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WO1990000623A1 - Analyse par fluorescence a resolution temporelle et marquage multiple de sequences d'acide nucleique, a l'aide de chelates de lanthanide - Google Patents

Analyse par fluorescence a resolution temporelle et marquage multiple de sequences d'acide nucleique, a l'aide de chelates de lanthanide Download PDF

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Publication number
WO1990000623A1
WO1990000623A1 PCT/SE1989/000378 SE8900378W WO9000623A1 WO 1990000623 A1 WO1990000623 A1 WO 1990000623A1 SE 8900378 W SE8900378 W SE 8900378W WO 9000623 A1 WO9000623 A1 WO 9000623A1
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WIPO (PCT)
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different
ligand
excitation
lanthanide
label
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PCT/SE1989/000378
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English (en)
Inventor
Marek Kwiatkowski
Erkki Soini
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Wallac Oy
Pharmacia Ab
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Publication of WO1990000623A1 publication Critical patent/WO1990000623A1/fr

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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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
    • C07C237/36—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having the nitrogen atom of the carboxamide group bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
    • C07D213/46—Oxygen atoms
    • C07D213/50—Ketonic radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/65—One oxygen atom attached in position 3 or 5

Definitions

  • nucleic acid sequence analysis has a central role in molecular biology.
  • the presently available techniques for nucleic acid sequence analysis are laborious and expensive and often involve the use of radioisotopes. For this reason, and for sequencing an endless number of genes including the human genome with 3 x 10 9 bases, the development of an automated non-isotopic method for nucleic acid sequence analysis is extremely important.
  • the strategy of the present invention is to provide an automatic real time nucleic acid sequence analyser for a single column electrophoresis system using four fluorescent probes for a simultaneous read-out of each of the four base specific reactions.
  • the nucleic acid fragments generated in each reaction are identified on the basis of the specific excitation and emission wavelengths of the fluorescent probes.
  • the conventional fluorescent probes however, have strongly overlapping excitation and emission spectra as shown in the work of Smith et al. In order to overcome this problem it was decided to investigate the possibility of using fluorescent lanthanide chelates in automatic real time nucleic acid sequencing.
  • Lanthanide chelates have become a very promising alternative as labeling or dye material for biospecific assays and cell analyses (Hemmila, I., 1985, Anal. Chem. 57: 1676-1681; Hemmila, I., Dakubu, S., Mukkala, V-M. , Siitari, H. and Lövgren, T. 1984, Anal. Biochem. 137: 335-343).
  • Europium and terbium have emission bands at 614 nm and 545 nm, respectively, and they also provide relatively strong fluorescence when they form appropriate chelates.
  • Samarinium has its specific emission band at 640 nm, which can be efficiently separated from the emission of Eu and Tb, and consequently the use of Eu, Tb and Sm allows a three parameter analysis in the same sample.
  • the fluorescence emission of the other lanthanides is too weak or their chelates currently known are not stable in water solutions. Therefore, it is difficult to find for example four lanthanides with different emission wavelengths for multiparameter analysis using four labels in the same sample.
  • This invention is related to the discovery of new chelate structures which include an active functional group for covalent binding to modified nucleic acid fragments and which almost equally bind europium and terbium but have different excitation wavelengths.
  • ligand A has the excitation maximum at 274 nm and ligand B at 307 nm.
  • the following combinations of four different lanthanide chelates are then useful for analysing the sample with four parameters.
  • the band widths of the excitation and emission spectra of these chelates as shown in Figures 1-4 - are narrow enough for the detection of each of these four chelates in the same sample using a filter fluorometer. In actual practice, this is achieved by changing the excitation and emission wavelengths according to the above table, and recording four signals for each measurement.
  • DNA sequencing the recording of each of the four DNA terminals (A, C, G and T) directly from the electrophoresis in the same column can be carried out using an appropriate double excitation/double emission wavelength fluorometer where recording of each wavelength combination is fast enough when compared with the movement of the DNA bands in the electrophoresis gel.
  • the sensitivity is of importance in the detection of nucleic acid bands from electrophoresis.
  • the most important sources of the background of biochemical and biological samples are the "autofluorescence" and the scattering.
  • the light emitted from these sources appears simultaneously with or promptly after the excitation as the fluorescence decay time of bio-organic substances is very short.
  • the use of lanthanide chelates as fluorescent probes offers a possibility for efficient reduction of the background and scattering because the fluorescence decay time of lanthanides is long.
  • Time-resolved fluorometry efficiently discriminates the prompt background from the long decay fluorescence of the probe because measurement does not occur until a certain time has elapsed from the moment of excitation.
  • it offers an almost ideal way of measuring probes with long fluorescence decay and considerably improves the sensitivity compared with the conventional measurement methods with short decay time organic fluorescent probes.
  • Lanthanides and their chelates are a special group of compounds which display ion fluorescence with decay times in the order of 50 to 1000 ⁇ sec. and these compounds are very useful for time-resolved microsecond fluorometry.
  • the lanthanide chelates can provide comparable, or better overall sensitivity when used in time-resolved fluorescence detection (Soini & Kojola, Clin. Chem. 29, 65, 1983). This is the case despite the lower quantum yield which is around one tenth or less that of the best organic fluorescent compounds. The low quantum yield stems largely from the wide Stokes shift and the long decay time of the fluorescent signal.
  • a simple fluorometric system based on the use of a Xenon flash lamp as an excitation source and interference filters for selecting the proper excitation and emission bands.
  • Small Xenon flash lamps with a pulse duration of 1 ⁇ s are commercially available and well suited for this purpose.
  • the operation of the time-resolved fluorometric system is shown schematically in Fig. 6.
  • the light source is pulsed having a width of t p .
  • the signal has a maximum instantaneous intensity I p at the end of the excitation pulse.
