WO1997042340A1 - Isoformes de recepteur du gene ob et acides nucleiques les codant - Google Patents

Isoformes de recepteur du gene ob et acides nucleiques les codant Download PDF

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Publication number
WO1997042340A1
WO1997042340A1 PCT/US1997/007521 US9707521W WO9742340A1 WO 1997042340 A1 WO1997042340 A1 WO 1997042340A1 US 9707521 W US9707521 W US 9707521W WO 9742340 A1 WO9742340 A1 WO 9742340A1
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WIPO (PCT)
Prior art keywords
isoform
rat
isoforms
receptor
mouse
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PCT/US1997/007521
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English (en)
Inventor
C. Thomas Caskey
John W. Hess
Patricia Hey
Michael S. Phillips
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Merck & Co., Inc.
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.)
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Publication date
Priority claimed from GBGB9610995.4A external-priority patent/GB9610995D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to CA002253832A priority Critical patent/CA2253832A1/fr
Priority to JP09540094A priority patent/JP2000512486A/ja
Priority to EP97922646A priority patent/EP0900282A4/fr
Publication of WO1997042340A1 publication Critical patent/WO1997042340A1/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
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat

Definitions

  • This invention relates to ob receptor protein isoforms, to DNA and RNA sequences encoding them, and to assays using the receptor isoform proteins.
  • Two different ob alleles have been identified: one mutation causes the premature termination of the leptin peptide resulting in a truncated protein, and the other mutation changes the transcriptional activity of the obesity ⁇ ob) gene, resulting in a reduced amount of circulating leptin.
  • leptin Although the synthesis of leptin occurs in the adipocyte, its ability to decrease food intake and increase metabolic rate appears to be mediated centrally by the hypothalamus. Injection of recombinant leptin into, the third ventricle of the brain elicits a similar response as peripheral administration of leptin. Furthermore, the recent cloning of the human receptor for the leptin, the ob-receptor (OB-R), reveals that it is transcribed in the hypothalamus (Tartaglia et al. 1995, Cell 83: 1263-1271 ; Stephens et al. 1995, Nature 311: 530-532).
  • OBP ob-receptor
  • isoforms of the OB-Rs have also been identified. These isoforms are due to alternative splicing. For example, in the mouse the a form has 5 amino acids following the Lysine at 889; the b form has 273 amino acids after Lysine 889; the c form has 3 amino acids after Lysine 889; and the d form contains 1 1 amino acids after Lysine 889.
  • This invention relates to novel ob receptor isoforms designated c ⁇ f and g which are substantially free from associated membrane proteins. It also relates to substantially purified ob receptor isoform c', f and g proteins. These isoforms are present in various species, including rat, mouse and human.
  • Another aspect of this invention is to nucleic acids which encode OB receptor isoforms c', f or g.
  • the nucleic acid may be any nucleic acid which can encode a protein, such as genomic DNA, cDNA, or any of the various forms of RNA.
  • the nucleic acid is cDNA.
  • This invention also includes vectors containing a OB-R isoform c', f or g gene, host cells containing the vectors, and methods of making substantially pure OB-R isoform c', f or g protein comprising the steps of introducing a vector comprising a OB-R isoform c', f or g gene into a host cell, and cultivating the host cell under appropriate conditions such that OB-R isoform c', f or g is produced.
  • the OB-R isoform c', f or g so produced may be harvested from the host cells in conventional ways.
  • Yet another aspect of this invention are assays which employ OB-R isoform c', f or g.
  • various molecules, suspected of being OB-R isoform c', f or g ligands are contacted with a OB-R isoform c', f or g, and their binding is detected.
  • agonists, antagonists, and ligand mimetics may be identified.
  • a further aspect of this invention are the ligands so indentified.
  • FIGURE 1 is the amino acid sequence of wild type rat OB-R.
  • FIGURE 2 is the cDNA sequence of wild type rat OB-R.
  • FIGURE 3 is the cDNA sequence encoding rat isoform.
  • FIGURE 4 is the cDNA specific for Rat isoform c'.
  • Substantially free from associated membrane proteins means that the receptor protein is not in physical contact with any membrane proteins.
