EP0377624A1 - Sondes moleculaires d'adn specifique du genome male du genre bos - Google Patents

Sondes moleculaires d'adn specifique du genome male du genre bos

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Publication number
EP0377624A1
EP0377624A1 EP19880907713 EP88907713A EP0377624A1 EP 0377624 A1 EP0377624 A1 EP 0377624A1 EP 19880907713 EP19880907713 EP 19880907713 EP 88907713 A EP88907713 A EP 88907713A EP 0377624 A1 EP0377624 A1 EP 0377624A1
Authority
EP
European Patent Office
Prior art keywords
formulas
dna
fragments
chromosome
fragment
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Withdrawn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
EP19880907713
Other languages
German (de)
English (en)
French (fr)
Inventor
Colin Bishop
Corinne Cotinot
Marc Fellous
Marek Kirszenbaum
Marcel Vaiman
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.)
Institut National de la Recherche Agronomique INRA
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Institut Pasteur
Original Assignee
Commissariat a lEnergie Atomique CEA
Institut National de la Recherche Agronomique INRA
Institut Pasteur
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 Commissariat a lEnergie Atomique CEA, Institut National de la Recherche Agronomique INRA, Institut Pasteur filed Critical Commissariat a lEnergie Atomique CEA
Publication of EP0377624A1 publication Critical patent/EP0377624A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination

