EP0618975A1 - Adn, constructions d'adn, et cellules et plantes derivees - Google Patents

Adn, constructions d'adn, et cellules et plantes derivees

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Publication number
EP0618975A1
EP0618975A1 EP92907487A EP92907487A EP0618975A1 EP 0618975 A1 EP0618975 A1 EP 0618975A1 EP 92907487 A EP92907487 A EP 92907487A EP 92907487 A EP92907487 A EP 92907487A EP 0618975 A1 EP0618975 A1 EP 0618975A1
Authority
EP
European Patent Office
Prior art keywords
dna
ptom75
plants
dna constructs
fruit
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP92907487A
Other languages
German (de)
English (en)
Inventor
Rupert George Fray
Donald Grierson
Grantley Walter Lycett
Wolfgang Walter Schuch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Ltd
Original Assignee
Zeneca Ltd
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 Zeneca Ltd filed Critical Zeneca Ltd
Publication of EP0618975A1 publication Critical patent/EP0618975A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life

Definitions

  • This application relates to novel DNA constructs, plant cells containing them and plants derived therefrom. In particular it involves the control of gene expression in plants .
  • a cell manufactures protein by transcribing the DNA of the gene for that protein to produce messenger RNA (mRNA), which is then processed (eg by the removal of introns) and finally translated by ribosomes into protein.
  • mRNA messenger RNA
  • antisense RNA an RNA sequence which is complementary to a sequence of bases in the mRNA in question: complementary in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense.
  • RNA Ribonucleic acid
  • RNA Ribonucleic acid
  • Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to transcribe backwards part of the coding strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).
  • the use of this technology to downregulate the expression of specific plant genes has been described, for example in European Patent publication no 271988 to ICI (corresponding to US serial 119614).
  • DNA constructs comprising a DNA sequence homologous to some or all of the gene encoded by the clone pTOM75.
  • the homologous DNA sequence may be preceded by a transcriptional initiation region operative in plants, so that the construct can generate mRNA in plant cells.
  • the present invention provides DNA constructs comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA complementary to a substantial run of bases showing substantial homology to an mRNA encoding the protein produced by the gene in pTOM75.
  • the invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; and fruit and seeds of such plants.
  • the constructs of the invention may be inserted into plants to regulate the production of enzymes encoded by genes homologous to pTOM75.
  • the production of the enzymes may be increased, or reduced (downregulated) , either throughout or at particular stages in the life of the plant.
  • production of the enzyme is enhanced only by constructs which express RNA homologous to the substantially complete endogenous pTOM75 mRNA.
  • the plants to which the present invention can be applied include commercially important fruit-bearing plants, in particular the tomato.
  • plants can be generated which may have one or more of the following characteristics: Novel flavour and aroma due to changes in the concentrations and ratios of the many aromatic compounds that contribute to fruit flavour;
  • Fruit e.g. tomatoes
  • changed or intensified flavour e.g. sweeter or sharper, or both
  • acids e.g. citric or malic acid
  • DNA constructs for downregulating gene expression preferably comprise a homologous base sequence at least 20, usually at least 50 bases in length. There is no theoretical upper limit to the base sequence - it may be as long as the relevant mRNA produced by the cell - but for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length. The preparation of such constructs is described in more detail below.
  • RNA for use in the present invention is DNA derived from the clone pTOM75.
  • the required DNA can be obtained in several ways, including: by cutting with restriction enzymes an appropriate sequence of such DNA; by synthesising a DNA fragment using synthetic oligonucleotides which are annealed and then ligated together in such a way as to give suitable restriction sites at each end; by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to generate the required fragment with suitable restriction sites at each end.
  • PCR polymerase chain reaction
  • the DNA is then cloned into a vector containing upstream promoter and downstream terminator sequences, the cloning (if so desired) being carried out so that the DNA sequence is inverted with respect to its orientation to the promoter in the strand from which it was cut.
  • the new .vector is intended to produce antisense RNA, the strand that was formerly the template strand becomes the coding strand, and vice versa.
  • Such a new vector will encode RNA in a base sequence which is complementary to the sequence of pTOM75 mRNA.
  • the two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3' ) .
  • pTOM75 As source of the DNA base sequence for transcription, it is convenient to use a cDNA clone such as pTOM75.
  • the base sequence of pTOM75 is set out in Figure