  • Substantially purified OB-receptor isoform c', f or g means that the protein isoform is at least 90% and preferably at least 95% pure.
  • Wild type means that the gene or protein is substantially the same as that found in an animal which is not considered to have a mutation for that gene or protein.
  • “fa” means that the gene or protein is substantially the same as that found in a rat homologous for ⁇ t fatty mutation.
  • nucleic acid or amino acid sequence means either it is the same as the reference sequence, or if not exactly the same, contains changes which do not affect its biological activity or function.
  • OB-R exists in a large variety of isoforms, including three novel ones, form c', f and g.
  • isoforms apply to all species, but for convenience, throughout the specification and claims, numberings of amino acids and nucleotides will use the rat wild type sequences (FIGURES 1 and 2) as a reference.
  • this invention is not limited to rat wild type proteins and nucleic acids and specifically includes rat (wild type and fatty), mouse, and human OB-R isoform c', f and g proteins and nucleic acids.
  • OB-R isoform f differs from wild type protein in that after the Lysine at position 889 (referring to the rat sequence in
  • FIGURE 1 there are six amino acids, ending at an Asparagine residue at position 895.
  • the codons are then followed by a Stop codon.
  • One cDNA for rat isoform f is shown in FIGURE
  • Lysine 891 corresponds to the rat Lysine 889, the same six amino acids follow Lysine 889.
  • the OB-R isoform f is from rat origin.
  • OB-R isoform g differs from the wild type in that it is much shorter that the wild type sequence.
  • the following eighteen amino acids are found at the beginning of the protein with the superscript numbers indicating their position.
  • the Arginine at position 18 is spliced to a large fragment of the wild type molecule, beginning at the Proline at position 166 (in both mouse and human). This isoform then extends for the remainder of the wild type molecule.
  • the remainder of the protein may be the same as wild type, or, alternatively it could also contain another isoform variation, such as isoform a, b, c, d, e, or f.
  • a particularly preferred embodiment is the rat isoform g.
  • OB-R isoform c' is similar to the OB-R isoform c which was previously described [Lee et al., Nature 379: 632-635]. After Lysine at position 889, it only has three amino acids, Val 8 90 ⁇ hr891 Phe 8 92 St op. As can be seen, isoform c' differs from isoform c in that the final amino acid is phenylalanine rather than valine found in isoform c. Further, there are untranslated sequences in the DNA encoding isoform c' which do not appear to be present in isoform c. The cDNA encoding the rat isoform c' is given in FIGURE 4. In humans, the Val, Thr, Phe follow Lysine 891.
  • One aspect of this invention is the molecular cloning of these various isoforms of OB-R.
  • the wild type and/ ⁇ receptor proteins contain an extracellular, a transmembrane domain. In the rat, the extracellular domain extends from amino acids 1 -830; the transmembrane domain is from amino acids 839-860; and the cytoplasmic domain is from amino acids 860-1 162. Similar domains have bene identified for the mouse and human proteins.
  • This invention also includes isoform c', f and g proteins which lack one or more of these domains. Such deleted proteins are useful in assays for identifying ligands and their binding activity.
  • the mature protein isoforms form yet another aspect of this invention. This differs somewhat from the signal sequence of 1 - 22 reported for mouse and human OB-R; the mature mouse and human isoforms form yet another aspect of this invention.
  • the OB-R isoform c', f or g gene can be introduced into virtually any host cell using known vectors. Preferred host cells include E. coli as well as mammalian and yeast cell lines.
  • the OB-R isoform c', f or g gene may be present in the vector in its native form, or it may be under the control of a heterologous promoter, and if desired, one or more enhancers, or other sequences known to regulate transcription or translation.
  • the host cell containing the OB-R isoform c ⁇ f or g gene is cultured, and the OB-R isoform c', f or g gene is expressed. After a suitable period of time the OB-R c', f or g isoform protein may be harvested from the cell using conventional separation techniques.
  • a further aspect of this invention is the use of an OB-R c', f or g isoform in assays to identify OB-R c', f or g isoform ligands.
