WO1993025691A1

WO1993025691A1 - Nucleotide sequences coding for a thermostable dna polymerase, dna polymerase and uses thereof

Info

Publication number: WO1993025691A1
Application number: PCT/IT1993/000058
Authority: WO
Inventors: Francesca Maria PISANI; Mosé ROSSI; Mario De Rosa
Original assignee: Consiglio Nazionale delle Richerche CNR
Current assignee: Consiglio Nazionale delle Richerche CNR
Priority date: 1992-06-11
Filing date: 1993-06-10
Publication date: 1993-12-23
Anticipated expiration: 1994-12-11
Also published as: ITRM920438A0; IT1255666B; AU4512693A; ITRM920438A1

Abstract

Nucleotide sequences coding a polypeptide or fragments thereof having a thermostable and thermophilic DNA polymerase activity, preferably derived from DNA of bacteria of Sulfolobus genus, DNA polymerase and uses thereof.

Description

NϋCLEOTiDE SEQUENCES CODING FOR A THERMOSTABLE DNA POLYMERASE, DNA POLYMERASE AND USES THEREOF

SPECIFICATION

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.

The authors of the instant invention have demonstrated that 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. Moreover 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. 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.

According to the invention the term "thermofilic" 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.

It is an object of the invention a 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. Preferably said nucleotide sequence is derived from DNA of bacteria of the ArcΛaeadomain, preferably of the Sulfolobus genus, more preferably of the S. solfataricusspecies . In a preferred embodiment said polypeptide or fragments thereof have also a 3" -5' exonuclease activity.

Preferably said 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.

Further object of the invention is a 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. Alternatively said nucleotide sequence lacks or is substituted of one or more nucleotides so that said DNA polymerase activity is maintained. Another object of the invention are 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. It is another object 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.

According to the invention said polypeptide has a sequence comprised in SEQ ID N2.

It is a further object of the invention recombinant cloning or expression vectors, having a plasmid or viral derivation, comprising the nucleotide sequences of the invention, preferably said vector is the plasmid pFCpolS (DSM N.7091) .

Another object are cells transformed with said vectors. The invention will be described in the following examples, with reference to the following figures: 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.

Example 1 Partial aminoacid sequence of DNA polymerase from S. solfataricus

30 μg of DNA polymerase purified from S. solfataricus, as described in Rossi M. et al. 1986,

System. Appl. Microbiol. 7, 337-341, is loaded on a 10% polyacrylammide gel in denaturing conditions. The gel is then electro-transferred on a PVDF membrane (Problott, Applied Biosystems) , as described in Matsudaira, P. (1987) J. Biol. Chem.

262, 10035-10038. 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

A, Applied Biosystems) , with an analyzer PTH 120 A.

N-terminal sequences of 50 e 40 kDa peptides are:

50 kDa GYKGAWIDP 40 kDa SAPVEEKKWR

Example 2 Isolation and sequence of the DNA polymerase gene of S. solfataricus

By using aminoacid sequences the following degenerated oligonucleotides are sinthesized: 29-mer SSDP50K corresponding to the N- terminal sequence of the 50 kDa fragment: 5'-GGATA(T/C) GG(T/A) GG(T/A) GC(T/A) GT(T/A) GT(T/A) AT(T/A) GAT CC-3'

23-mer SSDP40K corresponding to the N- terminal sequence of the 40 kDa fragment: 5'-GC(T/A) CC(T/A) GT(T/A) GA(A/G) AA(A/G)

AA(A/G) GT-3'.

Each oligonucleotide is labelled at its 5¹ end with χP³²ATP by means of T4 polynucleotide kinase and used to screen a genomic library of S. solfataricus, strain MT4 (ATCC n. 49155) , in the λ 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. 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.

