WO2000034324A1 - Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations - Google Patents

Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations Download PDF

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WO2000034324A1
WO2000034324A1 PCT/US1999/029412 US9929412W WO0034324A1 WO 2000034324 A1 WO2000034324 A1 WO 2000034324A1 US 9929412 W US9929412 W US 9929412W WO 0034324 A1 WO0034324 A1 WO 0034324A1
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dna
fluorescent protein
isolated
encodes
organism
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PCT/US1999/029412
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Sergey Anatolievich Lukyanoy
Arcady Fedorovich Fradkov
Yulii Aleksandrovich Labas
Mikhail Vladimirovich Matz
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Clontech Laboratories, Inc.
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Publication of WO2000034324A1 publication Critical patent/WO2000034324A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to the field of molecular biology. More specifically, this invention relates to novel fluorescent proteins , cDNAs encoding the proteins and uses thereof.
  • Fluorescence labeling is a particularly useful tool for marking a protein, cell, or organism of interest.
  • a protein of interest is purified, then covalently conjugated to a fluorophore derivative.
  • the protein-dye complex is then inserted into cells of interest using micropipetting or a method of reversible permeabilization.
  • the dye attachment and insertion steps make the process laborious and difficult to control .
  • An alternative method of labeling proteins of interest is t o concatenate or fuse the gene expressing the protein of interest to a gene expressing a marker, then express the fusion product.
  • Typical markers for this method of protein labeling include _-galactosidase, firefly luciferase and bacterial luciferase. These markers, however, require exogenous substrates or cofactors and are therefore of limited use for in vivo studies.
  • a marker that does not require an exogenous cofactor o r substrate is the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a protein with an excitation maximum at 395 nm, a second excitation peak at 475 nm and an emission maximum at 5 1 0 nm.
  • GFP is a 238-amino acid protein, with amino acids 65-67 involved in the formation of the chromophore.
  • GFP GFP-binding protein
  • GFP expression in plant cells is discussed by Hu and Cheng in Febs Letters 369 (1995), 331-334, while GFP expression in Drosophila embryos is described by Davis et al. in Dev. Biology 170 ( 1995), 726- 729.
  • GFP tertiary structure resembles a barrel (Orm ⁇ et al., Science 273 ( 1996), 1392- 1395 ; Yang, et al. , Nature Biotechnol. 14 ( 1996), 1246- 1251).
  • the barrel consists o f beta sheets in a compact structure, where, in the center, an alpha helix containing the chromophore is shielded by the barrel.
  • the compact structure makes GFP very stable under diverse and/or harsh conditions such as protease treatment, making GFP an extremely useful reporter in general. However, the stability of GFP makes it sub- optimal for determining short-term or repetitive events.
  • GFP GFP reagents useful and optimized for a variety of research purposes.
  • New versions of GFP have be en developed, such as a "humanized" GFP DNA, the protein product o f which has increased synthesis in mammalian cells (Haas, et al. , Current Biology 6 ( 1996), 315-324; Yang, et al., Nucleic Acids Research 24 (1996), 4592-4593).
  • One such humanized protein is "enhanced green fluorescent protein” (EGFP).
  • EGFP enhanced green fluorescent protein
  • Other mutations to GFP have resulted in blue-, cyan- and yellow-green light emitting versions .
  • Novel fluorescent proteins result in possible new colors, o r produce pH-dependent fluorescence.
  • Other benefits of novel fluorescent proteins include fluorescence resonance energy transfer (FRET) possibilities based on new spectra and better suitability for larger excitation.
  • FRET fluorescence resonance energy transfer
  • the prior art is deficient in novel fluorescent proteins wherein the DNA coding sequences are known.
  • the present invention fulfills this long-standing need in the art.
  • the present invention is directed to DNA sequences encoding fluorescent proteins selected from the group consisting of: (a) an isolated DNA from an organism from the Class Anthozoa which encodes a fluorescent protein; (b) an isolated DNA which hybridizes to the isolated DNA of (a) and which encodes a fluorescent protein ; and (c) an isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of the genetic code and that encodes a fluorescent protein.