  • a ligand binds to the OB-R isoform receptor, and in vivo may or may not result in an activation of the receptor.
  • Ligands may be agonists of the receptor (i.e. stimulate its activity), antagonists (inhibit its activity) or they may bind with little or no effect upon the receptor activity.
  • an OB-R isoform of this invention is exposed to a putative ligand, and the amount of binding is measured.
  • the amount of binding may be measured in many ways; for example, a ligand or the OB-R isoform being investigated may be labeled with a conventional label (such as a radioactive or fluorescent label) and then put in contact with the OB-R isoform under binding conditions. After a suitable time, the unbound ligand is separated from the OB-R isoform and the amount of ligand which has bound can be measured. This can be performed with any of the OB-R isoforms of this invention; alternatively the amount of binding of the various isoforms can be compared.
  • both the putative ligand and a known ligand are present, and the amount of binding of the putative ligand is compared to the amount of binding to a known ligand.
  • the putative ligand's ability to displace previously bound known ligand may be measured.
  • the assay may be a heterogeneous one, where the OB-R isoform may be bound to a surface, and contacted with putative ligands. Dectection of binding may be by a variety of methods, including labelling, reaction with antibodies, and chomophores.
  • the OB-R isoforms of this invention may be used in a "trans" activation assay.
  • Such assays are described in U.S. Application Serial No. , Attorney Docket No. 19686PV, which was filed on April 22, 1996 and which is hereby inco ⁇ orated by reference.
  • a cell which expresses an OB-R isoform of this invention (either naturally or through recombinant means) is transfected with a reporter gene construct comprising a minimal promoter, a leptin activation element and a reporter gene. Transcription of the reporter gene is dependant upon activation of the leptin activation element. Binding of a ligand to the receptor isoform activates the leptin activation element, which then allows transcription of the reporter gene.
  • Tissues were collected from lean and fa/fa Zucker rats and snap frozen in liquid nitrogen.
  • the tissues collected included: hypothalamus, pituitary, lung, liver, kidney, heart, adrenal glands, smooth muscle, skeletal muscle, and adipose tissue.
  • the tissues were homogenized with a Brinkmann Polytron homogenizer in the presence of guanadinium isothiocyanate.
  • mRNA was prepared from hypothalamus, lung, and kidney according to the instructions provided with the messenger RNA isolation kit (Stratagene, La Jolla, CA).
  • cDNA was prepared from approximately 2 ⁇ g of mRNA with the SuperscriptTM choice system (Gibco/BRL Gaithersburg, MD).
  • the first strand cDNA synthesis was primed using 1 ⁇ g of oligo(dT)i 2-18 primer and 25 ng of random hexamers per reaction.
  • Second strand cDNA sythesis was performed according to the manufacturer's instructions.
  • the quality of the cDNA was assessed by labeling an aliqout (l/10 tn ) of the second strand reaction with approximately 1 ⁇ Ci of [ ⁇ - 32 P]dCTP (3000 Ci/mmol).
  • the labeled products were separated on an agarose gel and detected by autoradiography.
  • phosphorylatedBitXI adapters (Invitrogen, San Diego, CA) were ligated to approximately 3 ⁇ g of cDNA prepared as described in Example 1. The ligation mix was then diluted and size-fractionated on a cDNA sizing column (Gibco/BRL Gaithersburg, MD). Drops from the column were collected and the eluted volume from the column was determined. An aliqout from each fraction was analyzed on an agarose gel. Fractions containing cDNA of greater than or equal to 1 kb were pooled and precipitated.
  • the size -fractionated cDNA with the Bst XI adapters was ligated into the prokaryotic vector pcDNA II (Invitrogen, San Diego, CA).
  • the vector (4 ⁇ g) was prepared for ligation by first cutting with the restriction endonuclease Bst XI, gel purifying the linearized vector, and then dephosphorylating the ends with calf intestinal phosphatase (Gibco/BRL, Gaithersburg, MD) according to the manufacturers instructions.
  • the ligation contained approximately 10-20 ng of cDNA and approximately 100 ng of vector and was incubated overnight at 14°C.
  • the ligation was transformed into 1 ml of XL-2 Blue Ultracompetent cells (Stratagene, La Jolla, CA) according to the manufacture's intructions. The transformed cells were spread on 133 mm
