EP1261738A2 - Acide nucleique et oligonucleotides derives de celui-ci pour l'amplification specifique et pour la detection specifique de genes de l'aquaporine provenant de l'espece vitis - Google Patents

Acide nucleique et oligonucleotides derives de celui-ci pour l'amplification specifique et pour la detection specifique de genes de l'aquaporine provenant de l'espece vitis

Info

Publication number
EP1261738A2
EP1261738A2 EP01913861A EP01913861A EP1261738A2 EP 1261738 A2 EP1261738 A2 EP 1261738A2 EP 01913861 A EP01913861 A EP 01913861A EP 01913861 A EP01913861 A EP 01913861A EP 1261738 A2 EP1261738 A2 EP 1261738A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
dna
nucleic acids
genes
vitis
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Withdrawn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
EP01913861A
Other languages
German (de)
English (en)
Inventor
Isabel Maria Baiges Blanco
Anton Rudolf SCHÄFFNER
Albert Mas
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.)
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Original Assignee
Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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 Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH filed Critical Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Publication of EP1261738A2 publication Critical patent/EP1261738A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to nucleic acids and oligonucleotides derived therefrom for the amplification and for the qualitative and quantitative detection of aquaporin genes, gene fragments, RNA sequences derived therefrom or the expression of these genes from Vitis spec, in particular from Vitis vinifera.
  • Aquaporins are transmembrane proteins that are present in plants in the plasma membrane and in the vacuolar membrane (tonoplast). They belong to a family of major intrinsic proteins (MIPs) and are called PIP (plasma intrinsic protein) or TIP (tonoplast intrinsic protein).
  • the invention is therefore based on the object of providing nucleic acids and oligonucleotides derived therefrom which are specific for aquaporin genes from Vitis spec. are and with which a specific amplification, a specific quantitative and / or qualitative detection of aquaporin genes and gene fragments and of RNA sequences derived therefrom or the expression of these genes from Vitis spec. is made possible.
  • the object is achieved by nucleic acids which are disclosed in SEQ ID NO: 1-28, and by derivatives of these sequences.
  • the invention also encompasses the complementary, reverse and reverse-complementary sequences, it being possible for one or more of these sequences in each case to be used in detection or amplification methods.
  • the sequences are each specific for aquaporin genes or fragments of aquaporin genes from Vitis spec. at the DNA or RNA level.
  • Figures 1-3 show the expression of aquaporin genes in different tissues from Vitis spec. using probes according to the invention.
  • Figure 4 shows the sequence of specific probes (small letters), ie including the flanking sections, which are used for the synthesis of specific oligonucleotides (capital letters) in the 5'-3 'direction (S' end) (forward direction) or in 3 '-5' direction (3 'end) (reverse) were used.
  • nucleic acids and derivatives derived therefrom are thus provided, which can be used, for example, as probes and primers, for example for the specific detection of aquaporin genes from Vitis spec. and to enable amplification of the genes coding therefor and gene fragments derived therefrom and the detection of the expression of aquaporin proteins.
  • “derivatives” and “modifications” are to be understood as those nucleic acids in which at least 1, but also 2, 3, 4 or more nucleotides are missing at one or both ends of the nucleic acids or also inside the nucleic acids or are replaced by other nucleotides were.
  • the prerequisite here is that a specific binding to aquaporin genes and gene fragments from Vitis vinifera is maintained, so that the object of the present invention can be achieved.
  • the nucleic acids can also be parts of larger DNA units and can therefore also be integrated in vectors, for example in plasmids.
  • the invention also includes fragments of the nucleic acids provided according to the invention and the nucleic acids provided therefrom and the oligonucleotides derived therefrom.
  • fragments are understood to mean nucleic acids which are more than 12, preferably more than 15, more than 20, more than 25 or more than 30 and up to 50 nucleotides.
  • oligonucleotides comprises fragments of 12-50 nucleotides and partial regions These sequences can be combined as desired from the nucleic acid sequences presented here, provided that they have at least 12 consecutive nucleotides.
  • the fragments and oligonucleotides described above are to be subsumed under the term “derivatives”.
  • nucleic acid sequences disclosed above the person skilled in the art can determine by testing which derivatives and modifications, in particular which oligonucleotides, which can be derived from these nucleic acids, are suitable for special detection methods, for example hybridization methods and PCR methods, and which are not or less suitable.