Another genomic library obtained in the λ EMBL3 vector with Mbol partially digested DNA of S. solfataricus, producing fragments of around 15 Kb, according to standard methods. 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 λ 4B and λ 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 pEMBLδ 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 ArcΛaeabacterial promoters (Reiter, W.D., Palm, P., and Zillig, W. (1988) Nucleic Acids Res. 16, 1-19; Reiter, W.D., Hudepohl, U. , and Zillig, W. (1990) Proc. Natl. Acad. Sci USA 87, 9509-9513. 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) .

Example 3 Sequence homology with other DNA polymerases

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. 61, 125-133; Earl, P.L., Jones, E.V., and Moss, B. (1986) Proc. Natl. Acad. Sci. USA 83, 3659-3663), human replicases (Wong, S.W. , Wahl, 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) and DNA polymerase ct ofi S. cerevisiae (Pizzagalli, A. , Valsasnini, P., Plevani, P., and Lucchini, G. (1988) Proc. Natl. Acad. Sci. USA 85, 3772-3776) . Few homologies are evident with E. coli DNA polymerases (Joyce, CM., Kelley, W.S., and Grindley, N.D. F. (1982) J. Biol. Chem. 257, 1958- 1964) .

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. 226, 24-33; Larder, B.A. , Kemp, S.D., and Darby, G. (1987) EMBO J. 6, 169-175; Bernard, A., Zaballos, A., Salas, , M. , and Blanco, L. (1987) EMBO J. 6, 4219-4225; Blanco, L. , Bernard, A., Blasco, M.A. , and Salas, M. (1991) Gene 100, 27-38), which are found also in the sequence of the invention, as shown inFigs. 2a and regions 1-8.

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. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Consiglio Nazionale delle Ricerche

(B) STREET: P.le Aldo Moro 5

(C) CITY: Roma

(D) STATE: Italy

(E) COUNTRY : Italy

(F) POSTAL CODE (ZIP) : 00185

(ii) TITLE OF INVENTION: Nucleotide sequences coding for a DNA polymerase, DNA polymerase and uses thereof

(iii) NUMBER OF SEQUENCES : 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3112 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DΝA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Sulfolobus solfataricus

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 198..2846

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATCTGGTGTT TTTCTTTCTC ATGCATATTA ATAATGTTTA CTAAGATTCA AGGCATATCT

CTTAAGAAAT GGCTAGATGA ATGAGAGGAG CAGGAGTAGC TTAAGAATCT TAAAACTTAG 1

GTTCTTCATA AATGTCTATT TTTTCTCCCG CATTAAAACT TATAGCGTAT TTCTCAGAAA 1

ATAATATATG TTAGAAA ATG ACT AAG CAA CTT ACC TTA TTT GAT ATT CCT 2

Met Thr Lys Gin Leu Thr Leu Phe Asp lie Pro 1 5 10

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

AGT GCT CCC GTT GAG GAA AAA AAG GTA GTT AGG AGG GAA TGG CTT GAA 3 Ser Ala Pro Val Glu Glu Lys Lys Val Val Arg Arg Glu Trp Leu Glu 30 35 40

GAG GCT CAG GAA AAT AAG ATA TAC TTC CTA TTG CAA GTA GAT TAT GAT 3 Glu Ala Gin Glu Asn Lys lie Tyr Phe Leu Leu Gin Val Asp Tyr Asp 45 50 55

GGT AAG AAA GGT AAG GCT GTA TGT AAG CTA TTC GAT AAA GAA ACT CAA 4 Gly Lys Lys Gly Lys Ala Val Cys Lys Leu Phe Asp Lys Glu Thr Gin 60 65 70 75 AAG ATC TAT GCC CTA TAT GAT AAT ACT GGA CAT AAG CCC TAC TTT CTA 47 Lys lie Tyr Ala Leu Tyr Asp Asn Thr Gly His Lys Pro Tyr Phe Leu

80 85 90

GTA GAT CTT GAA CCT GAT AAA GTA GGT AAA ATA CCT AAG ATT GTT AGA 51 Val Asp Leu Glu Pro Asp Lys Val Gly Lys lie Pro Lys lie Val Arg 95 100 105