  • the DNA is isolated from a non-bioluminescent organism from Class Anthozoa. More preferably, the DNA has the sequence shown in SEQ ID No. 55 and th e fluorescent protein has the amino acid sequence shown in SEQ ID No. 56.
  • a vector capable of expressing the DNA of the pre sent invention in a recombinant cell comprising said DNA and regulatory elements necessary for expression of the DNA in the cell.
  • the DNA encodes a fluorescent protein having the amino acid sequence shown in SEQ ID No. 56.
  • a host cell transfected with a vector of the present invention, such that the host cell expresses a fluorescent protein.
  • the cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells, insect cells and yeast cells.
  • a representative example of bacterial cell is an E. coli cell.
  • the present invention is also directed to an isolated and purified fluorescent protein coded for by DNA selected from the group consisting of: (a) isolated DNA from an organism from Class Anthozoa which encodes a fluorescent protein; (b) isolated DNA which hybridizes to the isolated DNA of (a) and which encodes a fluorescent protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to the degeneracy of th e genetic code, and which encodes a fluorescent protein.
  • the protein has the amino acid sequence shown in SEQ ID No. 56.
  • the present invention is also directed to a DNA sequence encoding a fluorescent protein selected from the group consisting of: (a) an isolated DNA which encodes a fluorescent protein, wherein said DNA is from an organism from Class Anthozoa and wherein s aid organism does not exhibit bioluminescence; (b) an isolated DNA which hybridizes to isolated DNA of (a) and which encodes a fluorescent protein; and (c) an isolated DNA differing from th e isolated DNAs of (a) and (b) in codon sequence due to degeneracy o f the genetic code and which encodes a fluorescent protein.
  • the organism is from Sub-class Zoantharia, Order Corallimorpharia.
  • the organism is from Family Discosomatidae, Genus Discosoma. Even more preferably, the organism is Discosoma sp. "magenta ". Most particularly, the present invention is drawn to a novel fluorescent protein from Discosoma sp. "magenta " , dmFP592.
  • the present invention is further directed to an amino acid sequence which can be used as a basis for designing a n oligonucleotide probe for identification of a DNA encoding a fluorescent protein by means of hybridizaton, wherein the amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8 , 11 , 12, 14.
  • amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8 , 11 , 12, 14.
  • such an oligonucleotide has a nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 6, 7, 9 , 10, 13, 15, 16.
  • Figure 1 shows the modified strategy of 3'-RACE used t o isolate the target fragments. Sequences of the oligonucleotides u s ed are shown in Table 2. Dpi and Dp2 are the degenerate primers u s ed in the first and second PCR, respectively (see Tables 3 and 4 for th e sequences of degenerate primers). In the case of Discosoma sp. "magenta ", the first degenerate primer used was NGH (SEQ ID No. 4 ) , and the second degenerate primer used was NFP (SEQ ID No. 1 3 ) . ⁇ Please confirm whether the degenerate primers used for dgFP512 are right. If not, please provide the right primers ⁇
  • Figure 2 shows the excitation and emission spectrum of the novel fluorescent protein from Discosoma sp. "magenta " , dmFP592.
  • GFP refers to the basic green fluorescent protein from Aequorea victoria, including prior ar t versions of GFP engineered to provide greater fluorescence o r fluoresce in different colors.
  • SEQ ID No. 54 The sequence of Aequorea victoria GFP (SEQ ID No. 54) has been disclosed in Prasher et al., Gene 111 ( 1992) , 229- 33.
  • EGFP refers to mutant variant o f
  • GFP having two amino acid substitutions: F64L and S65T (Heim et al. , Nature 373 (1995), 663-664).
