WO1993007275A1 - Adn, structures d'adn, cellules et plantes derivees de celles-ci - Google Patents

Adn, structures d'adn, cellules et plantes derivees de celles-ci Download PDF

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Publication number
WO1993007275A1
WO1993007275A1 PCT/GB1992/001806 GB9201806W WO9307275A1 WO 1993007275 A1 WO1993007275 A1 WO 1993007275A1 GB 9201806 W GB9201806 W GB 9201806W WO 9307275 A1 WO9307275 A1 WO 9307275A1
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WIPO (PCT)
Prior art keywords
dna
plants
dna constructs
cells
plant
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PCT/GB1992/001806
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English (en)
Inventor
Colin Roger Bird
Donald Grierson
John Anthony Ray
Wolfgang Walter Schuch
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Zeneca Limited
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Publication date
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Priority to EP92920571A priority Critical patent/EP0606323A1/fr
Publication of WO1993007275A1 publication Critical patent/WO1993007275A1/fr

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    • 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
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01009—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9)

Definitions

  • This application relates to novel DNA constructs, plant cells containing the constructs and plants derived therefrom.
  • it involves the use of antisense or sense RNA technology to control 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
  • 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).
  • antisense RNA has been proven to be useful in achieving downregulation of gene expression in plants.
  • antisense RNA can be used to downregulate the expression of genes encoding enzymes involved in respiration during ripening of tomato and other fruit.
  • the effect of this downr.gula ion in gene expression will be to reduce the respirative capacity of the fruit.
  • the consequent retardation of the metabolism will slow the ripening and subsequent deterioration of the fruit.
  • DNA constructs comprising a DNA sequence homologous to some or all of a gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase, under control of a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.
  • Such DNA constructs may comprise a DNA sequence homologous to some or all of a gene partially encoded by any of the clones pT0M38, M13T38S3.1 and M13T38S4.1 preceded by a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.
  • the 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 mRNA encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase.
  • the invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; and seeds of such plants.
  • Suitable DNA may be obtained from the genes which are partially encoded by the clones pT0M38, M13T38S3.1 and M13T38S4.1 or from cDNA of such genes.
  • constructs of the invention may be inserted into plants to regulate the production of the enzyme glyceraldehyde 3-phosphate dehydrogenase.
  • the production of the enzyme may be increased, or reduced, 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 mRNA.
  • the plants to which the present invention can be applied include commercially important fruit-bearing plants, in particular tomato.
  • plants can be generated which have modified expression levels of genes encoding respiration enzymes and which may have one or more of the following characteristics:
  • DNA constructs according to the invention preferably comprise a base sequence at least 10 bases in length for transcription into antisense RNA. 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.
  • the preferred DNA for use in the present invention is DNA derived from the clones pT0M38, M13T38S3.1 or M13T38S4.1.
  • the required antisense DNA can be obtained in several ways: 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.
  • the DNA is then cloned into a vector containing upstream promoter and downstream terminator sequences, the cloning being carried out so that the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut.
  • RNA in a base sequence which is complementary to the sequence of pT0M38, M13T38S3.1 or M13T38S4.1 mRNA will thus encode RNA in a base sequence which is complementary to the sequence of pT0M38, M13T38S3.1 or M13T38S4.1 mRNA.
  • the two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3').
  • cDNA clones such as pTOM38, M13T38S3.1 or M13T38S4.1.
  • the base sequences of pT0M38, M13T38S3.1 and M13T38S4.1 are set out in Figure 1.
  • cDNA clones similar to pT0M38 may be obtained from the mRNA of ripening tomatoes by the method described by Slater et al,
  • Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.
  • RNA for use in the present invention is DNA showing homology to the gene encoded by the clone pT0M38.
  • pT0M38 was derived from a cDNA library isolated from ripe tomato RNA (Slater et al Plant Molecular Biology 5, 137- 147, 1985). DNA sequence analysis has demonstrated that the cDNA insert of pT0M38 is 848 bases long.
  • M13T38S3.1 and M13T38S4.1 were derived from a cDNA library obtained from Clontech Laboratories Incorporated.
  • An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase.
