WO1996014403A1 - Gene de la bacterie rhodothermus marinus, codant pour la sequence d'acides amines de l'adn ligase, produite dans escherichia coli ou dans un autre organisme hote approprie - Google Patents

Gene de la bacterie rhodothermus marinus, codant pour la sequence d'acides amines de l'adn ligase, produite dans escherichia coli ou dans un autre organisme hote approprie Download PDF

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Publication number
WO1996014403A1
WO1996014403A1 PCT/IS1995/000001 IS9500001W WO9614403A1 WO 1996014403 A1 WO1996014403 A1 WO 1996014403A1 IS 9500001 W IS9500001 W IS 9500001W WO 9614403 A1 WO9614403 A1 WO 9614403A1
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Prior art keywords
ligase
dνa
dna
enzyme
gene
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PCT/IS1995/000001
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English (en)
Inventor
Sigridur Thorbjarnardottir
Zophanias O. Jonsson
Astridur Palsdottir
Gudmundur Eggertsson
Jakob K. Kristjansson
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Genis Hf.
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Priority to AU39430/95A priority Critical patent/AU3943095A/en
Publication of WO1996014403A1 publication Critical patent/WO1996014403A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • a gene from the bacterium Rhodothermus ma ⁇ nus coding for the amino acid sequence of DNA-ligase, produced in Escherichia col/ or some other suitable host organism.
  • This invention relates to a gene orriginating from. Rhodothermus m ⁇ rimis and the encoded thermostable DNA ligase enzyme, active over a wide range of temperatures (5-75°C), a unique property which allows the enzyme to be used in cycle sequencing of long D ⁇ A sequences, using computer-controlled automatic equipment.
  • the enzyme can ligate small oligomers during repeated thermal cycles, producing suitable primers for sequencing. This eliminates the need for time consuming subcloning procedures and primer synthesis.
  • DNA ligases catalyze the formation of phosphodiester linkages between adjacent 5 -phosphoryl and 3'-hydroxyl groups in double-stranded DNA.
  • the reaction proceeds in three steps, initiated by the formation of a stable enzyme-(lysine-e)-adenylate complex (for review see Lehmann, 1974) (1).
  • thermostable ligases Various analytical methods are based on the use of thermostable ligases.
  • LCR ligase chain reaction
  • RED repeat expansion detection
  • thermostable DNA ligase in localized D ⁇ A detection by circulari ration of oligodeoxynucleotides as described by ⁇ ilson et al. (4) and to construct sequencing primers from hexameric nucleotides as proposed by Szybalski (5) and Kaczorowski (6).
  • thermostable D ⁇ A ligases active over a wide range of temperatures, we have cloned the ligase gene of the thermophilic eubacterium, Rhodothermus m ⁇ rinus (7).
  • R m ⁇ rinus was originally isolated from a marine alkaline hot spring in Iceland (8) and deposited in "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Macheroder Weg lb, D- 38124 BraunschweigGermany" (strain number: DSM 4253). Analysis of 16S rR ⁇ A places it close to the root of the Flexibacter-Cytophaga-Bacteroides group (9).
  • This invention provides a new DNA ligase enzyme, active at temperatures between 5-75°C with half life of 7 min. at 90°C.
  • the invention constitutes the nucleotide sequence of the gene, coding for the enzyme, its expression in a productive host and purification of the protein to near homogeneity.
  • the properties of this enzyme make it suitable for use in the SPEL-6 method of primer walking (sequential primer elongation by ligation of 6-mers) initially described by Szybalski (5) and computer-controlled automatic sequencing of long DNA sequences. DESCRIPTION OF THE FIGURES Fig. 1.
  • the incubation temperatures for 2.5 finol of R. marinus were as follows: lane 1, 5°C; lane 2, 15°C; lane 3, 25°C; lane 4, 35°C; lane 5, 45°C; lane 6, 55°C; lane 7, 65°C; and lane 8, 75°C.
  • the incubation temperatures for 4 finol of T. scotoductus ligase were as follows: lane 9, 5°C; lane 10, 15°C; lane 11, 25°C; lane 12, 35°C; lane 13, 45°C; lane 14, 55°C; lane 15, 65°C; and lane 16, 75°C.
  • Lane A represents 400 ng of pUC19 plasmid DNA
  • lane B represents approximately 400 ng of nicked pUC19 plasmid DNA.
  • Lanes 1-8 represent R marinus ligase incubated with nicked pUC19 DNA at 5°C (lanes 1-4) and at 15°C (lanes 5-8). Lane 1 contains 5 finol ligase; lane 2 contains 10 finol ligase; lane 3 contains 20 finol ligase; lane 4 contains 40 finol ligase; lain 5 contains 5 finol Ugase; lain 6 contains 10 finol ligase; lain 7 contains 20 finol ligase, and lain 8 contains 40 finol ligase.
  • Lanes 9-16 represent T. scotoductus ligase incubated with nicked pUC19 DNA at 35°C (lanes 9-12) and 45°C (lanes 13-16).
  • Lane 9 contains 8 finol ligase;
  • lane 10 contains 16 finol ligase;
  • lane 11 contains 32 finol ligase;
  • lane 12 contains 64 finol ligase;
  • lane 13 contains 8 finol ligase;
  • laine 14 contains 16 finol ligase;
  • laine 15 contains 32 finol ligase, and lane 16 contains 64 finol Ugase.
  • Lane A represents 400 ng pUC19 plasmid DNA
  • lane B represents approximately 400 ng nicked pUC 19 plasmid DNA.
  • the Ugase encoding gene was cloned into E. coli by complementation of a temperature sensitive (ts) mutation.