  • the term "humanized” refers t o changes made to the GFP nucleic acid sequence to optimize the codons for expression of the protein in human cells (Yang et al., Nucleic Acids
  • NFP refers to novel fluorescent protein. Specifically, “NFP” refers to dmFP592 in the pre sent invention.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form o f deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromo somes .
  • a DNA "coding sequence” is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
  • a coding sequence can include, but is n o t limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, an d synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence may be located 3' to the coding sequence.
  • hybridization refers to th e process of association of two nucleic acid strands to form a n antiparallel duplex stabilized by means of hydrogen bonding between residues of the opposite nucleic acid strands.
  • oligonucleotide refers to a short (under 1 00 bases in length) nucleic acid molecule.
  • DNA regulatory sequences are transcriptional and translational control sequences, such a s promoters, enhancers, polyadenylation signals, terminators, and th e like, that provide for and/or regulate expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream ( 5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes .
  • Various promoters, including inducible promoters may be used t o drive the various vectors of the present invention.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cu t double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may b e maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell t o establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population o f cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • heterologous region of the DNA construct is a n identifiable segment of DNA within a larger DNA molecule that is no t found in association with the larger molecule in nature.
  • the heterologous region encodes a mammalian gene
  • the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • heterologous DNA includes coding sequence in a con struct where portions of genes from two different sources have been brought together so as to produce a fusion protein product. Allelic variations or naturally-occurring mutational events do not give rise t o a heterologous region of DNA as defined herein.
  • reporter gene refers to a coding sequence attached to heterologous promoter or enhancer elements and whose product may be assayed easily and quantifiably when th e construct is introduced into tissues or cells.
  • the amino acids described herein are preferred to be in the "L" isomeric form.
  • amino acid sequences are given in one- letter code (A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L leucine; M: metionine; N: asparagine; P: proline; Q: gluetamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophane; Y: tyrosine; X: any residue).
  • NH2 refers to the free amino group present at th e amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J Biol. Chem., 243 ( 1969) , 3552-59 is used.
  • the present invention is directed to an isolated DNA selected from the group consisting of: (a) isolated DNA from a n organism from the Class Anthozoa which encodes a fluorescent protein; (b) isolated DNA which hybridizes to isolated DNA of (a) and which encodes a fluorescent protein; and (c) isolated DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to th e degeneracy of the genetic code, and which encodes a fluorescent protein.
  • the DNA has the sequence shown in SEQ ID No. 5 5 and the fluorescent protein has the amino acid sequence shown in SEQ ID No. 56.
  • a vector capable of expressing the DNA of the present invention in a recombinant cell comprising said DNA and regulatory elements necessary for expression of the DNA in the cell.
  • the DNA encodes a fluorescent protein having the amino acid sequence shown in SEQ ID No. 56.
  • a host cell transfected with the vector of the pre s ent invention, which expresses a fluorescent protein of the pre sent invention.
  • the cell is selected from the group consisting o f bacterial cells, mammalian cells, plant cells, insect cells and yeast cells.
  • a representative example of bacterial cell is an E. coli cell.
  • the present invention is also directed to a DNA sequence encoding a fluorescent protein selected from the group consisting of: (a) an isolated DNA which encodes a fluorescent protein, wherein said DNA is from an organism from Class Anthozoa and wherein said organism does not exhibit bioluminescence; (b) an isolated DNA which hybridizes to isolated DNA of (a) and which encodes a fluorescent protein; and (c) an isolated DNA differing from th e isolated DNAs of (a) and (b) in codon sequence due to degeneracy o f the genetic code and which encodes a fluorescent protein.
  • the organism is from Sub-class Zoantharia, Order Corallimorpharia. More preferably, the organism is from Family Discosomatidae, Genus Discosoma. Most preferably, the organism is Discosoma sp.