  • This gene may differ from the cDNA of, e.g. pT0M38 in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out). When using such a gene as the source of the base sequence for transcription 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 ab 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 pT0M38
  • restriction enzymes to cut the sequence out.
  • the DNA strand so obtained is cloned (if desired, in reverse orientation) into a second vector containing the desired promoter sequence (for example cauliflower mosaic virus 35S RNA promoter or the tomato polygalacturonase gene promoter sequence - UK patent application 9024323.9) and the desired terminator sequence (for example the 3' of the Agrobacteriu tumefaciens nopaline synthase gene, the nos 3' end).
  • the desired promoter sequence for example cauliflower mosaic virus 35S RNA promoter or the tomato polygalacturonase gene promoter sequence - UK patent application 9024323.9
  • the desired terminator sequence for example the 3' of the Agrobacteriu tumefaciens nopaline synthase gene, the nos 3' end.
  • inducible or developmentally regulated promoters such as the ripe-fruit-specific polygalacturonase promoter
  • a constitutive promoter will tend to affect respiration in all parts of the plant which may inhibit growth and development of the whole plant.
  • respiration may be controlled more selectively.
  • PG gene UK patent application 9024323.9
  • ripening-specific promoters that could be used include the ripening- specific E8 promoter (Diekman & Fischer, 1988 EMBO 7, 3315-3320) and the fruit-specific 2A11 promoter (Pear et al, 1989 Plant Molecular Biology 13, 639-651).
  • 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 and melon, may be transformed by Agrobacteriu Ti plasmid technology.
  • tomato transformation is described by Fillatti et al. , Biotechnology, Vol. 5, July 1987, pp 726-730.
  • Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • the degree of production of antisense 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 of such as mangoes, peaches, apples, pears, strawberries, bananas and melons.
  • Figure 1 shows the base sequence of the clones pT0M38, M13T38S3.1 and M13T38S4.1.
  • Figure 2 shows the base sequence of oligonucleotide primers for amplification of a fragment of pT0M38 by PCR.
  • the vector pJR138A is constructed using the sequences corresponding to the complete insert of M13T38S3.1. This 626bp fragment is synthesised by polymerase chain reaction using synthetic primers ( Figure 2). The ends of the fragment are made flush with T4 polymerase and it is cloned into the vector pJRl which has previously been cut with Smal.
  • 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 (nos) 3' end termination sequence.
  • the fragment of the M13T38S3.1 cDNA that was described in Example 1 is also cloned into the vector pJR3 to give pJR338A.
  • pJR3 is a Binl9 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter.
  • This vector includes approximately 5 kb of promoter sequence and 1.8 kb of 3' sequence from the PG promoter separated by a multiple cloning site.
  • vectors with the correct orientation of pT0M38 sequences are identified by DNA sequence analysis.
  • EXAMPLE 3 Construction of sense RNA vectors with the CaMV 35S promoter The fragment of M13T38S3.1 cDNA that was described in Example 1 is also cloned into the vector pJRl in the sense orientation to give pJR138S.
  • the vectors with the sense orientation of pT0M38 sequence are identified by DNA sequence analysis.
  • the vectors with the sense orientation of pT0M38 sequence are identified by DNA sequence analysis.
  • Vectors are transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants. Transformation of tomato stem segments follow standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants are identified by their ability to grow on media containing the antibiotic kana ycin. Plants are regenerated and grown to maturity. Ripening fruit are analysed for modifications to their ripening characteristics.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
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  • Physics & Mathematics (AREA)
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  • Plant Pathology (AREA)
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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract

Des structures d'ADN comprennent une séquence d'ADN homologue à tout ou partie d'un gène codant l'enzyme déshydrogénase glycéraldéhyde 3-phosphate de la plante, obéissant à une région d'amorçage de transcription active dans des plantes pour transcrire cette séquence d'ADN, éventuellement dans des sens non-codant afin de produire l'ARN complémentaire à l'ARNm du gène. A partir de ces structures, on peut dériver des cellules de plantes transformées et des plantes dans lesquelles on inhibe l'expression du gène déshydrogénase glycéraldéhyde 3-phosphate: le fruit des plantes (tel que des tomates) peut présenter des propriétés de mûrissement modifiées, telles que l'arrêt de la respiration.
PCT/GB1992/001806 1991-10-03 1992-10-01 Adn, structures d'adn, cellules et plantes derivees de celles-ci WO1993007275A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP92920571A EP0606323A1 (fr) 1991-10-03 1992-10-01 Adn, structures d'adn, cellules et plantes derivees de celles-ci