  • the nucleic acids and Oligonucleotides of the invention can also be present, for example, in larger DNA or RNA sequences, for example flanked by restriction enzyme cuts.
  • Amplification and detection methods are known from the prior art. For example, reference is made here to laboratory manuals known to the person skilled in the art that describe this method, for example to Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor and all subsequent editions. PCR methods are e.g. described in Newton, PCR, BIOS Scientific Publishers Limited, 1994 and subsequent editions.
  • the invention also includes such modifications of the nucleic acids and derivatives and oligonucleotides claimed in the present case which hybridize with them, preferably under stringent conditions.
  • Stringent hybridization conditions are understood in particular to be 0.2 ⁇ SSC (0.03 M NaCl, 0.003 M sodium citrate, pH 7) at 65 ° C.
  • the hybridization temperature is below 65 ° C., for example above 55 ° C., preferably above 50 ° C., but in each case below 65 ° C.
  • Stringent hybridization temperatures depend on the size or length of the probe and its nucleotide compositions and are to be determined by a person skilled in the art by manual tests.
  • the oligonucleotides of the invention which are derived from the nucleic acids described here, preferably in detection methods and amplification methods of aquaporin genes from Vitis spec. used. However, they can also be used in detection methods of aquaporin genes from other types of Vitis, for example V. cinerea, V. coignetiae, V. labrusca, V. riparia, V. orientalis, V. rupestris, V. berlandieri, V. candicans. Other important types of wine are known to those skilled in the art, and the oligonucleotides presented here are suitable for the detection of aquaporin genes in all of these types due to the relationship between the Vitis types used for wine production.
  • the person skilled in the art can use the methods known per se with the present oligonucleotides.
  • the DNA or the RNA of a sample to be examined for the presence of said genes is brought into contact with at least one of the oligonucleotides.
  • the detection of the hybridization of the oligonucleotides with the RNA or the DNA of the sample is then carried out using, for example, PCR and hybridization techniques in order to show the presence of specific DNA and / or RNA sequences. All disclosed oligonucleotides can also be used in reverse complementary orientation as primers for reverse transcription of mRNA.
  • the nucleic acids and oligonucleotides preferably have a label.
  • a label examples of this are radioactive labeling, fluorescent labeling, biotin labeling, digoxigenin labeling, peroxidase labeling or labeling with labels detectable by alkaline phosphatase.
  • the oligonucleotides are bound to a solid phase matrix. Further chemical modifications of the nucleic acids and oligonucleotides are possible, for example to facilitate the PCR method, hybridization method, etc. Such modifications are known to the person skilled in the art.
  • oligonucleotides according to the invention are particularly suitable in detection methods such as a polymerase chain reaction (PCR), a reverse polymerase chain reaction (RT-PCR), an in situ PCR, a dot blot, a northern blot, a reverse northern blot, an in situ hybridization, a Differential display or a Southern blot.
  • PCR polymerase chain reaction
  • RT-PCR reverse polymerase chain reaction
  • in situ PCR a dot blot
  • a northern blot a reverse northern blot
  • an in situ hybridization a Differential display or a Southern blot.
  • the invention relates to the use of the oligonucleotides as described above as markers in transformation and / or breeding processes.
  • This makes it possible in an advantageous manner to be able to make statements by means of such genetic markers in a breeding process by Vitis spec, which has the goal of providing plants which are more resistant to drought, for example, which would otherwise only be possible by testing the plants under the respective growth conditions is possible.
  • the breeding process can be brought to success faster and more economically.
  • the DNA or RNA of the sample to be examined is either isolated directly from the plant cell or the plant cells are disrupted in a suitable manner or made permeable, so that direct contact of the primers and probes with the DNA and / or the RNA the samples is made possible without extensive and time-consuming cleaning procedures having to be carried out beforehand.
  • the oligonucleotides of the invention can also be used in in-situ hybridizations and in-situ PCR methods.
  • the application is not only restricted to plant cells, but can also be applied to all cells which are to be examined for the presence of aquaporin genes or fragments or for the expression of aquaporins, in each case from Vitis spec.
  • reaction buffer and wash buffer are composed of the following functional components:
  • Buffer system for adjusting and stabilizing the pH value between 7 and 8 (e.g. Tris / HCl or citrate buffer)
