WO1993025691A1 - Sequences de nucleotides codant une adn polymerase thermostable, adn oolymerase et ses utilisations - Google Patents

Sequences de nucleotides codant une adn polymerase thermostable, adn oolymerase et ses utilisations Download PDF

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Publication number
WO1993025691A1
WO1993025691A1 PCT/IT1993/000058 IT9300058W WO9325691A1 WO 1993025691 A1 WO1993025691 A1 WO 1993025691A1 IT 9300058 W IT9300058 W IT 9300058W WO 9325691 A1 WO9325691 A1 WO 9325691A1
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PCT/IT1993/000058
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Francesca Maria PISANI
Mosé ROSSI
Mario De Rosa
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Consiglio Nazionale Delle Ricerche
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Priority to AU45126/93A priority Critical patent/AU4512693A/en
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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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention concerns the isolation and the identification of sequences coding a DNA polymerase from bacteria belonging to the Archaea domain (Woese C.R. et al. 1990, Proc. Natl. Acad.Sci. USA 87, 4576-4579), to the protein coded by said sequence and to uses thereof.
  • DNA polymerases are enzymes responsible of the duplication of genomic DNA and, therefore, of the inheritance of the genetic material. Sequences coding DNA polymerase from bacteria belonging to the Archaea domain are not known in the prior art. Such bacteria are adapted to grow at high temperatures, and are evolutionary far from Eubacteria.
  • DNA polymerases may be classified in two classes (Ito, J., and Braithwaite, D.K. (1991) Nucleic Acids Res. 19, 4045-4057).
  • Class A comprises dideoxynucleotide inhibition sensitive and aphidicolin resistant enzymes, as pol I from E. coli (Joyce, CM., Kelley, W.S., and Grindley, N.D. F. (1982) J. Biol. Chem. 257, 1958-1964); class B is more heterogeneous, comprising aphidicolin sensitive and partially dideoxynucleotide inhibition resistant enzymes.
  • DNA polymerase extracted from bacteria of thermostable and thermofilic Sulfolobus solfataricus species has a molecular weight of around 100 kDa, by means of gel filtration chromatoghraphy and of glycerol gradient centrifugation.
  • An electrophoresis in denaturing conditions on polyacrylammide gel shows, other than the 100 kDa protein, two major bands, respectively of 50 e 40 kDa. These bands represent proteolytic cleavage fragments of the 100 kDa protein, being able to react with antisera raised against the native 100 kDa protein.
  • the 50 kDa fragment keeps a DNA polymerase activity (Karawya, E., Swack, J.A. , and Wilson, S.H. (1983) Anal. Biochem. 135, 318-325) .
  • the authors of the present invention have isolated and sequenced the gene coding the DNA polymerase from S. solfataricus, and have deduced the aminoacid sequence of the protein.
  • the gene Upon insertion into procaryotic or eucaryotic expression vectors and transformation of suitable hosts, the gene makes possible the production through recombinant DNA techniques of the DNA polymerase enzyme.
  • thermoofilic refers to enzymes with a peak of activity at temperatures comprised between 50°C and 85°C, preferably 75°C, when a substrate of DNA from activated calf thymus is used; the term “thermostable” refers to the fact that the enzyme keeps 100% of activity after incubation for 40 min at 75°C.
  • nucleic acid of natural, recombinant or synthetic origin comprising a nucleotide sequence coding a polypeptide or fragments thereof having a thermostable and thermofilic DNA polymerase activity.
  • nucleotide sequence is derived from DNA of bacteria of the Arc ⁇ aeadomain, preferably of the Sulfolobus genus, more preferably of the S. solfataricusspecies .
  • said polypeptide or fragments thereof have also a 3" -5' exonuclease activity.
  • nucleotide sequence codes the polypeptide having the aminoacid sequence of
  • SEQ ID N2 or fragments thereof, alternatively deleted or substituted for one or more aminoacids, so that said DNA polymerase activity is maintained.
  • nucleic acid comprised in the sequence of SEQ ID Nl characterized in that from nucleotide 1 to nucleotide 197 is a non coding sequence, from nucleotide 198 to nucleotide 2843 coding a polypeptide with a thermostable and thermofilic DNA polymerase activity and from nucleotide 2844 to nucleotide 3112 is a non coding sequence.