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GB919121074A GB9121074D0 (en) 1991-10-03 1991-10-03 Dna,dna constructs,cells and plants derived therefrom
GB9121074.0 1991-10-03

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WO1993007275A1 true WO1993007275A1 (fr) 1993-04-15

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078629A1 (fr) 2002-03-20 2003-09-25 Basf Plant Science Gmbh Produit de synthese et procede de regulation de l'expression genique
WO2003095655A3 (fr) * 2002-05-08 2004-08-26 Basf Plant Science Gmbh Procede pour augmenter la teneur en huile dans des vegetaux
EP2267138A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés et moyens d'obtention de phénotypes modifiés
WO2011001434A1 (fr) 2009-06-30 2011-01-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Introduction d'adn dans des cellules végétales
EP2436769A1 (fr) 2006-06-07 2012-04-04 Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. Constructions d'expression végétale et leurs procédés d'utilisation
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
WO2015162608A1 (fr) 2013-04-25 2015-10-29 Morflora Israel Ltd. Procédés et compositions pour l'administration d'acides nucléiques dans des semences
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264067A2 (fr) * 1986-10-08 1988-04-20 The General Hospital Corporation Production de glycéraldéhyde-3 phosphate déhydrogenase
WO1991009128A1 (fr) * 1989-12-13 1991-06-27 Imperial Chemical Industries Plc Constructions d'adn, et les cellules et plantes derivees

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0264067A2 (fr) * 1986-10-08 1988-04-20 The General Hospital Corporation Production de glycéraldéhyde-3 phosphate déhydrogenase
WO1991009128A1 (fr) * 1989-12-13 1991-06-27 Imperial Chemical Industries Plc Constructions d'adn, et les cellules et plantes derivees

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOTECHNOLOGY vol. 9, no. 7, July 1991, NEW YORK US pages 635 - 639 BIRD, C.R., ET AL. 'Using antisense RNA to study gene function:Inhibition of carotenoid biosynthesis in transgenic tomatoes' *
J. MOL. BIOL. vol. 208, 1989, pages 551 - 565 MARTINEZ, P., ET AL. 'Structure, evolution and anaerobic regulation of a nuclear gene encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase from maize' *
PLANT MOLECULAR BIOLOGY. vol. 13, no. 9, September 1989, DORDRECHT, THE NETHERLANDS. pages 303 - 311 SCHUCH, W., ET AL. 'Control and manipulation of gene expression during tomato fruit ripening' *
THEOR. APPL. GENET. vol. 76, no. 2, August 1988, pages 285 - 292 MUTSCHLER, M., ET AL. 'Changes in ripening related-processes in tomato conditioned by the alc mutant' *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2267138A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés et moyens d'obtention de phénotypes modifiés
EP2267139A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés ét moyens d'obtention de phénotypes modifies
EP3214177A2 (fr) 1998-04-08 2017-09-06 Commonwealth Scientific and Industrial Research Organisation Procédés et moyens pour obtenir des phénotypes modifiés
WO2003078629A1 (fr) 2002-03-20 2003-09-25 Basf Plant Science Gmbh Produit de synthese et procede de regulation de l'expression genique
WO2003095655A3 (fr) * 2002-05-08 2004-08-26 Basf Plant Science Gmbh Procede pour augmenter la teneur en huile dans des vegetaux
US7579517B2 (en) 2002-05-08 2009-08-25 Basf Plant Science Gmbh Methods for increasing oil content in plants
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes
EP2436769A1 (fr) 2006-06-07 2012-04-04 Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. Constructions d'expression végétale et leurs procédés d'utilisation
WO2011001434A1 (fr) 2009-06-30 2011-01-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Introduction d'adn dans des cellules végétales
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
WO2015162608A1 (fr) 2013-04-25 2015-10-29 Morflora Israel Ltd. Procédés et compositions pour l'administration d'acides nucléiques dans des semences

Also Published As

Publication number Publication date
GB9121074D0 (en) 1991-11-13
EP0606323A1 (fr) 1994-07-20
AU2650892A (en) 1993-05-03

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