Definitions

  • the present invention relates to molecular probes specific DNA useful for sex determination - or sexing - embryos or fetuses of ruminants, no ⁇ MENT of the subfamily of cattle, particularly of the genus Bos, having DNA sequences specific for the male sex, as well as flanking sequences, or primers, for the gene amplification of these probes; the present invention also encompasses the applications of said probes, preferably amplified using the above flanking sequences, and in particular their applications to the determination of the sex of embryos and fetuses of the aforementioned ruminants, and to the control of the presence of the Y chromosome in a population of spermatozoa as well as in the separation of said population into two groups respectively comprising the Y chromosome and the X chromosome, for the purpose of artificial insemination.
  • RNA is mentioned in this PCT International Application (see d) above), it seems that the preferred nucleic acids are DNA and consist of the EcoRI fragment of 6.2 kbp, 4, 0 kbp or 2.2 kbp of the genome of -ES6.0.
  • the process for sexing embryos or fetuses of the genus Bos consists in: - bringing the DNA of cells of the embryo or fetus into contact, under hybridization conditions, with one or more probes hybridization as defined above, each detectably marked;
  • the hybridization probes are obtained by nicktranslation and the DNA of embryos or fetuses would only come from at least 4, but preferably from at least 10, embryonic cells. .
  • PCT International Application 86/07095 further describes a method of isolating a DNA clone from a genomic library of males of the species concerned which consists of:
  • nucleic acids used as probes are marked by radioactive atoms and it also seems preferred to jointly use two of the probes defined above.
  • the applicants have, for their part, isolated a DNA molecular probe specific for the male genome of mammals, in particular of the genus Bos - which the inventors have moreover managed to synthesize -, the hybridization profile of which (determined by hybridization of said probe with male genomic DNA giganted by EcoRI) reveals the presence of a band of the order of 7 kb specific for the male genome of the genus Bos (and absent from the female genomic DNA), which probe - which has received the designation bc.1.2 - comprises 49 base pairs, the nucleotide sequence (A) is as follows:
  • the Inventors have managed to identify the flanking segments of said probes, which has enabled them to amplify the latter and has the consequence of increasing ter their sensitivity, to ensure a reliability of the sex determination of embryos of the order of 100%, to reduce the time necessary for such a determination.
  • the inventors have been able to establish that the segments or fragments of short male-specific DNA which they have identified and synthesized, can play the role of probes as well as flanking segments, or primers.
  • the subject of the present invention is synthetic oligonucleotides which are characterized in that they consist of fragments of a molecular probe comprising a nucleotide sequence of 49 base pairs, of formula A, below: known in itself, and constituting a DNA segment specific to the male genome of ruminant mammals, in particular of the genus Bos, which fragments are themselves characterized in that they comprise at least 10 bases of said sequence A, including at least 5 consecutive bases thereof, as well as their complementary or any fragment having at least 60% homology with said oligonucleotides.
  • the synthetic oligonucleotides according to the invention are characterized in that they consist of fragments of the molecular probe (A) which comprise 17 bases, including at least 5 consecutive bases of this one and in that they respond to formulas 1 to 8 below:
  • the synthetic oligonucleotides according to the invention are characterized in that they consist of fragments of the molecular probe (A) which comprise 20 bases each and have the nucleotide sequences represented by formulas (9) to (11), below:
  • sequences comprising at least 5 consecutive nucleotides or any fragment having at least 60% homology with said sequences.
  • sequence (9) corresponds to a fragment of the 49 bp probe of formula (A) which goes from base 1 to base 20
  • sequence (10) corresponds to a fragment of the probe 49 bp of formula (A) which goes from base 21 to base 40 and that the sequence (11) corresponds to a fragment of said probe which goes from base 11 to base 30.
  • the inventors have, for their part, synthesized a certain number of fragments of the bovine DNA sequence and have been able to establish that these fragments, on the one hand, are male-specific, and are capable of constituting molecular probes for sexing. of embryos and on the other hand, are capable of playing the role of primers - or flanking segments - and, consequently, of amplifying the fragments of the DNA sequence which play the role of probes, in particular the molecular probe (A), which is also the case for fragments (1) to (8) and (9) to (11) each of which can just as easily act as primers - or flanking segment - of another of these fragments used as probe, that the role of probed amplified by each of said fragments.
  • the molecular probe (A) which is also the case for fragments (1) to (8) and (9) to (11) each of which can just as easily act as primers - or flanking segment - of another of these fragments used as probe, that the role of probed amplified by
  • said fragments of the bovine DNA sequence are characterized in that they have the nucleotide sequences represented by the formulas (12) to (29) below:
  • the present invention further relates to a method for gene amplification of a DNA molecular probe specific for the male genome of mammals, especially ruminants, more particularly of the subfamily of cattle and in a genus-specific manner.