GAT CCA TCT TTT GAT CAC ATA GAG ACT GTG AGT AAG ATA GAC CCG TAT 56 Asp Pro Ser Phe Asp His lie Glu Thr Val Ser Lys lie Asp Pro Tyr 110 115 120

ACT TGG AAT AAA TTC AAA TTA ACT AAA ATC GTT GTT AGA GAT CCC CAT 61 Thr Trp Asn Lys Phe Lys Leu Thr Lys lie Val Val Arg Asp Pro His 125 130 135

GCA GTG AGA AGA TTA AGG AAT GAT GTT CCA AAA GCG TAT GAG GCT CAC 66 Ala Val Arg Arg Leu Arg Asn Asp Val Pro Lys Ala Tyr Glu Ala His 140 145 150 155

ATA AAA TAT TTT AAC AAC TAC ATG TAT GAC ATA GGT CTA ATC CCC GGT 71 lie Lys Tyr Phe Asn Asn Tyr Met Tyr Asp lie Gly Leu lie Pro Gly

160 165 170

ATG CCT TAT GTT GTT AAG AAT GGG AAG TTA GAA AGT GTC TAT TTG TCT 75 Met Pro Tyr Val Val Lys Asn Gly Lys Leu Glu Ser Val Tyr Leu Ser 175 180 185

TTG GAC GAG AAA GAT GTT GAG GAG ATT AAG AAA GCC TTC GCT GAT TCA 80 Leu Asp Glu Lys Asp Val Glu Glu lie Lys Lys Ala Phe Ala Asp Ser 190 195 200

GAT GAA ATG ACT AGA CAA ATG GCA GTC GAT TGG CTT CCC ATA TTT GAA 85 Asp Glu Met Thr Arg Gin Met Ala Val Asp Trp Leu Pro lie Phe Glu 205 210 215 ACT GAA ATA CCT AAA ATA AAA AGG GTT GCG ATA GAT ATT GAG GTA TAT 9 Thr Glu lie Pro Lys lie Lys Arg Val Ala lie Asp lie Glu Val Tyr 220 225 230 235

ACA CCA GTT AAG GGT AGA ATC CCA GAC TCT CAG AAG GCT GAG TTT CCA 9 Thr Pro Val Lys Gly Arg lie Pro Asp Ser Gin Lys Ala Glu Phe Pro

240 245 250

ATT ATA AGT ATA GCA TTA GCG GGG AGT GAT GGA TTA AAG AAG GTT CTT 9 lie lie Ser lie Ala Leu Ala Gly Ser Asp Gly Leu Lys Lys Val Leu 255 260 265

GTA TTA AAT AGG AAT GAT GTC AAT GAA GGG AGT GTA AAA CTT GAT GGA 10 Val Leu Asn Arg Asn Asp Val Asn Glu Gly Ser Val Lys Leu Asp Gly 270 275 280

ATA TCG GTT GAG AGA TTT AAT ACA GAG TAC GAA CTG TTA GGG AGA TTT 10 lie Ser Val Glu Arg Phe Asn Thr Glu Tyr Glu Leu Leu Gly Arg Phe 285 290 295

TTT GAT ATA CTG TTA GAA TAT CCG ATA GTT CTT ACA TTC AAT GGA GAC 11 Phe Asp lie Leu Leu Glu Tyr Pro lie Val Leu Thr Phe Asn Gly Asp 300 305 310 315

GAT TTT GAT TTA CCT TAC ATT TAC TTT AGG GCG TTA AAG TTA GGT TAT 11 Asp Phe Asp Leu Pro Tyr lie Tyr Phe Arg Ala Leu Lys Leu Gly Tyr

320 325 330

TTT CCA GAG GAA ATT CCC ATA GAT GTA GCT GGT AAG GAT GAA GCC AAG 12 Phe Pro Glu Glu lie Pro lie Asp Val Ala Gly Lys Asp Glu Ala Lys 335 340 345