  • the present invention is also directed to an isolated and purified fluorescent protein coded for by DNA selected from th e group consisting of: (a) an isolated protein encoded by a DNA which encodes a fluorescent protein wherein said DNA is from an organism from Class Anthozoa and wherein said organism does not exhibit bioluminescence; (b) an isolated protein encoded by a DNA which hybridizes to isolated DNA of (a); and (c) an isolated protein encoded by a DNA differing from the isolated DNAs of (a) and (b) in codon sequence due to degeneracy of the genetic code.
  • th e isolated and purified fluorescent protein is dmFP592.
  • the present invention is further directed to an amino acid sequence which can be used as a basis for designing a n oligonucleotide probe for identification of a DNA encoding a fluorescent protein by means of hybridizaton, wherein the amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8 , 11 , 12, 14.
  • the amino acid sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 8 , 11 , 12, 14.
  • such an oligonucleotide has a nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 6, 7, 9 , 10, 13, 15, 16 and is used as a primer in polymerase chain reaction.
  • it can be used as a probe for hybridization screening o f the cloned genomic or cDNA library.
  • Novel fluorescent proteins were identified from several genera of Anthozoa which do not exhibit any bioluminescence b u t have fluorescent color as observed under usual white light o r ultraviolet light. Six species were chosen (see Table 1).
  • Amplified cDNA samples were then prepared a s described in the protocol provided except the two primers used for PCR were the TS primer (5'-AAGCAGTGGTATCAACGCAGAGT, SEQ ID No. 2) (Table 2) and the TN3 primer (Table 2), both in 0.1 ⁇ M concentration. Twenty to twenty-five PCR cycles were performed t o amplify a cDNA sample. The amplified cDNA was diluted 20-fold in water and 1 ⁇ l of this dilution was used in subsequent procedures.
  • T7-TN3 5'-GTAATACGACTCACTATAGGGCCGCAGTCGACCG(T) 13
  • TS-primer 5'-AAGCAGTGGTATCAACGCAGAGT
  • T7-TS 5'-GTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
  • the modified strategy of 3'-RACE was used to isolate th e target fragments (see Figure 1).
  • the RACE strategy involved two consecutive PCR steps.
  • the first PCR step involved a first degenerate primer (Table 4) and the T7-TN3 primer (SEQ ID No. 17) which has a 3' portion identical to the TN3 primer used for cDNA synthesis (for sequence of T7-TN3, Table 2).
  • the reason for substituting the longer T7-TN3 primer in this PCR step was that background amplification which occurred when using the shorter TN3 primer was suppres sed effectively, particularly when the T7-TN3 primer was used at a low concentration (0.1 _M) (Frohman et al., (1998) PNAS USA, 85, 8998 - 9002).
  • the second PCR step involved the T ⁇ 3 primer (SEQ ID No. 1 , Table 2) and a second degenerate primer (Table 4).
  • NFP SEQ ID No. 13
  • PVMb SEQ ID No. 16
  • the first PCR reaction was performed as follows: 1 ⁇ l of 20-fold dilution of the amplified cDNA sample was added into the reaction mixture containing IX Advantage KlenTaq Polymerase Mix with provided buffer (CLONTECH), 200 ⁇ M dNTPs, 0.3 ⁇ M of first degenerate primer (Table 4) and 0.1 ⁇ M of T7-TN3 (SEQ ID No. 1 7 ) primer in a total volume of 20 ⁇ l.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 1 cycle for 95°C, 1 0 sec; 55°C, 1 min.; 72°C, 40 sec; 24 cycles for 95°C, 10 sec; 62°C, 3 0 sec; 72°C, 40 sec.
  • the reaction was then diluted 20-fold in water and 1 ⁇ l of this dilution was added to a second PCR reaction, which contained IX Advantage KlenTaq Polymerase Mix with the buffer provided by the manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.3 ⁇ M o f the second degenerate primer (Table 4) and 0.1 ⁇ M of TN3 primer .
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 1 cycle for 95°C, 10 sec; 55°C (for GEG/GNG or PVM) or 52°C (for NFP), 1 min.; 72°C, 40 sec; 13 cycles for 95°C, lOsec; 62°C (for GEG/GNG or PVM) or 58°C (for NFP), 30 sec; 72°C, 40 sec.
