PH27026A - Expression and secretion of heterologous proteins by yarrowia lipolytica transformants - Google Patents

Expression and secretion of heterologous proteins by yarrowia lipolytica transformants Download PDF

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Publication number
PH27026A
PH27026A PH34383A PH34383A PH27026A PH 27026 A PH27026 A PH 27026A PH 34383 A PH34383 A PH 34383A PH 34383 A PH34383 A PH 34383A PH 27026 A PH27026 A PH 27026A
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PH
Philippines
Prior art keywords
lipolytica
sequence
gene
atcc
transformant
Prior art date
Application number
PH34383A
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English (en)
Inventor
Lance Steven Davidow
John Robert Dezeeuw
Arthur Ernest Franke
Original Assignee
Pfizer
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer filed Critical Pfizer
Priority to PH36896A priority Critical patent/PH27171A/en
Priority to PH36903A priority patent/PH27198A/en
Publication of PH27026A publication Critical patent/PH27026A/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478Aspartic endopeptidases (3.4.23)
    • C12N9/6481Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52Genes encoding for enzymes or proenzymes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90Stable introduction of foreign DNA into chromosome
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/55Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Magnetic Ceramics (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Treatment Of Sludge (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Paper (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
  • Measurement Of Velocity Or Position Using Acoustic Or Ultrasonic Waves (AREA)
  • Fodder In General (AREA)
  • Noodles (AREA)
PH34383A 1985-10-18 1986-10-17 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants PH27026A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PH36896A PH27171A (en) 1985-10-18 1988-05-06 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants
PH36903A PH27198A (en) 1985-10-18 1988-05-06 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US78920685A 1985-10-18 1985-10-18
US06/841,121 US4937189A (en) 1985-10-18 1986-03-18 Expression and secretion of heterologous proteins by Yarrowia lipolytica transformants

Publications (1)

Publication Number Publication Date
PH27026A true PH27026A (en) 1993-02-01

Family

ID=27120892

Family Applications (3)

Application Number Title Priority Date Filing Date
PH34383A PH27026A (en) 1985-10-18 1986-10-17 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants
PH36903A PH27198A (en) 1985-10-18 1988-05-06 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants
PH36896A PH27171A (en) 1985-10-18 1988-05-06 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants

Family Applications After (2)

Application Number Title Priority Date Filing Date
PH36903A PH27198A (en) 1985-10-18 1988-05-06 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants
PH36896A PH27171A (en) 1985-10-18 1988-05-06 Expression and secretion of heterologous proteins by yarrowia lipolytica transformants

Country Status (24)

Country Link
US (1) US4937189A (xx)
EP (1) EP0220864B1 (xx)
JP (1) JP3096297B2 (xx)
KR (1) KR900008950B1 (xx)
CN (3) CN1034428C (xx)
AT (1) ATE86302T1 (xx)
AU (1) AU586041B2 (xx)
CA (1) CA1340837C (xx)
DD (7) DD276886A5 (xx)
DE (1) DE3687878T2 (xx)
DK (1) DK496886A (xx)
EG (1) EG18142A (xx)
ES (1) ES2038966T3 (xx)
FI (1) FI90352C (xx)
HU (1) HU209150B (xx)
IE (1) IE59774B1 (xx)
IL (7) IL80297A (xx)
NO (2) NO177318C (xx)
NZ (1) NZ217985A (xx)
PH (3) PH27026A (xx)
PL (2) PL155850B1 (xx)
PT (1) PT83562B (xx)
RU (2) RU2157845C2 (xx)
YU (2) YU46412B (xx)