  • Colony/Plaque Screen filters (Dupont/NEN, Boston, MA), plated at a density of 30,000 to 60,000 colonies per plate on Luria Broth agar plates containing 100 ⁇ g/ml Ampicillin (Sigma, St. Louis, MO).
  • Colonies on filters were replica plated onto a second filter set.
  • the master filter was stored at 4°C for subsequent isolation of regions containing colonies that gave a positive hybridization signal.
  • the replica filters were grown for several hours at 37°C until colonies were visible and then processed for in situ hybridization of colonies according to established procedures (Maniatis, et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, NY, which is hereby inco ⁇ orated by reference).
  • a Stratalinker (Stratagene, La Jolla, CA) was used to crosslink the DNA to the filter.
  • the filters were washed at 55°C for 2 hours in 2x SSC and 0.5% SDS to remove bacterial debris.
  • the probe a 2.2 kb fragment encoding the extracellular portion of the Ob-R was labeled by random priming with [alpha 32 P]dCTP (3000 Ci/mmole, Amersham, Arlington Heights, IL) using redi-prime (Amersham, Arlington Heights, IL).
  • the probe was purified from uninco ⁇ orated nucleotides using a Probequant G-50 spin column (Pharmacia Biotech, Piscataway, NJ). Filters were washed two times with O.lx SSC 0.1 % SDS at 60°C for 30 min and then subjected to autoradiography. Individual regions containing hybridization positive colonies were lined up with the autoradiogram of the hybridized filter.
  • the rat OB receptor was initially obtained by PCR using degenerate primers based on the mouse and human OB- receptor amino acid sequences.
  • a set of oligonucleotide primers, were designed to regions with low codon degeneracy.
  • the fragments of interest were amplified as long polymerase chain reaction (PCR) products by a modifying the method of Barnes (1994, Proc. Natl. Acad. Sci. 91 :2216-2220, which is hereby inco ⁇ orated by reference).
  • PCR polymerase chain reaction
  • Taq Extender Stratagene, La Jolla CA
  • Expand Long Template PCR System Boehringer Mannheim, Indianapolis, IN
  • the standard PCR reaction mix in a final volume of 20 ⁇ l, contained 5 ng of template (lean rat cDNA), 100 ng of primers, 500 ⁇ M dNTPs, 1 X Buffer 3 from the Expand kit, 0.1 ⁇ l each of Taq Polymerase and Taq Expander. Reactants were assembled in thin walled reaction tubes.
  • the amplification protocol was: 1 cycle of 92°C for 30 sec, followed by 32 cycles at 92°C for 30 sec, 45°C for 1 min. and 68°C for 3 min. using a Perkin-Elmer (Norwalk, CT) 9600 Thermal Cycler.
  • This strategy produced a series of PCR products with the largest being approximately 2.2 Kbp amplified from primers ROBR 2 and ROBR 9. These products were subcloned for DNA sequence analysis as described below.
  • the insert was excised from the cloning vector with the restriction endonuclease Ec ⁇ RI, and fragments were separated from the vector by agarose gel electrophoresis. The fragments were eluted from the gel using a Prep-A-Gene kit (BioRad, Richmond CA) according to the manufacturer's instructions and radiolabeled as described above.
  • PCR products of the appropriate size were prepared for subcloning by separation on an agarose gel, excising the band, and extracting the DNA using Prep-A-Gene (BioRad, Richmond, CA). PCR products were ligated into pCRTMII (Invitrogen, San Diego, CA) according to the instructions provided by the manufacturer. The ligation was transformed into INVaF' cells and plated on Luria- Bertani plates containing 100 ⁇ g/ml ampicillin and X-Gal (32 ⁇ l of 50 mg/ml X-Gal (Promega, Madison, WI). White colonies were picked and grown overnight in Luria-Bertani broth plus 100 ⁇ g/ml ampicillin. Plasmid DNAs were prepared using the Wizard miniprep kit (Promega, Madison, WI). Inserts were analyzed by digesting the plasmid DNA with EcoRI and separating the restriction endonulease digestion products on an agarose gel,
  • Plasmid DNA was prepared for DNA sequencing by ethanol precipitation of Wizard miniprep plasmid DNA and resuspending in water to achieve a final DNA concentration of 100 ⁇ g/ml. DNA sequence analysis was performed using the ABI
  • PRISMTM dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase, FS.
  • the initial DNA sequence analysis was performed with Ml 3 forward and reverse primers, subsequently primers based on the rat OB-R sequence were utilized.
  • the extension products were purified and analyzed on an ABI PRISM 377 automated sequencer (Perkin Elmer, Norwalk, CT). DNA sequence data was analyzed with the Sequencher program.