  • the excited states of the fluorescent label relax with a decay time ⁇ and after a suitable delay time t d the signal from the detector is sampled for a certain period of gate time, t 2 .
  • the fluorescence signal will be integrated during the gate time t p over a definite number of measurement cycles (n).
  • Instrumentation which is available and applicable to time-resolved fluorometric studies with lanthanide chelates should be equipped with a pulsed light source producing very short light pulses in comparison with the actual decay times.
  • the interferences between different fluorescent complexes can be reduced by optimizing the delay time t d and gate time t g , because the complexes have different decay times ⁇ . Therefore, in addition to optimizing the excitation and emission bands in multiparameter analysis, it is important to control the t d and t g .
  • the control should be made automatic and synchronous with the wavelength control.
  • FIG. 7 illustrates the principle of the nucleic acid sequencing device according to the invention.
  • the electrophoresis column (3) communicates with an upper buffer reservoir (1) and a lower buffer reservoir (2).
  • the excitation light sources producing light pulses of wavelength 274 nm (5) and wavelength 307 nm (6) incorporate an appropriate interference filter and lens system.
  • the light pulses are focused in point (4) in the column (3). Fluorescence emissions from the column are collected to the detectors (7) and (8).
  • the detectors include an appropriate lens system and interference filters for wavelengths 544 nm and 614 nm.
  • the excitation light sources and detectors are controlled by the electronics unit, and activated periodically for example as follows
  • 4th cycle light source 6 detector 8 in such a way that emissions at both wavelengths are recorded for both excitation wavelengths and these 4 cycles are repeated until a sufficient number of photon counts has been recorded for acceptable statistical precision.
  • the electronic control system is also adjusted to record the fluorescence emission in a time-resolved mode.
  • ligands A and B and for terbium and europium are presented as examples.
  • the invention is not limited to the use of terbium and europium as a pair, or ligand A (274 nm max excitation) and ligand B (307 nm max excitation) as a pair, but any other pairs with appropriate wavelengths are within the scope of this invention.
  • 6,6'-Dimethyl-2,2'-bipyridine-N-oxide (1.50 g, 0.00749 moles) was dissolved in concentrated sulphuric acid (8.0 ml) and fuming nitric acid (6.0 ml) and the mixture was heated at 100oC for four hours. The solution was poured slowly into ice-water and the pH was adjusted to 5.5 with 10 % sodium hydroxide. The product was filtered and dried.
  • 6-Bromo-2-pyridinecarboxaldehyde J.Am.Chem.Soc. 91, (11), 3500 (1970)
  • J.Am.Chem.Soc. 91, (11), 3500 (1970) (11.38 g, 62.2 mmoles) was dissolved in a mixture of dry methanol (200 ml) and trimethyl orthoformate (26.5 g, 250 mmoles).
  • p-toluenesulfonic acid monohydrate 250 mg
  • the mixture was refluxed for 1 h, cooled and neutralized by addition of pyridine (5 ml). Evaporation of solvent and distillation of the product under reduced pressure yielded the pure dimethylacetal as a colorless liquid.
  • 6-Bromo-2-dimethoxymethyl pyridine (14) (14.2 g, 61 mmoles) was dissolved in dry diethyl ether (200 ml) and cooled down to -70oC in a round bottom three-necke flask equipped with reflux condenser and dropping funnel while a gentle flow o dry argon was passing through the magnetically stirred mixture.
  • Butyllithium (25.1 ml (2.6M), 65.3 mmoles) was added dropwise and the tempera ture of the reaction mixture was kept below -60oC.
  • the mixture was stirred fo an additional 1 h after accomplised addition, and ethyl chloroformate (4.52 g 41.7 mmoles) dissolved in dry diethyl ether was introduced at such a rate tha the temperature did not exceed -60 C.
  • the yellow suspension was stirred for 4 min at -60oC and then additionally for 15 min at -40oC.
  • the ether phase wa separated and the aqueous phase was extracted twice with 100 ml of dichloromethane.
  • the combined organic phase was evaporated and coevaporated with toluene which yielded crude title compound (15).
  • This compound was synthesized following the general procedure for tetraester synthesis from Example 14b using compound (6) and diethyl iminodiacetate as starting materials.
  • This compound was synthesized using the general procedure for nitrogroup reduction from Example 14c having compound (8) as a starting material.
  • the monobromomethyl diester (19) was synthesized starting from compound (18) and one molecular equivalent of iminodiacetic acid diethyl ester, in a reaction analogous to that of Example 14 a. Yield: 62 %.
  • 1 H NMR (60 MHz, CDCl 3 ) 1.21 (t, 6 H), 3.60 (s, 4 H), 4.08 (s, 2 H), 4.15 (q, 4 H), 4.58 (s, 2 H), 7.22-8.15 (m, 6 H) .
  • the hexadecamer oligonucleotide of M 13 phage sequencing primer was synthesized using Pharmacia Gen Assembler solid phase nucleic acid synthesizer (Pharmacia, Sweden) following the recommended conditions. When the oligonucleotide synthesis had been accomplished the detritylated compound was subjected to the reaction with 1,1'-carbonyldiimidazole and diaminohexane according to Nucleic Acids Research 14,, 7987-7994 (1986).
  • the reactions were monitored by FPLC, and labeled products from each reaction were separated in ion-pair mode using the reverse phase FPLC system.
  • the purified sequences were characterized by their fluorescent spectra which were found to be identical to the unbound labels.
  • the four differently labeled primers did not show any differences in their electrophoretic mobilities when run under the real sequencing conditions on a 20 % acrylamide gel. This reflects the fact that all labels employed are indeed very closely structure related. No differences were found even when electrophoresis was used for running free labels.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédé d'analyse séquentielle fluorométrique simultanée en temps réel, de fragments d'acide nucléique de différentes longueurs et de quatre terminaisons différentes, lesdits fragments étant marqués par différentes marques fluorescentes pour les bases de terminaison respectives. Les fragments sont marqués à l'aide de quatre marques différentes comprenant les deux mêmes lanthanides et les deux mêmes ligands comme suit: marque 1 = ligand 1 + lanthanide 1, marque 2 = ligand 2 + lanthanide 1, marque 3 = ligand 1 + lanthanide 2, marque 4 = ligand 2 + lanthanide 2. Les ligands 1 et 2 ont des longueurs d'ondes d'excitation différentes, et les lanthanides 1 et 2 ont des longueurs d'ondes d'émission différentes. La lumière d'excitation est produite sous la forme d'impulsions lumineuses alternant entre les différentes longueurs d'ondes d'excitation des ligands. Les photons émis par les marques sont détectés périodiquement pour les différentes longueurs d'ondes d'émission des lanthanides.
PCT/SE1989/000378 1988-07-08 1989-07-03 Analyse par fluorescence a resolution temporelle et marquage multiple de sequences d'acide nucleique, a l'aide de chelates de lanthanide WO1990000623A1 (fr)