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JPH082304B2 (ja) * 1987-12-17 1996-01-17 オリエンタル酵母工業株式会社 プラスミドとその製造方法
FR2626584B1 (fr) * 1988-01-28 1990-07-13 Agronomique Inst Nat Rech Sequence ars efficace chez yarrowia lipolytica et procede pour sa preparation
EP0382332A3 (en) * 1989-01-06 1990-11-14 Collaborative Research Inc. Improved supersecreting mutants of saccharomyces cerevisiae
JPH0669365B2 (ja) * 1989-05-22 1994-09-07 株式会社ミドリ十字 アルブミン遺伝子を含むプラスミド、形質転換体、形質転換体の製造方法、アルブミンの製造方法
EP0402226A1 (en) * 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
EP0716704A1 (en) * 1993-09-02 1996-06-19 Pfizer Inc. MULTIPLE INTEGRATIVE VECTORS AND $i(YARROWIA LIPOLYTICA) TRANSFORMANTS
US5807824A (en) * 1993-12-06 1998-09-15 Ciba-Geigy Corporation C5A receptor antagonists having substantially no agonist activity
US5837499A (en) * 1993-12-06 1998-11-17 Ciba-Geigy Corporation DNA encoding C5A receptor antagonists having substantially no agonist activity and methods of expressing same
DE19513152A1 (de) 1995-04-07 1996-10-10 Bundesrep Deutschland Verwendung eines "Immundefizienzvirus-supprimierenden Lymphokins (ISL)" zur Hemmung der Virusvermehrung, insbesondere von Retroviren
EP0832223A1 (en) * 1995-06-05 1998-04-01 Novartis AG C5a RECEPTOR ANTAGONISTS HAVING SUBSTANTIALLY NO AGONIST ACTIVITY AND METHODS FOR PREPARATION
EP0747484A1 (en) 1995-06-08 1996-12-11 Institut National De La Recherche Agronomique (Inra) Upstream activator sequences and recombinant promoter sequences functional in yarrowia and vectors containing them
WO1997044470A1 (en) 1996-05-21 1997-11-27 Novo Nordisk A/S Novel yeast promoters suitable for expression cloning in yeast and heterologous expression of proteins in yeast
FR2782733B1 (fr) * 1998-09-01 2002-02-08 Agronomique Inst Nat Rech Procede de transformation non-homologue de yarrowia lipolytica
US7459546B2 (en) 2003-06-25 2008-12-02 E.I. Du Pont De Nemours And Company Glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase regulatory sequences for gene expression in oleaginous yeast
US20110059496A1 (en) * 2003-06-25 2011-03-10 E. I. Du Pont De Nemours And Company Glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase promoters for gene expression in oleaginous yeast
US7259255B2 (en) * 2003-06-25 2007-08-21 E. I. Du Pont De Nemours And Company Glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate mutase promoters for gene expression in oleaginous yeast
US7202356B2 (en) * 2003-11-14 2007-04-10 E. I. Du Pont De Nemours And Company Fructose-bisphosphate aldolase regulatory sequences for gene expression in oleaginous yeast
US7264949B2 (en) * 2004-09-15 2007-09-04 E.I. Du Pont De Nemours And Company Glycerol-3-phosphate o-acyltransferase promoter for gene expression in oleaginous yeast
US20060094102A1 (en) * 2004-11-04 2006-05-04 Zhixiong Xue Ammonium transporter promoter for gene expression in oleaginous yeast
WO2006102342A2 (en) 2005-03-18 2006-09-28 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US8691555B2 (en) 2006-09-28 2014-04-08 Dsm Ip Assests B.V. Production of carotenoids in oleaginous yeast and fungi
FR2927089B1 (fr) * 2008-02-05 2011-03-25 Inst Nat De La Rech Agronomique Inra Procede d'integration ciblee de multicopies d'un gene d'interet dans une souche de yarrowia
WO2010004141A2 (fr) * 2008-07-11 2010-01-14 Institut National De La Recherche Agronomique (Inra) Nouvelles souches de levure mutantes capables d'accumuler une grande quantité de lipides
FR2962133B1 (fr) 2010-07-01 2014-09-12 Agronomique Inst Nat Rech Optimisation de la synthese et de l'accumulation de lipides
BR112013016769A2 (pt) * 2010-12-30 2016-10-11 Du Pont yarrowia lipolytica transformada e método para produzir pelo menos um produto não nativo de interesse
WO2012135773A1 (en) 2011-03-31 2012-10-04 E. I. Du Pont De Nemours And Company Yarrowia diacylglycerol acyltransferase promoter regions for gene expression in yeast
EP2694657A1 (en) 2011-04-05 2014-02-12 E. I. Du Pont de Nemours and Company Yarrowia n-alkane-hydroxylating cytochrome p450 promoter regions for gene expression in yeast
US8609369B2 (en) 2011-04-07 2013-12-17 E I Du Pont De Nemours And Company Yarrowia peroxisomal 2,4-dienoyl-CoA reductase promoter regions for gene expression in yeast
GB201106188D0 (en) 2011-04-12 2011-05-25 Csir Production of extracellular polypeptides
FR3002774A1 (fr) 2013-03-04 2014-09-05 Agronomique Inst Nat Rech Levures mutantes ayant une production accrue de lipides et d'acide citrique
FR3005317B1 (fr) 2013-05-02 2016-03-18 Agronomique Inst Nat Rech Levures mutantes capables de produire un acide gras inhabituel
WO2015116781A2 (en) 2014-01-31 2015-08-06 Dsm Ip Assets B.V. Promoters suitable for heterologous gene expression in yeast
EP3155094A1 (en) 2014-06-11 2017-04-19 Institut National De La Recherche Agronomique (INRA) Improved lipid accumulation in yarrowia lipolytica strains by overexpression of hexokinase and new strains thereof
WO2016075314A1 (fr) 2014-11-13 2016-05-19 Institut National De La Recherche Agronomique Identification de facteurs de transcription de yarrowia lipolytica affectant la production de proteines
FR3028527A1 (fr) 2014-11-13 2016-05-20 Pivert Identification de facteurs de transcription de yarrowia lipolytica
WO2017100158A1 (en) 2015-12-11 2017-06-15 Danisco Us Inc. Methods and compositions for enhanced nuclease-mediated genome modification and reduced off-target site effects
EP3390631B1 (en) 2015-12-18 2020-04-08 Danisco US Inc. Methods and compositions for t-rna based guide rna expression
EP3455362A1 (en) 2016-05-10 2019-03-20 Institut National De La Recherche Agronomique Mutant yeast strains with enhanced production of erythritol or erythrulose
EP3348647A1 (en) 2017-01-13 2018-07-18 Institut National De La Recherche Agronomique Mutant yeast strain capable of producing medium chain fatty acids