  • detergent for lowering the surface tension of aqueous solutions e) Nonionic, aprotic compounds (eg formamide) weaken the binding energy of duplex nucleic acids.
  • Salt functional units are cations which neutralize the negative charges of the nucleic acid - phosphate groups and thereby facilitate the duplex formation of two single stranded nucleic acids (eg Na + in NaCl).
  • Components e) and f) influence the binding strengths of nucleic acid duplex molecules. Increasing the monovalent cations in the reaction or washing solution stabilizes the duplex molecules formed, while with an increasing content of e.g. Formamide the duplex bonds are weakened.
  • a suitable nucleic acid or oligonucleotide concentration must be used.
  • the hybridization must take place at a suitable temperature (the higher the temperature, the weaker the binding of the hybrids).
  • Stringent hybridization and washing conditions are the reaction conditions (the right choice of the four factors) under which only duplex molecules between the oligonucleotides and the desired target molecules (perfect hybrids) are formed or only the desired target organism is detected.
  • Stringent reaction conditions for oligonucleotides mean, for example, a hybridization temperature of approximately 5-10 ° C. below the respective oligonucleotide melting point.
  • the stability of the DNA / DNA or RNA / DNA hybrids must be guaranteed even at low salt concentrations corresponding to 0.1 x SSC / 0.5% SDS. In this way, undesirable cross-reactions with other genes can be prevented.
  • the respective temperature conditions can, however depending on the chosen test conditions and depending on the nucleic acid sample to be examined, and must then be adapted accordingly.
  • the hybridization product can be detected, for example, by autoradiography in the case of radioactively labeled primer molecules or by fluorimetry when using fluorescence-labeled oligonucleotides.
  • Suitable stringency conditions can be determined, for example, using reference hybridizations.
  • the oligonucleotides are used as forward and backward primers for a PCR reaction.
  • the PCR method has the advantage that very small amounts of DNA can be detected. Depending on the material to be detected, the temperature conditions and the cycle numbers of the PCR have to be modified. The optimal reaction conditions can be determined by hand tests in a manner known per se.
  • An example of a PCR is as follows:
  • the respective PCR conditions are determined by the person skilled in the art on the basis of known regulations.
  • the hybridization temperatures / annealing temperatures for the PCR are thus calculated from the length and sequence of the oligonucleotides according to algorithms known to the person skilled in the art. For example, a hybridization temperature of 55 ° C. for 30 seconds (typical for short fragments) was used for the nucleic acids (oligonucleotides) and their complementary forms specified in SEQ ID NO 8-21.
  • the initial denaturation takes place, for example, at 94 ° C. for 2 minutes, whereby these conditions can of course be varied depending on the nucleic acids used; for example, the temperature can also be 95 ° C or above and / or the denaturation times can be longer. Denaturation is preferably carried out for 30 seconds during the cycles.
  • the polymerization time preferably at 72 ° C., is preferably 30 seconds, since the nucleic acid sequences SEQ LD NO 8-21 are relatively short DNA fragments.
  • the conditions have to be adapted.
  • the characteristic, species-specific DNA marker fragments formed in the course of the PCR amplification by the extension of the primer sequences can be detected, for example, by gel electrophoresis or fluorimetrically using fluorescence-labeled oligonucleotides. Of course, other detection methods known to the person skilled in the art can also be used.
  • the probes are hybridized with the DNA or RNA sample to be investigated, stringent hybridization conditions being selected. Stringent conditions must be met for the the respective application can be determined empirically. The conditions described above can serve as guidelines.
  • the DNA or RNA of the sample to be examined can be present either in the PCR reaction or RT-PCR as well as in the hybridization reaction either in extracted form or in the form of complex mixtures in which the DNA or RNA to be examined has only a very small proportion on the fraction of the special biological sample.
  • the cells to be examined can thus either be in purified form or, for example, "contaminated” in the tissue. Both in situ PCR and in situ RT-PCR can be carried out with the oligonucleotides described here.
  • the oligonucleotides of the invention are used for the PCR amplification of fragments on cDNA matrices which are generated after reverse transcription with oligo (dT) or with these oligonucleotides at the 3 'end.
  • the expression can then be evaluated qualitatively, in conjunction with suitable standards and techniques, such as an internal standard and a quantitative PCR, also quantitatively.