TAT CTA GCT GGT CTT CAT ATA GAC TTG TAC AAA TTC TTC TTT AAT AAG 12 Tyr Leu Ala Gly Leu His lie Asp Leu Tyr Lys Phe Phe Phe Asn Lys 350 355 360 GCA GTG AGG AAT TAT GCA TTT GAG GGA AAG TAT AAT GAA TAC AAT TTA 133 Ala Val Arg Asn Tyr Ala Phe Glu Gly Lys Tyr Asn Glu Tyr Asn Leu

365 370 375

GAT GCA GTT GCA AAG GCC TTA TTA GGG ACA TCA AAA GTT AAG GTA GAT 138 Asp Ala Val Ala Lys Ala Leu Leu Gly Thr Ser Lys Val Lys Val Asp 380 385 390 395

ACG CTA ATA TCT TTC TTA GAT GTA GAA AAA TTA ATA GAA TAT AAC TTT 143 Thr Leu lie Ser Phe Leu Asp Val Glu Lys Leu lie Glu Tyr Asn Phe

400 405 410

AGG GAT GCC GAA ATC ACA CTT CAG CTT ACT ACA TTT AAT AAC GAC CTA 147 Arg Asp Ala Glu lie Thr Leu Gin Leu Thr Thr Phe Asn Asn Asp Leu 415 420 425

ACT ATG AAG TTA ATT GTA TTG TTT TCT AGA ATT TCT AGA CTA GGA ATT 152 Thr Met Lys Leu lie Val Leu Phe Ser Arg lie Ser Arg Leu Gly lie 430 435 440

GAG GAA TTA ACT CGG ACA GAA ATA TCT ACT TGG GTA AAG AAT TTA TAT 157 Glu Glu Leu Thr Arg Thr Glu lie Ser Thr Trp Val Lys Asn Leu Tyr 445 450 455

TAT TGG GAA CAT AGA AAA AGA AAT TGG TTA ATT CCT CTT AAG GAA GAA 162 Tyr Trp Glu His Arg Lys Arg Asn Trp Leu lie Pro Leu Lys Glu Glu 460 465 470 475

ATC TTA GCG AAA TCC TCT AAT ATA AGA ACT TCT GCT CTA ATA AAG GGA 167 lie Leu Ala Lys Ser Ser Asn lie Arg Thr Ser Ala Leu lie Lys Gly

480 485 490

AAA GGA TAT AAA GGC GCA GTA GTT ATA GAC CCA CCT GCT GGA ATA TTC 171 Lys Gly Tyr Lys Gly Ala Val Val lie Asp Pro Pro Ala Gly lie Phe 495 500 505 TTT AAC ATA ACT GTT TTA GAT TTT GCA TCA CTA TAT CCT TCA ATA ATT 17 Phe Asn lie Thr Val Leu Asp Phe Ala Ser Leu Tyr Pro Ser lie lie •» 510 515 520

* AGA ACG TGG AAT CTT AGT TAC GAG ACT GTA GAC ATT CAA CAA TGT AAG 18 Arg Thr Trp Asn Leu Ser Tyr Glu Thr Val Asp lie Gin Gin Cys Lys 525 530 535

AAG CCC TAT GAA GTA AAG GAT GAG ACA GGG GAG GTG CTA CAT ATA GTT 18 Lys Pro Tyr Glu Val Lys Asp Glu Thr Gly Glu Val Leu His lie Val 540 545 550 555

TGC ATG GAT AGG CCA GGT ATA ACA GCA GTA ATA ACT GGG TTA CTA AGA 19 Cys Met Asp Arg Pro Gly lie Thr Ala Val lie Thr Gly Leu Leu Arg

560 565 570

GAC TTC AGA GTA AAG ATA TAC AAA AAG AAA GCG AAG AAC CCT AAT AAT 19 Asp Phe Arg Val Lys lie Tyr Lys Lys Lys Ala Lys Asn Pro Asn Asn 575 580 585

AGT GAG GAA CAA AAA CTA CTC TAT GAC GTA GTA CAG AGA GCA ATG AAA 20 Ser Glu Glu Gin Lys Leu Leu Tyr Asp Val Val Gin Arg Ala Met Lys 590 595 600