  • Bos which probe has one of the nucleotide sequences of formulas (A) and (1) to (29) above, which amplification method consists in attaching to each of the ends, respectively, of said molecular probe, a flanking sequence judiciously chosen from the DNA fragments of formulas (1) to (29) as defined above, by hybridization, then to implement an enzymatic extension process using DNA polymerase, followed a process of ⁇ enaturation, and to repeat the hybridization-extension-denaturation cycle, known as the PCR cycle, a number of times sufficient to increase the quantity of the sequence constituting the starting molecular probe in a e exponential proportion compared to the number of cycles implemented.
  • Bovine DNA fragments which can be used as molecular DNA probes specific for the male genome of mammals mentioned above, or as flanking segments of these probes, identified above and, where appropriate, advantageously amplified in accordance with the present invention, and duly marked with a radioisotope or with an appropriate non-radioactive substance, are used as described in French Patent Application N ° 86 02811 and / or in its Certificate of Addition N ° 86 12616, for the sexing of embryos or fetuses or for checking the presence of the Y chromosome in a sperm population, or for the separation of spermatozoa into two populations of spermatozoa carrying respectively the Y chromosome and the X chromosome with a view to their use for artificial insemination.
  • the present invention also relates to a process for the preparation of monoclonal or polyclonal antibodies by immunization of rodent mammals by sperm fractions carrying the Y- or X-chromosome controlled by hybridization with a bovine DNA fragment of formula (1) to (29), or a probe of formula (A), amplified by one or more primers - or flanking segments - of formulas (1) to (29).
  • the present invention further relates to specific immunological reagents recognizing antigenic determinants controlled by chromosomes X or Y, which have served to obtain said reagents and expressed on the surface of the spermatozoa, characterized in that they consist of the antibodies obtained by implementing the above process, optionally purified.
  • the invention also comprises other arrangements which will emerge from the description which follows.
  • oligonucleotides of 17 base pairs each, comprising at least 5 consecutive bases of the molecular probe (A), which correspond respectively to formula (1) are fragments of the 49 bp sequence which has the nucleotide composition (A) indicated above.
  • Their chemical synthesis was carried out using an automatic synthesizer "APPLIED BIOSYSTEMS” according to the technique of phophoramidite in solid phase.
  • 10 to 20 ⁇ M of the fragments (1) to (8) in a volume of 3 ⁇ l are added to 23 ⁇ 1 of the reaction mixture composed of: 100 mM dithiothreitol (2.5 ⁇ 1), 10 mM spermidine (2.5 ⁇ 1), buffer d incubation 500 mM Tris-HC1 pH 7.5 + 100 mM MgCl2 (2.5 ⁇ 1), 100 ⁇ Ci ( ⁇ -32p) adenosine triphosphate-ATP 3000Ci / mmole (Amersham) and 20 U T4 polynucleotide kinase (Boehringer). After incubation for 30 min at 37 ° C, the reaction is stopped with 2 ⁇ 1 400 mM EDTA and the labeled oligonucleotide is separated from ATP by column chromatography with Sephadex G-25.
  • the reaction mixture composed of: 100 mM dithiothreitol (2.5 ⁇ 1), 10 mM sperm
  • the filters or blots are prehybridized at 40 to 65 ° C for 1 to 4 hours with a 5xSSPE mixture containing 0.9 M - NaCl; 50 mM NaHPO 4 pH 7.4; 5 mM EDTA pH 7.4; 5 x Denhart; 0.3 mg / ml of sonicated salmon sperm DNA and hydrolysis; 0.5% sodium dodecyl sulfate.
  • Hybridization is carried out in the same mixture.
  • Each of the 32 P radiolabelled probes, denatured at 100 ° C. for two minutes, is added to the hybridization solution at 1 to 6 ⁇ 10 6 cpm / ml.
  • Hybridization is carried out from 40 to 65 ° C for at least 1 hour.
  • the filters are washed in a 2 x SSC buffer plus 0.5% SDS, once or twice at 45 ° C for 30 minutes.
  • the filters are then placed in a cassette fitted with "CRONEX” intensifiers (DUPONT), placed in contact with a Kodak XAR-5 film for autoradiography for 1 to 16 hours at -70 ° C.
  • DUPONT "CRONEX" intensifiers
  • the reaction was 95-97% efficient at each stage, it was necessary to purify the final product (subfragments (9) to (11) - 20 mer) from the intermediate oligonucleotides.
  • About 7 mg of the mixture of oligonucleotides in 60 ⁇ 1 H 2 O are separated by electrophoresis in the gel of $ 20 polyacrylamide + 8 M urea at 700 V, 38mA for 3 hours.
  • the bands corresponding to the sea are identified using an aluminum sheet coated with silica gel, fluorescent at 254 nm (Merck). This part of the gel is cut, then the DNA eluted in 1.5 ml of the 0.5 M CH 3 COONH 4 + 5 mM EDTA solution with stirring at 37 ° C. for 16 hours.
  • 10 to 20 ⁇ M of fragments 9 to 11 in a volume of 3 ⁇ l are added to 23 ⁇ 1 of the reaction mixture composed of: 100 mM dithiothreitol (2.5 ⁇ 1), 10 mM spermidine (2.5 ⁇ 1), incubation buffer 500 mM Tris-HC1 pH 7.5 + 100 mM MgCl2 (2.5 ⁇ 1), 100 ⁇ Ci (-32P) adenosine triphosphate-ATP 3000Ci / mole (Amersham) and 20 U T4 polynucleoide kinase (Boehringer).
  • the reaction mixture composed of: 100 mM dithiothreitol (2.5 ⁇ 1), 10 mM spermidine (2.5 ⁇ 1), incubation buffer 500 mM Tris-HC1 pH 7.5 + 100 mM MgCl2 (2.5 ⁇ 1), 100 ⁇ Ci (-32P) adenosine triphosphate-ATP 3000Ci / mole
  • Hybridization is carried out at 55 ° C for at least 1 hour. After hybridization, the filters are washed in a 6 x SSC buffer plus 0.1% SDS, once or twice at 65 ° C for 20 minutes. The filters are then placed in a cassette fitted with “CRONEX” intensifiers (DUPONT), placed in contact with a Kodak XAR-5 film for autoradiography for 7 to 36 hours at -70 ° C.
  • DUPONT “CRONEX” intensifiers
  • the biotinylation of said fragments is carried out by adding 3 ′ of biotinated dUTP or any other biotinized deoxynucleotide using the enzymeterminal transferase (Boehringer).
  • 10 to 20 ⁇ M respectively of the synthesized fragments are added to 100 ⁇ l of the reaction mixture composed of: 140 mM potassium cacodylate; 30 mM Tris-HCl pH 7.6; 0.1 mM dithiothreitol; 1 mM CoC1 2 + 0.02 mM biotinylated dUTP and 1 to 2 units of terminal transferase enzyme.
  • the labeled oligonucleotide is separated by column chromatography with Sephadex 25 superfine.