  • Gene libraries were made by Ugating Sau3Al partially digested genomic DNA from the thermophilic bacterium into BamHl digested pUC18 plasn ⁇ ds. Plasmids complementing the ts mutation were selected and the presence of the ligase genes confirmed by complementation of two additional ts ligase mutations. Further proof was obtained by assaying nick-closing enzyme activity in heat treated crude extracts.
  • the cloned gene was subcloned into M13mpl8/19 and completely sequenced in both directions (Fig. 1). The sequence information was used to clone the gene into the high expression vector pET23, using PCR and restriction enzymes.
  • For expression we used an E. coli strain carrying a ts ligase mutation and chromosomally inserted DE3 prophage.
  • the prophage contains a T7 polymerase gene under the control of an IPTG inducible lac promoter. This setup secures high expression of Ugase. After induction with IPTG for 4 hours, ligase can make up as much as 15% of total cell protein.
  • the enzyme has been purified to near homogeneity using a simple method relying on heating at 80°C for 30 minutes to denature most of the proteins produced by the host, DEAE-sepharose chromatography to remove contaminating nucleic acids, a Cibacron Blue column for further purification and gel filtration for desalting.
  • a novel assay for ligase activity, used for measuring DNA ligase thermostability was applied on the R marinus enzyme and two other reference enzymes (Thermus thermophilus (Stratagene, La Jolla, CA, USA) and Thermus scotoductus (our own clone)) at 91°C (Figs. 2a and 2b). Dilutions of the enzymes were incubated at 91 °C in ligase reaction buffer and aliquots taken at regular intervals. The aliquots were added to a reaction mix containing two differentially end labeled primers and the complementary template. After thermal cycling, the amount of ligated doubly labeled product was assayed with ELISA. The half life of the R.
  • marinus enzyme was estimated to be 7 min. (Fig. 2a) but both the T. thermophilus and T. scotoductus ligases are somewhat more heat stable, their half lives being around 26 min. at 91°C (Fig. 2b). Our estimate of the half life of T. thermophilus ligase is in agreement with the half life of 30 min. at 90°C reported by the manufacturer.
  • nick closing activity 10
  • T. scotoductus ligase activity could not be detected below 25°C under the same conditions.
  • ligation was not detected with the R. marinus ligase at 5°C.
  • the R marinus ligase is suitable for the SPEL- 6 method of primer walking (6) and permits even cycling as in cycle sequencing (Szybalski and Kaczorowski, personal communication).
  • the SPEL abbreviation stands for "Sequential Primer Elongation by Ligation”.
  • the SPEL-6 method of primer walking described by Szybalsky (5), represents an original strategy of using hexameric DNA of different sequences for production of primers that can be used in DNA sequencing procedures.
  • the SPEL method provides a fast and simple way of constructing primers for sequencing, and eliminates the need for synthesis of individual primers.
  • the key to the SPEL-6 method of primer walking is a DNA Ugase, sufficiently active at low temperatures for ligation of the selected complimentary hexameres, but stable enough to survive subsequent thermal dissociation of produced primers from the template DNA. Consequently, the R marinus Ugase can ideally be used in computer-controlled automatic cycle sequencing, incorporating a ligation step in each cycle.
  • the SPEL-6 method of primer walking eliminates the need for subcloning, permits direct sequencing of large DNA fragments, is ideally suited for automation, and should accelerate the sequencing of large genomes by more than one order of magnitude (6).
  • the 3' end of the oUgo complementary to the 5' site of the template was labeled with biotin and the 5' end phosphorylated (pCCAGGGCTICACACGTGCTACAATG-Bio).
  • the other oUgo complementary to the 3' she of the 50-nt template was 5' labeled with fluorescein (Flu) (Flu-CGTCAAGTCATCATGGCCCTTACGC).
  • Flu fluorescein
  • the heat-treated ligases were mixed with 230 finol template and 10 pmol of each 25-nt primer in 40 ⁇ l of standard assay buffer (see section (d)) and overlayed with parafin oil. The reactions were cycled 20 times between 90°C and 60°C in a thermal cycler and kept for 20 sec at each temperature.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Nouvelle enzyme dite ADN ligase dérivée de la bactérie thermophile Rhodothermus marinus, et active aux températures comprises entre 5 et 75 °C, sa demi-vie à 90 °C étant de 7 minutes. L'invention concerne la séquence nucléotidique du gène codant pour cette enzyme, son expression dans un hôte productif, la purification de la protéine jusqu'à obtention d'une homogénéité quasi totale, et l'application de l'enzyme au séquençage de cycles. Les propriétés particulières de cette enzyme la rendent utilisable dans le procédé SPEL-6 de marche sur l'amorce (allongement séquentiel d'amorces par liaison de 6-mères) décrit en premier par Szybalski (5), et dans le séquençage automatique commandé par ordinateur des longues séquences d'ADN.
PCT/IS1995/000001 1994-11-04 1995-11-03 Gene de la bacterie rhodothermus marinus, codant pour la sequence d'acides amines de l'adn ligase, produite dans escherichia coli ou dans un autre organisme hote approprie WO1996014403A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39430/95A AU3943095A (en) 1994-11-04 1995-11-03 A gene from the bacterium rhodothermus marinus, coding for the amino acid sequence of dna-ligase, produced in escherichia coli or some other suitable host organism