GTA TTC ATA AAT GCT ACT TAC GGT GTA TTT GGA GCT GAA ACA TTT CCG 20 Val Phe lie Asn Ala Thr Tyr Gly Val Phe Gly Ala Glu Thr Phe Pro 605 610 615

TTA TAT GCG CCA CGT GTA GCG GAG AGT GTT ACT GCA CTG GGG AGA TAC 21 Leu Tyr Ala Pro Arg Val Ala Glu Ser Val Thr Ala Leu Gly Arg Tyr 620 625 630 635

GTT ATT ACC AGT ACC GTA AAG AAA GCT AGG GAA GAA GGT TTA ACT GTA 21 Val lie Thr Ser Thr Val Lys Lys Ala Arg Glu Glu Gly Leu Thr Val

640 645 650 TTA TAC GGT GAT ACT GAT TCT TTA TTC CTC CTT AAT CCT CCC AAG AAT 219 Leu Tyr Gly Asp Thr Asp Ser Leu Phe Leu Leu Asn Pro Pro Lys Asn 655 660 665

AGT TTA GAA AAT ATT ATA AAA TGG GTT AAA ACT ACT TTC AAT TTA GAT 224 Ser Leu Glu Asn lie lie Lys Trp Val Lys Thr Thr Phe Asn Leu Asp 670 675 680

TTG GAA GTT GAT AAA ACC TAC AAG TTT GTG GCT TTT TCT GGA TTG AAG 229 Leu Glu Val Asp Lys Thr Tyr Lys Phe Val Ala Phe Ser Gly Leu Lys 685 690 695

AAG AAT TAC TTT GGA GTA TAC CAA GAC GGG AAG GTT GAT ATA AAG GGG 234 Lys Asn Tyr Phe Gly Val Tyr Gin Asp Gly Lys Val Asp He Lys Gly 700 705 710 715

ATG TTA GTG AAG AAG AGA AAC ACG CCG GAA TTT GTA AAG AAG GTA TTT 239 Met Leu Val Lys Lys Arg Asn Thr Pro Glu Phe Val Lys Lys Val Phe

720 725 730

AAC GAG GTA AAG GAG CTA ATG ATC TCC ATA AAC TCG CCA AAC GAT GTG 243 Asn Glu Val Lys Glu Leu Met He Ser He Asn Ser Pro Asn Asp Val 735 740 745

AAG GAG ATT AAA AGA AAA ATT GTA GAC GTA GTT AAA GGA TCA TAT GAA 248 Lys Glu He Lys Arg Lys He Val Asp Val Val Lys Gly Ser Tyr Glu 750 755 760

AAA CTA AAA AAC AAA GGA TAC AAT CTG GAC GAA TTA GCG TTT AAA GTA 253 Lys Leu Lys Asn Lys Gly Tyr Asn Leu Asp Glu Leu Ala Phe Lys Val 765 770 775

ATG CTA TCG AAG CCT TTA GAT GCG TAC AAA AAG AAC ACT CCC CAA CAC 258 Met Leu Ser Lys Pro Leu Asp Ala Tyr Lys Lys Asn Thr Pro Gin His 780 785 790 795 GTA AAG GCA GCT CTA CAA CTT AGA CCA TTT GGA GTT AAC GTA TTA CCA 263 Val Lys Ala Ala Leu Gin Leu Arg Pro Phe Gly Val Asn Val Leu Pro

800 805 810

CGA GAT ATA ATA TAC TAT GTT AAG GTT AGA TCT AAA GAT GGA GTG AAA 267 Arg Asp He He Tyr Tyr Val Lys Val Arg Ser Lys Asp Gly Val Lys 815 820 825

CCA GTA CAA CTA GCT AAA GTT ACT GAA ATA GAC GCA GAG AAA TAT TTA 272 Pro Val Gin Leu Ala Lys Val Thr Glu He Asp Ala Glu Lys Tyr Leu 830 835 840