Applications Claiming Priority (2)

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SE8802573A SE8802573D0 (sv) 1988-07-08 1988-07-08 Multi-label time-resolved fluorescence analysis of nucleic acid sequences using lanthanide chelates
SE8802573-9 1988-07-08

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SE (1) SE8802573D0 (fr)
WO (1) WO1990000623A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0340675A3 (fr) * 1988-05-02 1991-08-14 The Perkin-Elmer Corporation Procédé de dosage de nucléotides marquées avec lanthanide par fluorométrie à résolution temporelle
WO1992014841A1 (fr) * 1991-02-14 1992-09-03 Baxter Diagnostics Inc. Nouveaux oligonucleotides conjugues a des chelates de lanthanide
FR2687687A1 (fr) * 1992-02-25 1993-08-27 Us Energy Procede pour mesurer la taille de fragments d'adn.
EP0516753A4 (en) * 1990-02-26 1994-05-18 Us Health A method for the fluorescent detection of a dna sequence in real time
WO2000039335A1 (fr) * 1998-12-24 2000-07-06 Aclara Biosciences, Inc. Surfaces solides adressables individuellement pour operations multiplexees
US6329205B1 (en) 1999-08-31 2001-12-11 Molecular Probes, Inc. Detection method using luminescent europium-based protein stains
EP1104491A4 (fr) * 1998-08-11 2003-01-29 Caliper Techn Corp Sequen age d'adn par differenciation des periodes de decroissance d'emission de sondes fluorescentes et systemes a cet effet
US6716394B2 (en) 1998-08-11 2004-04-06 Caliper Technologies Corp. DNA sequencing using multiple fluorescent labels being distinguishable by their decay times
WO2007104834A1 (fr) * 2006-03-13 2007-09-20 Wallac Oy Substrats terminateurs pour adn polymérases
US7482444B2 (en) 2006-03-13 2009-01-27 Wallac Oy Terminating substrates for DNA polymerases
US9062342B2 (en) 2012-03-16 2015-06-23 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1504599A (en) * 1997-12-17 1999-07-05 Shionogi & Co., Ltd. Novel pyridine compounds