  • a cDNA clone similar to pTOM75 may be obtained from the mRNA of ripening tomatoes by the method described by Slater et al , Plant Molecular Biology 5, 137-147, 1985. In this way may be obtained sequences coding for the whole, or substantially the whole, of the mRNA produced by pTOM75. Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.
  • An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the pTOM75 protein.
  • a suitable gene may differ from the cDNA of pTOM75 in that introns may be present.
  • the introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out).
  • part of such a gene as the source of the base sequence for transcription (with the object of downregulating gene expression) it is possible to use either intron or exon regions.
  • a further way of obtaining a suitable DNA base sequence for transcription is to synthesise it a_b initio from the appropriate bases, for example using Figure 1 as a guide.
  • Recombinant DNA and vectors according to the present invention may be made as follows.
  • a suitable vector containing the desired base sequence for transcription for example pTOM75
  • restriction enzymes to cut the sequence out.
  • the desired terminator sequence for example the 3' sequence of the desired promoter sequence
  • Agrobacterium tumefaciens nopaline synthase gene with the nos 3' end, or the 'long' PG promoter, with the PG gene 3' end, as described in ICI's UK patent application 9024323.9, filed 11 November 1990).
  • constitutive promoters such as cauliflower mosaic virus 35S RNA
  • inducible or developmentally regulated promoters such as the ripe-fruit-specific polygalacturonase promoter
  • Vectors according to the invention may be used to transform plants as desired, to make plants according to the invention.
  • Dicotyledonous plants such as tomato
  • Agrobacterium Ti plasmid technology for example as described by Bevan (1984) Nucleic Acid Research, 12, 8711-8721.
  • Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • RNA in the plant cells can be controlled by suitable choice of promoter sequences, or by selecting the number of copies, or the site of integration, of the DNA sequences according to the invention that are introduced into the plant genome. In this way it may be possible to modify ripening or senescence to a greater or lesser extent.
  • the constructs of our invention may be used to transform cells of both monocotyledonous and dicotyledonous plants in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to regenerate whole plants which subsequently reproduce to give successive generations of genetically modified plants. Examples of genetically modified plants according to the present invention include, as well as tomatoes, fruits such as mangoes, peaches, apples, pears, strawberries, bananas and melons; and carnations and other ornamental flowers.
  • RNA for use in the present invention is DNA showing homology to the gene encoded by the clone pTOM75.
  • pTOM75 was derived from a cDNA library isolated from ripe tomato RNA (Slater et al Plant Molecular Biology _5, 137-147, 1985).
  • pTOM75 has been characterised by hybrid select translation. it may not contain the full length coding sequence for the gene. Slater et al (Plant
  • MIP bovine lens fibre major intrinsic protein
  • soybean nodulin-26 soybean nodulin-26
  • the glycerol facilitator protein of E. coli the Drosophila big brain gene
  • a turgor- responsive gene expressed in wilted pea shoots a seed-specific tonoplast intrinsic protein amongst others.
  • MIP is believed to be involved in the formation of aqueous channels in lens tissue and has been shown to allow the passive transport of ions and small molecules when inserted into artificial membranes. Its function may be to allow transport of metabolites within the avascular tissue of the lens to regulate the volume of the extracellular space.
  • coli forms a passive channel allowing bi-directional transport of neutral molecules less than 0.4 nm in diameter across the inner cytoplasmic membrane.
  • Antisense RNA could be used to clarify whether these rather tentative sequence relationships imply a functional homology or whether they are not significant.
  • pTOM75 the mRNA for which pTOM75 codes is expressed in ripening tomato fruit, in roots and in senescing leaves of tomatoes. Almost no expression could be detected in mature green fruit. pTOM75 is expressed most strongly at the full orange stage of ripening. The level of mRNA then declines in line with the general decline in biosynthetic capacity of the ripening fruit. Expression of pTOM75 is also detected at high levels in immature green fruit. pTOM75 can also be induced by exposing mature green fruit to exogenous ethylene. The expression of pTOM75 is apparently increased in the ripening inhibitor (rin) tomato fruit ripening mutant which mature very slowly ( Knapp et al , Plant Mol .
  • Figure 1 shows the base sequence of the clone pTOM75
  • Figure 2 shows the construction of plant transformation antisense RNA vectors according to the invention.
  • Figure 2 also shows the construction of the pTOM75 expression vectors according to the invention.
  • the base sequence of pTOM75 has not previously been determined.
  • the sequence was determined by standard DNA sequencing procedures and is shown in Figure 1. Knowledge of this sequence is essential for determining the orientation of the open reading frame and for the subsequent construction of RNA antisense vectors.
