Akira CHIBA Coenzyme Activity of NAD Analogs for 3-Isopropylmalate Dehydrogenase from Thermus thermophilus HB8
Journal Volume Year Pages Concerned
Biosci. Biotechnol. Biochem. 63 1999 1647-1649

Author Title of Article
Nathaniel J. COSPER Structural Conservation of the Isolated Ainc Site in Archaeal Sinc-containing Ferredoxins as Revealed by X-ray Absorption
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J. Biol. Chem. 274 1999 23160-23168

Author Title of Article
Akira CHIBA Synthetic and Mechanistic Studies of 2R (3S)-3-Vinylmalic Acid as a Mechanism-Based Inhibitor of 3-Isopropylmalate Dehvdrogenase
Journal Volume Year Pages Concerned
J. Org. Chem. 64 1999 6159-6165

Author Title of Article
Mita, K. The cDNA and intron catalog of ribosomal protein genes of Schizosaccharomvces pombe.
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Nucl. Acids Res, In Press 1999  

Author Title of Article
Crawford, D. -J. G. Indirect regulation of termination efficiency at highly expressed genes and recoding sites by the factor recycling function of Escherichia coli
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EMBO J. 18 1999 727-732

Author Title of Article
Ebihara, K. C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved
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RNA 5 1999 739-750

Author Title of Article
Fujiwara, T. Amber mutations in ribosome recycling factors of Escherichia coli and Therm us thermophilus: evidence for C-terminal modulator element.
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FEBS Lett. 447 1999 297-302

Author Title of Article
Yoshimura, K. Amber (UAG) suppressors affected in UGA/UAA-specific polypeptide release factor 2 of bacteria: genetic prediction of initial binding to
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Genes Cells 4 1999 253-266

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Powell, B. Cloning and analysis of the rnc-era-recO operon from Pseudomonas aeruginosa.
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J. Bacteriol. 181 1999 5111-5113

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Karamyshev, A. Polypeptide release factor eRF1 from Tetrahymena thermophile: cDNA cloning, purification and complex form ation with yeast eRF3.
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FEBS Lett. 457 1999 483-488

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Karamyshev, A. Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila.
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Biochemistry (Moscow) 64 1999 1648-1658

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Wada, M. Type-II major-surface-glycoprotein family of Pneumocystis carinii.
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Wada, M. Immunological characterization of surface subtilisin-like protease (SSP) of Pneumocystis carinii.
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J. Euk. Microbiol. 46 1999 125S-126S

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Wada, M. Type-II major-surface-glycoprotein family of Pneumocystis carinii under the control of novel expression elements.
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DNA Res. 6 1999 211-217

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J. Biol. Chem. 274 1999 681-686

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Hasegawa, H. High frequency of antibody activity against ribosomal protein S10 in anti-Sm sera from patients with systemic lupus erythematosus
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Lupus 8 1999 439-443

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Uchiumi, T. Replacement of L7/L12. L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of
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J. Biol. Chem. 274 1999 27578-27582

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Nameki, N. Functional and structural analysis of a pseudoknot upstream of the tag-encoding sequence in E. coli tmRNA.
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J. Mol. Biol. 286 1999 733-744

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Nameki, N. Amino acid acceptor identity switch of Escherichia coli tmRNA from alanine to histidine in vitro.
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J. Mol. Biol. 289 1999 1-7

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Nameki, N. An NMR and mutational study of an RNA pseudoknot of E. coli tmRNA involved in trans-translation.
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Nucleic Acids Res. 27 1999 3667-3675

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J. Mol. Biol. 293 1999 1029-1038

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姫野俵太 tmRNAによるtrans-translationに関する研究
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生化学 79 1999 1119-1130

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Author Title of Article
Ryo Morishita Ribonuclease Activity of Rat Liver Perchloric Acid-Soluble Protein, A Potent Inhibitor of Protein Synthesis
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The EMBO J. 18 1999 6522-6531

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Genes to Cells 5 2000 689-698

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FEBS Letters 467 2000 175-178

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H. Murakami 1 2000 118-125

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Biochem. Biophys. Res. Commun. 270 2000 1136-1139

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Chem. Commun. 2000 2000 1199-1200

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Proc. Japan Acad. B76 2000 92-96

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Electrochemistry 68 2000 352-355

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Yokoyama, S. Structural genomics projects in japan
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生物物理 232 2000 391-394

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Cell 100 2000 551-560

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Nucleic Acid Symp. Ser 44 2000 167-168

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Genes Cells 5 2000 627-636

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Author Title of Article
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Author Title of Article
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Author Title of Article
T. Uchiumi A Covariant Change of the Two Highly Conserved Bases in the GTPase-associated Center of 28 S rRNA in Silkworms and Other
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J. Biol. Chem. 275 2000 35116-35121

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N. Nameki Three of four pseudoknots in tm RNA are interchangable and are substitutable with single-stranded RNAs.
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FEBS Lett. 470 2000 345-349

Author Title of Article
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Genes to Cells 5 2000 627-636

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Extremophiles 5 2001 17-22

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Clin. Immunol. 98 2001 249-257

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C. J. Anderson Auto antibodies to the 20-kDa Ribosomal Proteins: Identification, Characterization, and New Aspects on Prevalence in Systemic Lupus
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Clinical Immunology 98 2001 249-257

Author Title of Article
Sato, K. Localization of mitochondrial ribosomal RNAs on the chromatoid bodies of the marine planarian polyclad embryos.
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Develop. Growth Differ. in press    

Author Title of Article
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submitted      

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submitted      

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河原崎泰昌 化学と生物,
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Good-by“大腸菌"無細胞タンパク質合成系の挑戦58(8), 2000 250-257

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中野秀雄 バイオサイエンスとインダストリー
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IV. 無細胞系. 組替えタンパク質生産法(生物化学実験法), 塚越紀弘編 著, 2000 11-15

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姫野俵太 学会出版センター
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武藤晃 シュプリンガー・フェアラーク東京
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牛田千里 シュプリンガー・フェアラーク東京
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遠藤弥重太 シュプリンガー・フェアラーク東京
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遠藤彌重太 スプリンガー・フェアラーク東京
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遠藤彌重太 BIO INDUSTRY
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山根恒夫 (株)学会出版センター
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Madin, K. Springer-Verlag GmbH&Co. KG WIKOM-Publication Services
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遠藤彌重太 Springer-Verlag
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