  • said nucleotide sequence lacks or is substituted of one or more nucleotides so that said DNA polymerase activity is maintained.
  • nucleotide sequences able to hybridize at medium stringency to nucleotide sequences of the invention, preferably said sequences are complementary to the sequences of the invention.
  • a polypeptide with a thermostable and thermofilic DNA polymerase activity preferably produced through recombinant DNA techniques by nucleotide sequences according to the invention, preferably by the nucleotide sequence comprised in SEQ ID Nl.
  • polypeptide has a sequence comprised in SEQ ID N2.
  • figure 1 which represents a restriction map of the coding region of the DNA polymerase gene of S. solfataricus
  • figures 2a and 2b which represent a sequence analysis of DNA polymerase sequences from different organisms.
  • the membrane is stained with
  • Coomassie Brilliant Blue R-250 Three protein bands of 100, 50 e 40 kDa are cutted and loaded directly on a gas-phase aminoacid sequencer (M. 470
  • N-terminal sequences of 50 e 40 kDa peptides are:
  • Each oligonucleotide is labelled at its 5 1 end with ⁇ P 32 ATP by means of T4 polynucleotide kinase and used to screen a genomic library of S. solfataricus, strain MT4 (ATCC n. 49155) , in the ⁇ gtllvector, at the EcoRI site, according to standard methods. Filter hybridization are made at 45°C with the SSDP40K probe and at 50°C with the SSDP50K probe, in 6 x saline citrate buffer (SSC) as described in Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) in Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor.
  • SSC 6 x saline citrate buffer
  • Inserts of positive phages pAl, pC5 e pEl are subcloned into the EcoRI site of the pUC18 vector, and sequenced (Sequenase, USB) .
  • the inserts have partial overlapping regions and an open reading frame, as shown in Fig. 1.
  • the library is screened with the EcoRI insert of pC5 clone as probe.
  • Hybridizations are performed on filters at 65°C, 6 x SSC, according to Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) in Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor.
  • Two positive phages ⁇ 4B and ⁇ 2P are purified and digested with restriction enzymes (Fig. 1) .
  • the EcoRI-PstI fragment, present in both phages, and able to hybridize with pEl, pAl and pC5 clones is inserted into the pEMBL ⁇ vector, producing the plasmid named pFCpolS (DSM N. 7091) .
  • the sequence is shown in SEQ ID Nl.
  • the sequence shows a region of 882 codons with an open reading frame, in agreement with the 100 kDa molecular weight of the protein.
  • the 5* end non coding region does not comprise promoter sequences homologous to other Arc ⁇ aeabacterial promoters (Reiter, W.D., Palm, P., and Zillig, W. (1988) Nucleic Acids Res.
  • a pirimidine rich region comprising the TTTTTAT sequence is present at the 3'end of the termination codon, in analogy with other terminators from Archaea bacteria (Cubellis, M.V. , Rozzo, C, Nitti, G. , Arnone, M.I, Marino, G. , and Sannia, G. (1989) Eur. J. Biochem. 186, 375-381; Cubellis, M.V. , Rozzo, C, Montecucchi, P., and Rossi, M. (1990) Gene 94, 89-94; Reiter, W.D., Palm, P., and Zullig, W. (1989) Nucleic Acid Res. 16, 2445-2459) .
  • a sequence analysis shows homologies with class B DNA polymerases, as viral eucaryote replicases (Gibbs, J.S., Chiou, H.C., Hall, J.D., Mount, D.W. , Retondo, M.J., Weller, S.K., and Coen, D.M. (1985) Proc. Natl. Acad. Sci. USA 82, 7969- 7973; Kouzarides, T. , Bankier, A.T., Satchwell, S.C., Weston, K. , Tomlison, P., and Barrel, B.G. (1987) J. Virol.
  • Class B DNA polymerases show conserved motifs (Ito, J., and Braithwaite, D.K. (1991) Nucleic Acids Res. 19, 4045-4057; Wong. S.W. , Zahl, A.F., Yuan, P.-M., Arai, N. , Pearson, B. E., Arai, K.-I., Korn, D., Hunkapiller, M.W. , and Wang, T. S.-F. (1988) EMBO J. 7, 37-47; Iwasaki, H. , Ishino, Y., Toh, H., Nakata, A., and Shinagawa, H. (1991) Mol. Gen. Genet.
  • Regions 1, 2 e 3 correspond to EXO motifs found in DNA polymerases with 3'-5' exonuclease activity (Morrison, A., Bell, J.B., Kunkel, T.A. , and Sugino, A. (1991) Proc. Natl. Acad. Sci. USA 88, 9473-9477) , where three aspartic acid and one glutammic acid residues are maintained.
  • ORGANISM Sulfolobus solfataricus
  • TCA TCT AAA CCC GCT AAG AGT GAA CAA AAT ACT CAA CAA TCG CAA CAG 2 Ser Ser Lys Pro Ala Lys Ser Glu Gin Asn Thr Gin Gin Ser Gin Gin 15 20 25