  • DNA, RNA or PNA can be present, in particular in the form of oligonucleotides, are used as probes on dot blots or DNA microarrays for qualitative and / or quantitative analysis of the expression of the aquaporin genes from Vitis spec. used.
  • PNA means nucleic acids in which the heterocyclic bases are coupled to a protein backbone and not, as usual, to a sugar-phosphate backbone. This results in higher stability and better hybridization properties, and such PNA connections are particularly suitable for microarray analysis methods.
  • the fragments derived from the nucleic acids presented in the present invention are synthesized on a solid support and after prior isolation (for example via amplification after cloning into a vector or, preferably, via a PCR reaction) on a solid support , for example glass, nylon or nitrocellulose, applied.
  • the dot blot and microarray is then hybridized in a reverse Northern experiment with labeled cDNA, which was produced from RNA from Vitis plants.
  • Usual markings such as radioactive markings or
  • Fluorescence labels can be used for this. This makes it possible to measure and compare the transcription of the Vitis aquaporins defined by the nucleic acids.
  • the oligonucleotides of the invention are produced in a manner which is customary and known per se.
  • the probes can, for example, be inserted into vectors and cut out again after propagation, they can be produced as a template by PCR or else synthetically. Production in the form of, for example, PNA (peptide-based nucleic acid) or derivatized nucleic acids in order to change the stability, the hybridization properties or binding properties to solid supports is also possible.
  • the oligonucleotides presented here can be used for a quantitative and qualitative detection of aquaporin genes and their expression from Vitis spec. be used and then correlated with properties such as the drought tolerance and / or the salt tolerance of the cultivars. This means that these traits can also be tracked in breeding programs.
  • nucleic acids claimed in the present patent application and the derivatives derived therefrom, in particular also their fragments and oligonucleotides, were derived from aquitisin genes from Vitis vinifera by sequencing complete cDNA clones (SEQ ID NO 29-35).
  • Vitis vinifera cDNA fragments were used to screen a Vitis vinifera cDNA library.
  • These probes were obtained by an RT-PCR reaction using degenerate primers directed against conserved regions of plant genes encoding TIP or PIP. 7 complete nucleic acids were isolated, which are shown in the attached sequence listing under SEQ ID NO 29-35. The specific probes were derived from 3 'end sequences after comparison of these sequences.
  • the oligonucleotides shown in SEQ ID NO 8-21 were used to quantify the transcripts of the associated aquaporin genes from Vitis vinifera. For this purpose, either an RT or a PCR or a dot blot analysis with PCR fragments, which cover the entire nucleic acid sequences shown in SEQ LD NO 1-7, was used. The specificity of the probes is based on the following observations: - In all cases, only a single PCR product was amplified; all PCR products had the sizes predicted from the gene sequences, e.g. 160 bp for SEQ ID NO 1.
  • fragments thereof can also be derived according to rules that are known to the person skilled in the art and used in PCR methods. This also applies to use in microarrays in which fragments of the nucleic acids presented here, in particular DNA, RNA and / or DNA fragments, for example oligonucleotides, were fixed on the supports or synthesized thereon.
  • the detection of expression by means of dot blot / reverse Northem analysis was investigated, for example, to determine the tissue specificity of the expression in roots and leaves of the aquaporins from Vitis vinifera, as shown in SEQ ID NO 29-35.
  • two members were analyzed by RT-PCR to examine daily rhythms in expression.
  • the detection via RT-PCR was preferably carried out with 0.2-0.4 ⁇ g of total RNA from Vitis plants after reverse transcription with oligo (dT) and 25-30 cycles in the PCR amplification.
  • reverse transcription can also be carried out directly by an oligonucleotide which is specific for aquaporin genes from Vitis vinifera.
  • Vitis PIP1-1 (SEQ1) (Fig. 1) Vitis PIP2-1 (SEQ4) (Fig. 2) Vitis TIP 1 (SEQ6) (Fig. 3)
  • Vitis GDH Glutamate Dehydrogenase
  • Vitis-malic Malic Enzyme
  • sequences SEQ ID NO. 29 - 40 show:
  • SEQ ID NO. 29 Vitis vinifera plasma membrane intrinsic protein (Vitis PIP 1-1), cDNA, complete sequence
  • SEQ ID NO. 30 Protein sequence for SEQ ED NO. 29
  • SEQ ID NO. 31 Complete Vitis MIPl protein cDNA. potential
  • SEQ ID NO. 32 amino acid sequence for SEQ ID NO. 31
  • SEQ ID NO. 33 Complete Vitis MIPl protein cDNA. Possible water tunnel protein. Arabidopsis PIP 1 -like protein. (Vitis PIP1-3).
  • SEQ ID NO. 34 amino acid sequence to SEQ ID NO. 33rd
  • SEQ ID NO. 35 Complete citis from Vitis MIPl protein; potential
  • SEQ ID NO. 36 Amino acid sequence for SEQ ID NO. 35
  • SEQ ID NO. 37 Complete citis of Vitis MIPl protein. potential
  • SEQ ID NO. 38 amino acid sequence to SEQ ID NO. 37
  • SEQ ID NO. 39 TIP1 from Vitis vinifera
  • SEQ ID NO. 40 TIP 2 from Vitis vinifera