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Abstract

Le récepteur du gène ob présente de nombreuses isoformes résultant d'un épissage différent, ces nouvelles isoformes, appelées c', f et g, sont décrites. Les acides nucléiques codant ces isoformes sont présentés. De même, une partie de l'invention a trait à des vecteurs contenant l'acide nucléique codant les récepteurs, des cellules hôtes transformées à l'aide de ces gènes ainsi que des dosages utilisant les gènes ou les isoformes protéiques.
PCT/US1997/007521 1996-05-06 1997-05-02 Isoformes de recepteur du gene ob et acides nucleiques les codant WO1997042340A1 (fr)

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CA002253832A CA2253832A1 (fr) 1996-05-06 1997-05-02 Isoformes de recepteur du gene ob et acides nucleiques les codant
JP09540094A JP2000512486A (ja) 1996-05-06 1997-05-02 Ｏｂレセプターイソ型及びそれらをコードする核酸
EP97922646A EP0900282A4 (fr) 1996-05-06 1997-05-02 Isoformes de recepteur du gene ob et acides nucleiques les codant

Applications Claiming Priority (4)

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US1689996P 1996-05-06 1996-05-06
US60/016,899 1996-05-06
GBGB9610995.4A GB9610995D0 (en) 1996-05-24 1996-05-24 OB Receptor isoforms and nucleic acids encoding them
GB9610995.4 1996-05-24

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US6770444B2 (en) 1998-07-16 2004-08-03 Synaptic Pharmaceutical Corporation Methods of identifying or screening for agents that binds the Ob-Re
US7063958B1 (en) 1996-01-16 2006-06-20 The Rockefeller University Nucleic acids db, the receptor for leptin
US7084252B1 (en) 1996-01-16 2006-08-01 The Rockefeller University DB, the receptor for leptin
US7148004B1 (en) 1997-01-16 2006-12-12 The Rockefeller University Oligonucleotides of the OB-R isoforms and methods of diagnosing body weight
US7619079B2 (en) 1996-02-14 2009-11-17 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof

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CA2104996A1 (fr) * 1992-08-29 1994-03-01 Karlheinz Enssle Methode de detection et de determination de mediateurs
WO1996008510A1 (fr) * 1994-09-14 1996-03-21 Progenitor, Inc. L'Hu-B1.219, UN NOUVEAU RECEPTEUR HUMAIN DE L'HEMATOPOIETINE