  • Plasmid DNA vectors with the CaMV 35S promoter vector containing the 695 base pair Pstl-Dral fragment
  • a vector pBDH75A was constructed using the base pair Pstl-Dral fragment derived from pTOM75 cDNA by digestion of pTOM75 with Pstl and Dral , followed by isolation of the 695 base pair fragment after electrophoresis. The fragment was then cloned into the vector pDH51 or pjRl which had previously been cut with Pstl and Smal . Recombinant plasmids were isolated and characterised.
  • pDH75A derived from pDH51 - see Figure 2
  • pJR75A derived from pJRl
  • pDH51 is a pUC based cloning vector containing a CaMV35S promoter and terminator fragment.
  • pJRl (Smith et al Nature 334, 724-726, 1988) is a Binl9 ( Bevan, Nucleic Acids Research, 12, 8711-8721, 1984) based vector, which permits the expression of the antisense RNA under the control of the CaMV 35S promoter.
  • This vector includes a nopaline synthase (no ⁇ ) 3' end termination sequence.
  • the expression cassette was transferred to Binl9 (Bevan, Nucleic Acids Research, 12, 8711-8721, 1984) to yield pBDH75A.
  • Binl9 Bevan, Nucleic Acids Research, 12, 8711-8721, 1984
  • Vectors pJR75CA and pJR75CS are prepared as follows: the complete cDNA pTOM75 insert (889 bases) is inserted into pJRl as a Pstl fragment. This results in clones having the pTOM75 fragment either in the antisense or sense orientation inserted into the cloning vector pJR1.
  • the antisense vector is called pJR75CA.
  • the sense vector is called pJR75CS.
  • pTOM75 antisense RNA vector with the polygalacturonase promoter vector containing the 695 base pair Pstl-Dral fragment.
  • pJR2 is a Binl9-based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter.
  • This vector includes a nopaline synthase (nos) 3' end termination sequence.
  • the isolated fragment is made flush-ended with T4 poly erase and then cloned into the Hindi site of pJR2.
  • vectors are identified which have the pTOM75 insert fragment in the antisense (pJR275A) and sense (pJR275S) orientation.
  • Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and used to transform tomato plants ( Lycopersicon esculentum, var. Ailsa Craig) . Transformation of tomato stem segments and cotyledons followed standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. Plants were regenerated and grown to maturity. Ripening fruit were analysed biochemically and the presence of the antisense pTOM75 gene construct was verified by Southern and PCR analysis .
  • Similar plants may be produced using the vectors PJR275A and pJR275S from Example 3A in place of pBDH75A. These are also expected to show inhibition of expression of the pTOM75 gene.
  • Drought stress tests were carried out on leaves of a transformed plant produced in Example 4, found to contain the pBDH75A construct. Comparisons were made with the leaves of similar wild-type tomatoes. In each case total leaf RNA was probed with pTOM75 sense and antisense transcript, before or after 24 hours drought stress. Samples were hybridised with nick-translated pTOM75 insert 5xSSPE at 65°C, with a final wash at 65°C in 0.2x55PE. The wild-type plant showed no pTOM75 mRNA before drought-stress. After drought-stress the wild-type plant showed substantial amounts of pTOM75 mRNA, while pBDH75A showed only traces.
  • Example 4 A plant from Example 4 transformed with the pBDH75A construct was grown to maturity and produced fruit. individual fruit were sampled for malic acid content, using a commercially available food analysis kit (Boehringer Mannheim) . Results are shown in Table 1 below, compared with similar results for wild-type fruit. The mean malic acid level in transformed fruit was over 25% above the mean level in wild type fruit. Application of Student's T test suggests that the result was statistically significant at the 2% level.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
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  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
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  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Nutrition Science (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Des constructions d'ADN contiennent une séquence ADN homologue à tout ou partie du gène codé par le clone pTOM75. Lesdites constructions peuvent en outre comprendre une région d'initiation transcriptionnelle pouvant transcrire cette séquence d'ADN dans les plantes, éventuellement dans la direction non codante afin de produire de l'ARNm complémentaire au gène pTOM75. A partir de telles constructions il est possible de dériver des cellules de plantes ainsi que des plantes transformées, dans lesquelles l'expression du gène pTOM75 est inhibée: les fruits des plantes (par exemple les tomates) peuvent montrer des caractéristiques de maturation modifiées, comme par exemple une teneur en acide accrue.
EP92907487A 1991-03-28 1992-03-26 Adn, constructions d'adn, et cellules et plantes derivees Withdrawn EP0618975A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB919106713A GB9106713D0 (en) 1991-03-28 1991-03-28 Dna,dna constructs,cells and plants derived therefrom
GB9106713 1991-03-28
PCT/GB1992/000557 WO1992017596A1 (fr) 1991-03-28 1992-03-26 Adn, constructions d'adn, et cellules et plantes derivees