  • the product of PCR was cloned into PCR-Script vector (S tratagene) according to the manufacturer' s protocol.
  • the step-out reaction mixture contained lx Advantage KlenTaq Polymerase Mix using buffer provided by th e manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.2 ⁇ M of the first gene- specific primer (see Table 5), 0.02 ⁇ M of the T7-TS primer (SEQ ID No. 18), 0.1 ⁇ M of T7 primer (SEQ ID No.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 23-27 cycles for 95°C, 10 sec; 60°C, 30 sec; 72°C, 40 sec .
  • the product of amplification was diluted 50-fold in water and one ⁇ l of this dilution was added to the second (nested) PCR.
  • the reaction contained IX Advantage KlenTaq Polymerase Mix with provided buffer (CLONTECH), 200 ⁇ M dNTPs, 0.2 ⁇ M of the second gene- specific primer and 0.1 ⁇ M of TS primer (SEQ ID No.
  • Both primers had 5 ' - heels coding for a site for a restriction endonuclease; in addition, th e upstream primer was designed so as to allow the cloning of the PCR product into the pQE30 vector (Qiagen) in such a way that resulted in the fusion of reading frames of the vector-encoded 6xHis-tag and nFP.
  • the PCR was performed as follows: 1 ⁇ l of the 20-fold dilution of th e amplified cDNA sample was added to a mixture containing l x Advantage KlenTaq Polymerase Mix with buffer provided by th e manufacturer (CLONTECH), 200 ⁇ M dNTPs, 0.2 ⁇ M of upstream primer and 0.2 ⁇ M of downstream primer, in a final total volume of 20 ⁇ l.
  • the cycling profile was (Hybaid OmniGene Thermocycler, tube control mode): 23-27 cycles for 95°C, 10 sec; 60°C, 30 sec; 72°C, 40 sec .
  • the product of this amplification step was purified by phenol- chlorophorm extraction and ethanol precipitation and then cloned into pQE30 vector using restriction endonucleases corresponding t o the primers' sequence according to standard protocols.
  • All plasmids were amplified in XL-1 blue E. coli and purified by plasmid DNA miniprep kits (CLONTECH).
  • the recombinant clones were selected by colony color, and grown in 3 ml of LB medium (supplemented with 100 ⁇ g/ml of ampicillin) at 37°C overnight. 1 00 ⁇ l of the overnight culture was transferred into 200 ml of fresh LB medium containing 100 ⁇ g/ml of ampicillin and grown at 37°C, 200 rpm up to OD 600 0.6-0.7. 1 mM IPTG was then added to the culture and incubation was allowed to proceed at 37°C for another 16 hours .
  • the cells were harvested and recombinant protein, which incorporated 6x His tags on the N-terminus, was purified using TALONTM metal-affinity resin according to the manufacturer' s protocol (CLONTECH).
  • dmFP592 One of the full-length cDNAs encoding fluorescent proteins found is described herein (dmFP592).
  • the nucleic acid sequence and deduced amino acid sequence are SEQ ID Nos. 55 and 56, respectively.
  • dmFP592 The spectral properties of dmFP592 is listed in Table 7, and th e emission and excitation spectra for the dmFP592 is shown in Figure 2.
  • *relative brightness is extinction coefficient multiplied by qu antum yield divided by the same value for A. victoria GFP.

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Abstract

La présente invention concerne de nouvelles protéines fluorescentes d'organismes non bioluminescents de la classe des anthozoaires. L'invention concerne aussi des ADNc codant pour ces protéines fluorescentes.
PCT/US1999/029412 1998-12-11 1999-12-10 Proteines fluorescentes d'especes non bioluminescentes de la classe des anthozoaires, genes codant pour ces proteines et utilisations WO2000034324A1 (fr)

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