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IS4231 1994-11-04
IS4231 1994-11-04

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044481A1 (fr) * 1996-05-21 1997-11-27 Case Western Reserve University Compositions et procedes de criblage d'antimicrobiens
WO2005108583A1 (fr) * 2004-05-06 2005-11-17 Prokaria Ehf. Polypeptide thermostable a activite polynucleotide kinase et/ou activite phosphatase

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017239A1 (fr) * 1990-05-03 1991-11-14 Cornell Research Foundation, Inc. Systeme d'amplification d'adn induit par ligase thermostable, servant a detecter des maladies genetiques.
EP0571880A1 (fr) * 1992-05-23 1993-12-01 Roche Diagnostics GmbH Ligase thermostable d'Archeobacteria
WO1994002615A1 (fr) * 1992-07-23 1994-02-03 Stratagene Ligase d'adn du pyrococcus furiosus thermostable purifiee

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017239A1 (fr) * 1990-05-03 1991-11-14 Cornell Research Foundation, Inc. Systeme d'amplification d'adn induit par ligase thermostable, servant a detecter des maladies genetiques.
EP0571880A1 (fr) * 1992-05-23 1993-12-01 Roche Diagnostics GmbH Ligase thermostable d'Archeobacteria
WO1994002615A1 (fr) * 1992-07-23 1994-02-03 Stratagene Ligase d'adn du pyrococcus furiosus thermostable purifiee

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 101, No. 17, 22 October 1984, (Columbus, Ohio, USA), TAKAHASI MIHO et al., "Thermophilic DNA Ligase. Purification and Properties of the Enzyme from Thermus Thermophilus HB8", page 292, Abstract No. 146594x; & J. BIOL. CHEM., 1984, 259(16), 10041-10047. *
CHEMICAL ABSTRACTS, Volume 108, No. 17, 25 April 1988, (Columbus, Ohio, USA), ALFREDSSON GUDNI A. et al., "Rhodothermus Marinus, Gen. Nov., Sp. Nov., a Thermophilic, Halophilic Bacterium from Submarine Hot Springs in Iceland", page 428, Abstract No. 147065g; & J. GEN. MICROBIOL., 1988, 134(2), 299-306. *
CHEMICAL ABSTRACTS, Volume 117, No. 3, 20 July 1992, (Columbus, Ohio, USA), LAUER GAIL et al., "Cloning, Nucleotide Sequence and Engineered Expression of Thermus Thermophilus DNA Ligase, a Homolog of Escherichia Coli DNA Ligase", page 189, Abstract No. 21385d; & J. BACTERIOL., 1991, 173(16), 5047-5053. *
CHEMICAL ABSTRACTS, Volume 118, No. 13, 29 January 1993, (Columbus, Ohio, USA), BARANY FRANCIS et al., "Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-encoding Gene", page 353, Abstract No. 119741n; & GENE, 1991, 109(1), 1-11. *
CHEMICAL ABSTRACTS, Volume 123, No. 25, 18 December 1995, (Columbus, Ohio, USA), THORBJARNARDOTTIR SIGRIDUR H. et al., "Cloning and Sequence Analysis of the DNA Ligase-encoding Gene of Rhodothermus Marinus and Overproduction, Purification and Characterization of Two Thermophilic DNA Ligases", page 487, Abstract No. *
PATENT ABSTRACTS OF JAPAN, Vol. 9, No. 96, C-278; & JP,A,59 227 295 (KAGAKU GIJIYUTSUCHIYOU), 20 December 1984. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044481A1 (fr) * 1996-05-21 1997-11-27 Case Western Reserve University Compositions et procedes de criblage d'antimicrobiens
AU731074B2 (en) * 1996-05-21 2001-03-22 Case Western Reserve University Compositions and methods for screening antimicrobials
US6809180B2 (en) 1996-05-21 2004-10-26 Case Western Reserve University Compositions and methods for screening antimicrobials
WO2005108583A1 (fr) * 2004-05-06 2005-11-17 Prokaria Ehf. Polypeptide thermostable a activite polynucleotide kinase et/ou activite phosphatase

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