Publications (1)

Publication Number Publication Date
EP0618975A1 true EP0618975A1 (fr) 1994-10-12

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EP92907487A Withdrawn EP0618975A1 (fr) 1991-03-28 1992-03-26 Adn, constructions d'adn, et cellules et plantes derivees

Country Status (7)

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EP (1) EP0618975A1 (fr)
JP (1) JPH06506110A (fr)
AU (1) AU1434092A (fr)
BR (1) BR9205814A (fr)
CA (1) CA2106090A1 (fr)
GB (1) GB9106713D0 (fr)
WO (1) WO1992017596A1 (fr)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2158473A1 (fr) * 1993-03-22 1994-09-29 Umi Kalsom Abu-Bakar Adn, structures d'adn, et cellules et plantes derivees
GB9311346D0 (en) * 1993-06-02 1993-07-21 Zeneca Ltd Modified fruit
GB9318927D0 (en) * 1993-09-13 1993-10-27 Zeneca Ltd Regulation of senescence
US5612471A (en) * 1994-05-25 1997-03-18 The Regents Of The University Of California Nematode-induced genes in tomato
AU4722797A (en) * 1996-10-24 1998-05-15 Japan Tobacco Inc. Method for controlling water content of plant
CA2487328A1 (fr) 1998-03-20 1999-09-30 Benitec Australia Ltd. Sirna pour controler l'expression genique
AUPP249298A0 (en) 1998-03-20 1998-04-23 Ag-Gene Australia Limited Synthetic genes and genetic constructs comprising same I
US20040214330A1 (en) 1999-04-07 2004-10-28 Waterhouse Peter Michael Methods and means for obtaining modified phenotypes
JP5015373B2 (ja) 1998-04-08 2012-08-29 コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼイション 改良表現型を得るための方法及び手段
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
US6423885B1 (en) 1999-08-13 2002-07-23 Commonwealth Scientific And Industrial Research Organization (Csiro) Methods for obtaining modified phenotypes in plant cells
DE10212892A1 (de) 2002-03-20 2003-10-09 Basf Plant Science Gmbh Konstrukte und Verfahren zur Regulation der Genexpression
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes
US9315818B2 (en) 2006-06-07 2016-04-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem Plant expression constructs and methods of utilizing same
RU2012103038A (ru) 2009-06-30 2013-08-10 Йассум Ресерч Девелопмент Кампани Оф Зе Хибрю Юниверсити Оф Иерусалим Лтд. Введение днк в растительные клетки
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
US20150040268A1 (en) 2013-04-25 2015-02-05 Morflora Israel Ltd Methods and compositions for the delivery of nucleic acids to seeds

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8916213D0 (en) * 1989-07-14 1989-08-31 Ici Plc Dna constructs,cells and plants derived therefrom

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9217596A1 *

Also Published As

Publication number Publication date
JPH06506110A (ja) 1994-07-14
AU1434092A (en) 1992-11-02
GB9106713D0 (en) 1991-05-15
BR9205814A (pt) 1994-06-28
CA2106090A1 (fr) 1992-09-29
WO1992017596A1 (fr) 1992-10-15

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