GAA GCG TTA AGA AGT ACG TTT GAG CAA ATC TTA AGG GCA TTC GGA GTC 277 Glu Ala Leu Arg Ser Thr Phe Glu Gin He Leu Arg Ala Phe Gly Val 845 850 855

TCT TGG GAT GAG ATA GCA GCC ACA J-TG TCG ATA GAT TCG TTC TTT TCA 282 Ser Trp Asp Glu He Ala Ala Thr Met Ser He Asp Ser Phe Phe Ser 860 865 870 875

TAC CCA AGT AAA GGA AAT AGT TAATTAAGAA AGATAGCAAT TCTTCATAAT 287 Tyr Pro Ser Lys Gly Asn Ser

880

AAATTTTTAG AAGCAATTTT TACCCACATA AGTTATAAAG ATTTTTAGAA AATTTAAATC 293

GTATATTTTT ATTCTTCCTC CTCTTCCTCT AATTCTTCCT TTAATTCTTC TTGTTTCTGC 299

ATACCCAAGT AAAGGAAATA GTTAATTAAG AAAGATAGCA ATTCTTCATA ATAAATTTTT 305

AGAAGCAATT TTTACCCACA TAAGTTATAA AGATTTTTAG AAAATTTAAA TCGTATATT 311 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 882 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Thr Lys Gin Leu Thr Leu Phe Asp He Pro Ser Ser Lys Pro Ala 1 5 10 15

Lys Ser Glu Gin Asn Thr Gin Gin Ser Gin Gin Ser Ala Pro Val Glu 20 25 30

Glu Lys Lys Val Val Arg Arg Glu Trp Leu Glu Glu Ala Gin Glu Asn 35 40 45

Lys He Tyr Phe Leu Leu Gin Val Asp Tyr Asp Gly Lys Lys Gly Lys 50 55 60

Ala Val Cys Lys Leu Phe Asp Lys Glu Thr Gin Lys He Tyr Ala Leu 65 70 75 80

Tyr Asp Asn Thr Gly His Lys Pro Tyr Phe Leu Val Asp Leu Glu Pro

85 90 95

Asp Lys Val Gly Lys He Pro Lys He Val Arg Asp Pro Ser Phe Asp 100 105 110

His He Glu Thr Val Ser Lys He Asp Pro Tyr Thr Trp Asn Lys Phe 115 120 125 Lys Leu Thr Lys He Val Val Arg Asp Pro His Ala Val Arg Arg Leu 130 135 140

Arg Asn Asp Val Pro Lys Ala Tyr Glu Ala His He Lys Tyr Phe Asn 145 150 155 160

Asn Tyr Met Tyr Asp He Gly Leu He Pro Gly Met Pro Tyr Val Val

165 170 175

Lys Asn Gly Lys Leu Glu Ser Val Tyr Leu Ser Leu Asp Glu Lys Asp 180 185 190

Val Glu Glu He Lys Lys Ala Phe Ala Asp Ser Asp Glu Met Thr Arg 195 200 205

Gin Met Ala Val Asp Trp Leu Pro He Phe Glu Thr Glu He Pro Lys 210 215 220

He Lys Arg Val Ala He Asp He Glu Val Tyr Thr Pro Val Lys Gly 225 230 235 240

Arg He Pro Asp Ser Gin Lys Ala Glu Phe Pro He He Ser He Ala

245 250 255

Leu Ala Gly Ser Asp Gly Leu Lys Lys Val Leu Val Leu Asn Arg Asn 260 265 270

Asp Val Asn Glu Gly Ser Val Lys Leu Asp Gly He Ser Val Glu Arg 275 280 285

Phe Asn Thr Glu Tyr Glu Leu Leu Gly Arg Phe Phe Asp He Leu Leu 290 295 300

Glu Tyr Pro He Val Leu Thr Phe Asn Gly Asp Asp Phe Asp Leu Pro 305 310 315 320 Tyr He Tyr Phe Arg Ala Leu Lys Leu Gly Tyr Phe Pro Glu Glu He