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3501306A1 (de) * 1984-01-16 1985-07-25 California Institute Of Technology, Pasadena, Calif. Verfahren fuer die elektrophoretische analyse von dna - fragmenten
EP0252683A2 (fr) * 1986-07-02 1988-01-13 E.I. Du Pont De Nemours And Company Procédé et réactifs pour la détermination de séquences ADN
EP0268406A2 (fr) * 1986-11-06 1988-05-25 AMERSHAM INTERNATIONAL plc Appareil de séquençage biochimique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3501306A1 (de) * 1984-01-16 1985-07-25 California Institute Of Technology, Pasadena, Calif. Verfahren fuer die elektrophoretische analyse von dna - fragmenten
EP0252683A2 (fr) * 1986-07-02 1988-01-13 E.I. Du Pont De Nemours And Company Procédé et réactifs pour la détermination de séquences ADN
EP0268406A2 (fr) * 1986-11-06 1988-05-25 AMERSHAM INTERNATIONAL plc Appareil de séquençage biochimique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nature, Vol 321, 12 June 1986, LLOYD M. SMITH et al: "Fluorescence Detection in Automated DNA Sequence Analysis", p 674-679 see Abstract, figures 1-3 *
Nucleic Acids Research, Vol 15, No 11, 1987, WILHELM ANSORGE et al: "Automated DNA Sequencing: Ultrasensitive Detection of Fluorescent Bands during Electrophoresis", p 4593-4601 see Abstract, p 4600 line 23 - p 4601 line 21 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0340675A3 (fr) * 1988-05-02 1991-08-14 The Perkin-Elmer Corporation Procédé de dosage de nucléotides marquées avec lanthanide par fluorométrie à résolution temporelle
EP0516753A4 (en) * 1990-02-26 1994-05-18 Us Health A method for the fluorescent detection of a dna sequence in real time
WO1992014841A1 (fr) * 1991-02-14 1992-09-03 Baxter Diagnostics Inc. Nouveaux oligonucleotides conjugues a des chelates de lanthanide
US5955601A (en) * 1991-02-14 1999-09-21 E G & G Wallac Lanthanide chelate conjugated oligonucleotides
FR2687687A1 (fr) * 1992-02-25 1993-08-27 Us Energy Procede pour mesurer la taille de fragments d'adn.
US6716394B2 (en) 1998-08-11 2004-04-06 Caliper Technologies Corp. DNA sequencing using multiple fluorescent labels being distinguishable by their decay times
EP1104491A4 (fr) * 1998-08-11 2003-01-29 Caliper Techn Corp Sequen age d'adn par differenciation des periodes de decroissance d'emission de sondes fluorescentes et systemes a cet effet
WO2000039335A1 (fr) * 1998-12-24 2000-07-06 Aclara Biosciences, Inc. Surfaces solides adressables individuellement pour operations multiplexees
US6329205B1 (en) 1999-08-31 2001-12-11 Molecular Probes, Inc. Detection method using luminescent europium-based protein stains
WO2007104834A1 (fr) * 2006-03-13 2007-09-20 Wallac Oy Substrats terminateurs pour adn polymérases
GB2450454A (en) * 2006-03-13 2008-12-24 Wallac Oy Terminating substrates for DNA polymerases
US7482444B2 (en) 2006-03-13 2009-01-27 Wallac Oy Terminating substrates for DNA polymerases
GB2450454B (en) * 2006-03-13 2011-04-13 Wallac Oy Terminating substrates for DNA polymerases
US9062342B2 (en) 2012-03-16 2015-06-23 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
US9334528B2 (en) 2012-03-16 2016-05-10 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
US9757725B2 (en) 2012-03-16 2017-09-12 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module
US9914119B2 (en) 2012-03-16 2018-03-13 Stat-Diagnostica & Innovation, S.L. Test cartridge with integrated transfer module

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SE8802573D0 (sv) 1988-07-08
JPH03500249A (ja) 1991-01-24

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