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CA2246226A1 (fr) * 1996-02-22 1997-08-28 Merck & Co., Inc. Recepteurs de l'obesite du rat et nucleotides codant ces recepteurs
AU2408397A (en) * 1996-04-30 1997-11-19 Otsuka Pharmaceutical Co., Ltd. Ob protein receptor genes and use of the same

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CA2104996A1 (fr) * 1992-08-29 1994-03-01 Karlheinz Enssle Methode de detection et de determination de mediateurs
WO1996008510A1 (fr) * 1994-09-14 1996-03-21 Progenitor, Inc. L'Hu-B1.219, UN NOUVEAU RECEPTEUR HUMAIN DE L'HEMATOPOIETINE

Non-Patent Citations (7)

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Title
CHEN H., ET AL.: "EVIDENCE THAT THE DIABETES GENE ENCODES THE LEPTIN RECEPTOR: IDENTIFICATION OF A MUTATION IN THE LEPTIN RECEPTOR GENE IN DB/DB MICE.", CELL, CELL PRESS, US, vol. 84., 9 February 1996 (1996-02-09), US, pages 491 - 495., XP002029746, ISSN: 0092-8674, DOI: 10.1016/S0092-8674(00)81294-5 *
CHUA S C, ET AL.: "PHENOTYPES OF MOUSE DIABETES AND RAT FATTY DUE TO MUTATIONS IN THE OB (LEPTIN) RECEPTOR", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 271, 16 February 1996 (1996-02-16), US, pages 994 - 996, XP000941868, ISSN: 0036-8075, DOI: 10.1126/science.271.5251.994 *
CIOFFI J. A., ET AL.: "NOVEL B219/OB RECEPTOR ISOFORMS: POSSIBLE ROLE OF LEPTIN IN HEMATOPOIESIS AND REPRODUCTION.", NATURE MEDICINE., NATURE PUBLISHING GROUP, NEW YORK, NY., US, vol. 02., no. 05., 1 May 1996 (1996-05-01), US, pages 585 - 589., XP002019361, ISSN: 1078-8956, DOI: 10.1038/nm0596-585 *
HODGSON J.: "RECEPTOR SCREENING AND THE SEARCH FOR NEW PHARMACEUTICALS.", BIOTECHNOLOGY. THE INTERNATIONAL MONTHLY FOR INDUSTRIAL BIOLOGY, NATURE PUBLISHING GROUP, US, vol. 10., 1 September 1992 (1992-09-01), US, pages 973 - 977., XP002923250, ISSN: 0733-222X, DOI: 10.1038/nbt0992-973 *
LEE G.-H., ET AL.: "ABNORMAL SPLICING OF THE LEPTIN RECEPTOR IN DIABETIC MICE.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 379., 15 February 1996 (1996-02-15), United Kingdom, pages 632 - 635., XP002030818, ISSN: 0028-0836, DOI: 10.1038/379632a0 *
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TAOUIS M, ET AL.: "CLONING THE CHICKEN LEPTIN GENE", GENE., ELSEVIER, AMSTERDAM., NL, vol. 208, 1 January 1998 (1998-01-01), NL, pages 239 - 242, XP002939906, ISSN: 0378-1119, DOI: 10.1016/S0378-1119(97)00670-7 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US7063958B1 (en) 1996-01-16 2006-06-20 The Rockefeller University Nucleic acids db, the receptor for leptin
US7084252B1 (en) 1996-01-16 2006-08-01 The Rockefeller University DB, the receptor for leptin
US7612171B2 (en) 1996-01-16 2009-11-03 The Rockefeller University DB, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
US7812137B2 (en) 1996-01-16 2010-10-12 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
US7619079B2 (en) 1996-02-14 2009-11-17 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
US7148004B1 (en) 1997-01-16 2006-12-12 The Rockefeller University Oligonucleotides of the OB-R isoforms and methods of diagnosing body weight
US6770444B2 (en) 1998-07-16 2004-08-03 Synaptic Pharmaceutical Corporation Methods of identifying or screening for agents that binds the Ob-Re

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EP0900282A4 (fr) 2001-04-11

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