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Biotechnology (AREA)
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  • Wood Science & Technology (AREA)
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  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des acides nucléiques pour l'amplification et pour la détection qualitative et quantitative de gènes et de fragments de gènes de l'aquaporine, et de séquences d'ARN dérivées de ces acides nucléiques ou de l'expression de ces gènes provenant de l'espèce Vitis.
EP01913861A 2000-03-09 2001-03-09 Acide nucleique et oligonucleotides derives de celui-ci pour l'amplification specifique et pour la detection specifique de genes de l'aquaporine provenant de l'espece vitis Withdrawn EP1261738A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10011480A DE10011480A1 (de) 2000-03-09 2000-03-09 Nukleinsäure und hieraus abgeleitete Oligonukleotide zur spezifischen Amplifikation und zum spezifischen Nachweis von Aquaporin-Genen aus Vitis vinifera
DE10011480 2000-03-09
PCT/EP2001/002718 WO2001066793A2 (fr) 2000-03-09 2001-03-09 Acide nucleique et oligonucleotides derives de celui-ci pour l'amplification specifique et pour la detection specifique de genes de l'aquaporine provenant de l'espece vitis

Publications (1)

Publication Number Publication Date
EP1261738A2 true EP1261738A2 (fr) 2002-12-04

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EP01913861A Withdrawn EP1261738A2 (fr) 2000-03-09 2001-03-09 Acide nucleique et oligonucleotides derives de celui-ci pour l'amplification specifique et pour la detection specifique de genes de l'aquaporine provenant de l'espece vitis

Country Status (4)

Country Link
EP (1) EP1261738A2 (fr)
AU (1) AU3928901A (fr)
DE (1) DE10011480A1 (fr)
WO (1) WO2001066793A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10239585A1 (de) * 2002-08-28 2004-04-08 Biotecon Diagnostics Gmbh Verfahren und Nukleinsäuren zum Nachweis von Pflanzenmaterial

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741671A (en) * 1991-12-12 1998-04-21 The Johns Hopkins University Isolation cloning and expression of transmembrane water channel aquaporin 1(AQP1)
WO1998042842A1 (fr) * 1997-03-25 1998-10-01 Her Majesty In Right Of Canada, As Represented By The Minister Of Health And Welfare, Canada Gene de porine extrait de campylobacter jejuni, produits apparentes et leurs utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5741671A (en) * 1991-12-12 1998-04-21 The Johns Hopkins University Isolation cloning and expression of transmembrane water channel aquaporin 1(AQP1)
WO1998042842A1 (fr) * 1997-03-25 1998-10-01 Her Majesty In Right Of Canada, As Represented By The Minister Of Health And Welfare, Canada Gene de porine extrait de campylobacter jejuni, produits apparentes et leurs utilisations

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 11 September 1997 (1997-09-11), "vm34a03.r1 Knowles Solter mouse blastocyst B1 Mus musculus cDNA clone", Database accession no. (AA574989) *
SCHOFFNER ET AL: "aquaporin function, structure, and expression: are there more surprises to surface in water relations?", PLANTA, vol. 204, 1998, SPRINGER VERLAG, pages 131 - 139 *
See also references of WO0166793A3 *

Also Published As

Publication number Publication date
AU3928901A (en) 2001-09-17
DE10011480A1 (de) 2001-09-20
WO2001066793A2 (fr) 2001-09-13
WO2001066793A3 (fr) 2002-09-26

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