  • the slides are incubated with 90 to 100 ⁇ 1 of hybridization liquid and covered with a plastic film (to avoid evaporation).
  • the slides After denaturing for 10 min in an oven at 100 ° C, the slides are. put in a humid chamber and incubated at 55 ° C for 16 hours.
  • the final composition of the hybridization liquid is:
  • DAB SIGMA ref. D 8001 a diaminobenzidine solution
  • the precipitate formed by the action of peroxidase on DAB is amplified by successive precipitation of gold and silver salts according to the method described by BURNS et al (Journal Clinical Pathology 1985, 35, 1085-1092): Firstly, 50 to 100 ⁇ l are deposited per slide of a 2.5 mM solution of Na chloroaurate (NaAuCl 4 BDH ref. 30125 2R) and incubated for 5 min at room temperature. After washing for 5 min in distilled water, the slides are incubated for 5 min at room temperature with 50 to 100 ⁇ l of a 0.1 M solution of Na sulphide (Na 2 S, SIGMA ref. S 2006), rinsed 5 min with distilled water and incubated 4 to 8 minutes with 100 to 300 ⁇ l of silver reagent.
  • BURNS et al Journal Clinical Pathology 1985, 35, 1085-1092
  • composition of the silver reagent is as follows:
  • the silver reagent is prepared by successively mixing the solutions B1 - B2 - B3 - B4 with stirring. The mixture obtained is added 1: 1 to the. solution A and the reagent thus prepared is used immediately.
  • the cells used are frozen bull sperm stored in liquid nitrogen. We thaw a straw, about 13,000,000 sperm living. The straws are warmed for 30 seconds at 37 ° C. then the sperm diluted in 1 ml of sterile PBS.
  • a series of centrifugations is carried out with the aim of eliminating the freezing medium as much as possible.
  • the suspension of the spermatozoa in PBS is first centrifuged for 10 min at 500 g, the supernatant aspirated and the spermatozoa taken up in 1 ml of 0.11 M sodium citrate. Centrifuge for 10 min at 500 g. The supernatant is aspirated, the residue is taken up in 1 ml of 0.11 M sodium citrate + 5% DMSO, and centrifuged for 10 min at 500 g. We aspirate the supernatant, we resuspend in
  • the cells are either deposited on a slide (30 - 40 ⁇ l of sperm suspension per slide) and treated by in situ hybridization with one of the probes of formulas (1) to (29) biotinylated, or deposited on a filter and hybridized according to the dot-blot technique with one of the probes of formulas (1) to (29) labeled with radioactive nucleotides.
  • the in vitro gene amplification process is based on successive cycles of hybridization of the probes with oligonucleotide "primers" (the “primers” are flanking segments and, specifically, in the present case, any of the fragments of formulas (1) to (29)), -extension from “primers”, using the DNA polymerase enzyme and - denaturation, for example according to a process said PCR ("Polymerase chain reaction") described by the company CETUS CORPORATION (cf. in particular MULLIS et Al., COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, Vol. II, 1986, pages 263-272).
  • PCR Polymerase chain reaction
  • the cells of a biopsy are placed in a microtube in a volume of 1 O ⁇ l in PBS - 35 ⁇ 1 of H2 ⁇ are added, then the tube is placed at 95 ° C for 10 minutes to lyse the cells and denature the DNA.
  • 55 ⁇ l of PCR buffer 50 mM KC1, 10 mM Tris pH 8.3, 8 mM MgC1 2 , 1 ⁇ M of primer A to be fixed at one of the ends of the probe, 1 ⁇ M of primer are added to the lysate B to be fixed at the other end of the probe, 1.5 mM ATP, 1.5 mM dCTP, 1.5 mM dTTP, 1.5 mM dGTP
  • the samples are then transferred to an appropriate temperature for 2 minutes so that the primers are fixed
  • the strand extension is initiated by the addition of 4 units of "Taq Polymerase" (CETUS CORPORATION) and incubation at 69 ° C for 2 minutes.
  • the membranes are then neutralized and then dried at 80 ° C, prehybridized and hybridized at 40 or 65 ° C depending on the primer used, in a buffer 5 x SSPE, 5 x Denhart, 0.5% SDS containing the radiolabelled probes in 5 '- terminal with / ⁇ 32 p / ATP using the polynucleotide kinase enzyme.
  • the probes (49 sea or 17 sea or 20 sea) can be either radioactive. Drink marked with biotiné or another cold compound. Hybridization is carried out at 40 to 60 ° C for 1 hour. The membranes are then washed twice in
  • Primer B GATCAGTGC ⁇ GGGACCG (1) the PCR cycle takes place as follows: The biopsy cells are deposited in a microtube in a volume of 10 ⁇ l in PBS with addition of 35 ⁇ l of H 2 O, as described above above ; however the tube is placed at 95 ° C for only 2 minutes and the operations continue as described above, except that the samples are transferred for 2 minutes at 55 ° C and that the incubation with "Taq Polymerase” takes place at 69 ° C for 2 minutes and the cycle is repeated 40 times.
  • the probe used can be a probe chosen from the probes or fragments of formulas A and 1 to 29. It is possible, for example, with primers of formulas (7) and (1) to use a probe of formula (4):
  • the prehybridization is carried out, as is the hybridization, at 65 ° C.
  • the hybridization at 65 ° C.
  • a very intense male signal distinct from that of females, which is very weak, from 125 picograms of DNA, the equivalent of 25 cells and directly from cells, with 50 male cells, after 30 minutes of autoradiography.
  • a specifically male signal is obtained, from 125 picograms of DNA.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
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  • Genetics & Genomics (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Medicinal Chemistry (AREA)
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EP19880907713 1987-08-27 1988-08-25 Sondes moleculaires d'adn specifique du genome male du genre bos Withdrawn EP0377624A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8711975A FR2619819B1 (fr) 1987-08-27 1987-08-27 Sondes moleculaires d'adn specifique du genome male de ruminants, notamment de la sous-famille des bovines et en particulier du genre bos, sequences flanquantes pour l'amplification de ces sondes et applications desdites sondes
FR8711975 1987-08-27