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US4321365A (en) * 1977-10-19 1982-03-23 Research Corporation Oligonucleotides useful as adaptors in DNA cloning, adapted DNA molecules, and methods of preparing adaptors and adapted molecules
US4394443A (en) * 1980-12-18 1983-07-19 Yale University Method for cloning genes
US4666847A (en) * 1981-01-16 1987-05-19 Collaborative Research, Inc. Recombinant DNA means and method
US4775622A (en) * 1982-03-08 1988-10-04 Genentech, Inc. Expression, processing and secretion of heterologous protein by yeast
US4546082A (en) * 1982-06-17 1985-10-08 Regents Of The Univ. Of California E. coli/Saccharomyces cerevisiae plasmid cloning vector containing the alpha-factor gene for secretion and processing of hybrid proteins
NZ207926A (en) * 1983-04-25 1988-04-29 Genentech Inc Use of yeast #a#-factor to assist in expression of proteins heterologus to yeast
IN163393B (xx) * 1983-10-06 1988-09-17 Pfizer
US4628033A (en) * 1983-10-06 1986-12-09 Pfizer Inc. Novel host strain for transformation of Yarrowia lipolytica
DE3572688D1 (en) * 1984-06-26 1989-10-05 Agronomique Inst Nat Rech Transformation vector for yeast yarrowia lipolytica, transformation process and transformed yeast

Also Published As

Publication number Publication date
PL155850B1 (en) 1992-01-31
AU6416186A (en) 1987-05-14
KR870004138A (ko) 1987-05-07
YU177386A (en) 1989-04-30
CN1138632A (zh) 1996-12-25
NO177318B (no) 1995-05-15
IL99894A0 (en) 1992-08-18
AU586041B2 (en) 1989-06-29
NO864148L (no) 1987-04-21
IE59774B1 (en) 1994-04-06
ES2038966T3 (es) 1993-08-16
PH27171A (en) 1993-04-02
IL99893A0 (en) 1992-08-18
CA1340837C (en) 1999-12-07
CN1034428C (zh) 1997-04-02
IL80297A0 (en) 1987-01-30
DK496886A (da) 1987-06-11
NO923616D0 (no) 1992-09-17
DD276886A5 (de) 1990-03-14
IL99893A (en) 1993-02-21
NO178793C (no) 1996-06-05
FI864206A0 (fi) 1986-10-17
EG18142A (en) 1992-08-30
PT83562B (pt) 1989-05-31
CN1141339A (zh) 1997-01-29
DD269396A5 (de) 1989-06-28
DD267996A5 (de) 1989-05-17
IL99894A (en) 1993-02-21
EP0220864B1 (en) 1993-03-03
RU2069694C1 (ru) 1996-11-27
PL154427B1 (en) 1991-08-30
DD267995A5 (de) 1989-05-17
IL80297A (en) 1993-02-21
DE3687878D1 (de) 1993-04-08
HUT43632A (en) 1987-11-30
NO864148D0 (no) 1986-10-17
HU209150B (en) 1994-03-28
CN1069346C (zh) 2001-08-08
NO178793B (no) 1996-02-26
ATE86302T1 (de) 1993-03-15
DD267998A5 (de) 1989-05-17
FI864206A (fi) 1987-04-19
IE862754L (en) 1987-04-18
FI90352C (fi) 1994-01-25
NZ217985A (en) 1991-06-25
DK496886D0 (da) 1986-10-17
PT83562A (en) 1986-11-01
PL261912A1 (en) 1987-10-19
CN86106522A (zh) 1987-09-16
YU46571B (sh) 1993-11-16
FI90352B (fi) 1993-10-15
IL99892A (en) 1993-02-21
KR900008950B1 (ko) 1990-12-15
YU124588A (en) 1989-08-31
US4937189A (en) 1990-06-26
PH27198A (en) 1993-04-16
IL99892A0 (en) 1992-08-18
YU46412B (sh) 1993-10-20
DE3687878T2 (de) 1993-07-15
DD269397A5 (de) 1989-06-28
JP3096297B2 (ja) 2000-10-10
JPS63164889A (ja) 1988-07-08
RU2157845C2 (ru) 2000-10-20
EP0220864A1 (en) 1987-05-06
NO923616L (no) 1987-04-21
DD267997A5 (de) 1989-05-17
NO177318C (no) 1995-08-23

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