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  • Microbiology (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Enzymes And Modification Thereof (AREA)
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Abstract

Séquences de nucléotides codant un polypeptide ou des fragments de ce polypeptide, présentant une activité d'ADN polymérase thermostable et thermophile, obtenues de préférence de l'ADN de bactéries de l'espèce Sulfolobus, ADN polymérase et ses utilisations.
PCT/IT1993/000058 1992-06-11 1993-06-10 Sequences de nucleotides codant une adn polymerase thermostable, adn oolymerase et ses utilisations WO1993025691A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45126/93A AU4512693A (en) 1992-06-11 1993-06-10 Nucleotide sequences coding for a thermostable dna polymerase, dna polymerase and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITRM920438A IT1255666B (it) 1992-06-11 1992-06-11 Sequenze nucleotidiche codificanti per una dna polimerasi.
ITRM92A000438 1992-06-11

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WO1993025691A1 true WO1993025691A1 (fr) 1993-12-23

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648211A (en) * 1994-04-18 1997-07-15 Becton, Dickinson And Company Strand displacement amplification using thermophilic enzymes
EP0880590A1 (fr) * 1996-02-16 1998-12-02 Recombinant Biocatalysis Inc. Esterases
WO2000053772A1 (fr) * 1999-03-06 2000-09-14 Roche Diagnostics Gmbh Adn-polymerase obtenue a partir de $i(pyrobaculum islandicum)
US7288400B2 (en) 1996-02-16 2007-10-30 Verenium Corporation Nucleic acids encoding esterases and methods of making and using them

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0455430A2 (fr) * 1990-04-26 1991-11-06 New England Biolabs, Inc. DNA polymérase thermostable purifiée à obtenir de Thermococcus litoralis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0455430A2 (fr) * 1990-04-26 1991-11-06 New England Biolabs, Inc. DNA polymérase thermostable purifiée à obtenir de Thermococcus litoralis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
EMBL Database Accesion number X64466; 30 May 1992 *
NUCLEIC ACIDS RESEARCH. vol. 20, no. 11, 11 June 1992, ARLINGTON, VIRGINIA US pages 2711 - 2716 PISANI, F.M. ET AL. 'A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases' *
SYSTEM. APPL. MICROBIOL. vol. 7, 1986, pages 337 - 341 M. ROSSI ET AL. 'Struture and properties of a thermophilic and thermostable DNA polymerase isolated from Sulfolobus solfataricus' *
THE ITALIAN JOURNAL OF BIOCHEMISTRY vol. 39, no. 2, April 1990, pages 83 - 99 R. RELLA ET AL. 'Purification and properties of a thermophilic and thermostable DNA polymerase from the Archaebacterium Sulfolobus solfataricus' *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5648211A (en) * 1994-04-18 1997-07-15 Becton, Dickinson And Company Strand displacement amplification using thermophilic enzymes
EP0880590A1 (fr) * 1996-02-16 1998-12-02 Recombinant Biocatalysis Inc. Esterases
EP0880590A4 (fr) * 1996-02-16 2000-11-08 Diversa Corp Esterases
EP1550721A2 (fr) * 1996-02-16 2005-07-06 Diversa Corporation Esterases
EP1550721A3 (fr) * 1996-02-16 2007-04-18 Diversa Corporation Esterases
US7288400B2 (en) 1996-02-16 2007-10-30 Verenium Corporation Nucleic acids encoding esterases and methods of making and using them
WO2000053772A1 (fr) * 1999-03-06 2000-09-14 Roche Diagnostics Gmbh Adn-polymerase obtenue a partir de $i(pyrobaculum islandicum)

Also Published As

Publication number Publication date
AU4512693A (en) 1994-01-04
IT1255666B (it) 1995-11-09
ITRM920438A0 (it) 1992-06-11
ITRM920438A1 (it) 1993-12-11

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