Publications (1)

Publication Number Publication Date
EP0377624A1 true EP0377624A1 (fr) 1990-07-18

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP19880907713 Withdrawn EP0377624A1 (fr) 1987-08-27 1988-08-25 Sondes moleculaires d'adn specifique du genome male du genre bos

Country Status (8)

Country Link
EP (1) EP0377624A1 (en, 2012)
JP (1) JPH03503357A (en, 2012)
AU (1) AU621556B2 (en, 2012)
DK (1) DK51690A (en, 2012)
FR (1) FR2619819B1 (en, 2012)
IN (1) IN167595B (en, 2012)
NZ (1) NZ225953A (en, 2012)
WO (1) WO1989001978A1 (en, 2012)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2648151B1 (fr) * 1989-06-08 1991-12-20 Michel Georges Minisatellite bovin specifique au chromosome y des bovines
WO1991017188A1 (en) * 1990-05-08 1991-11-14 A.B. Technolody Pty. Limited Separation of mammalian semen into fractions enriched either for spermatozoa containing an x chromosome or for spermatozoa contai ning a y chromosome
JP2593021B2 (ja) * 1991-12-13 1997-03-19 伊藤ハム株式会社 ウシ胚の性の識別方法
CA2708751A1 (en) * 2007-12-14 2009-06-25 Minitube Of America, Inc. Gender-specific separation of sperm cells and embryos

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4448767A (en) * 1977-10-11 1984-05-15 Sumar Corporation Preparation of monospecific male-specific antibody and the use thereof for increasing the percentage of mammalian offspring of either sex
FR2566911A1 (fr) * 1984-06-29 1986-01-03 Pasteur Institut Sonde et procede pour le diagnostic du sexe d'un embryon humain
US4769319A (en) * 1985-05-31 1988-09-06 Salk Institute Biotechnology Industrial Associates, Inc. Nucleic acid probes for prenatal sexing
DE3680156D1 (de) * 1985-05-31 1991-08-14 Amoco Corp Verstaerkung von hybridisierungssignalen durch verwendung von komplementarischen dns-straengen.
FR2603701B2 (fr) * 1986-02-28 1990-09-21 Agronomique Inst Nat Rech Applications des sondes moleculaires d'adn specifique du genome male de mammiferes, notamment du genre bos, au controle de la presence du chromosome y dans une population de spermatozoides et a la separation des spermatozoides en deux populations de spermatozoides porteurs respectivement du chromosome y et du chromosome x pour leur utilisation pour l'insemination artificielle
AU614494B2 (en) * 1986-08-12 1991-09-05 Australian National University, The Sex determination in ruminants using y-chromosome specific polynucleotides
WO1989007154A1 (en) * 1988-01-29 1989-08-10 Advanced Riverina Holdings Limited Determination of genetic sex in ruminants using y-chromosome-specific polynucleotides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8901978A1 *

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Publication number Publication date
JPH03503357A (ja) 1991-08-01
AU2321888A (en) 1989-03-31
NZ225953A (en) 1991-04-26
IN167595B (en, 2012) 1990-11-17
DK51690D0 (da) 1990-02-27
WO1989001978A1 (fr) 1989-03-09
AU621556B2 (en) 1992-03-19
FR2619819A1 (fr) 1989-03-03
FR2619819B1 (fr) 1990-08-10
DK51690A (da) 1990-04-27

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