325 330 335

Pro He Asp Val Ala Gly Lys Asp Glu Ala Lys Tyr Leu Ala Gly Leu 340 345 350

His He Asp Leu Tyr Lys Phe Phe Phe Asn Lys Ala Val Arg Asn Tyr 355 360 365

Ala Phe Glu Gly Lys Tyr Asn Glu Tyr Asn Leu Asp Ala Val Ala Lys 370 375 380

Ala Leu Leu Gly Thr Ser Lys Val Lys Val Asp Thr Leu He Ser Phe 385 390 395 400

Leu Asp Val Glu Lys Leu He Glu Tyr Asn Phe Arg Asp Ala Glu He

405 410 415

Thr Leu Gin Leu Thr Thr Phe Asn Asn Asp Leu Thr Met Lys Leu He 420 425 430

Val Leu Phe Ser Arg He Ser Arg Leu Gly He Glu Glu Leu Thr Arg 435 440 445

Thr Glu He Ser Thr Trp Val Lys Asn Leu Tyr Tyr Trp Glu His Arg 450 455 460

Lys Arg Asn Trp Leu He Pro Leu Lys Glu Glu He Leu Ala Lys Ser 465 470 475 480

Ser Asn He Arg Thr Ser Ala Leu He Lys Gly Lys Gly Tyr Lys Gly

485 490 495

Ala Val val He Asp Pro Pro Ala Gly He Phe Phe Asn He Thr Val 500 505 510 Leu Asp Phe Ala Ser Leu Tyr Pro Ser He He Arg Thr Trp Asn Leu 515 520 525

Ser Tyr Glu Thr Val Asp He Gin Gin Cys Lys Lys Pro Tyr Glu Val 530 535 540

Lys Asp Glu Thr Gly Glu Val Leu His He Val Cys Met Asp Arg Pro 545 550 555 560

Gly He Thr Ala Val He Thr Gly Leu Leu Arg Asp Phe Arg Val Lys

565 570 575

He Tyr Lys Lys Lys Ala Lys Asn Pro Asn Asn Ser Glu Glu Gin Lys 580 585 590

Leu Leu Tyr Asp Val Val Gin Arg Ala Met Lys Val Phe He Asn Ala 595 600 605

Thr Tyr Gly Val Phe Gly Ala Glu Thr Phe Pro Leu Tyr Ala Pro Arg 610 615 620

Val Ala Glu Ser Val Thr Ala Leu Gly Arg Tyr Val He Thr Ser Thr 625 630 635 640

Val Lys Lys Ala Arg Glu Glu Gly Leu Thr Val Leu Tyr Gly Asp Thr

645 650 655

Asp Ser Leu Phe Leu Leu Asn Pro Pro Lys Asn Ser Leu Glu Asn He 660 665 670

He Lys Trp Val Lys Thr Thr Phe Asn Leu Asp Leu Glu Val Asp Lys 675 680 685

Thr Tyr Lys Phe Val Ala Phe Ser Gly Leu Lys Lys Asn Tyr Phe Gly 690 695 700 Val Tyr Gin Asp Gly Lys Val Asp He Lys Gly Met Leu Val Lys Lys 705 710 715 720

Arg Asn Thr Pro Glu Phe Val Lys Lys Val Phe Asn Glu Val Lys Glu

725 730 735

Leu Met He Ser He Asn Ser Pro Asn Asp val Lys Glu He Lys Arg 740 745 750

Lys He Val Asp Val Val Lys Gly Ser Tyr Glu Lys Leu Lys Asn Lys 755 760 765

Gly Tyr Asn Leu Asp Glu Leu Ala Phe Lys Val Met Leu Ser Lys Pro 770 775 780

Leu Asp Ala Tyr Lys Lys Asn Thr Pro Gin His Val Lys Ala Ala Leu 785 790 795 800

Gin Leu Arg Pro Phe Gly Val Asn Val Leu Pro Arg Asp He He Tyr

805 810 815

Tyr Val Lys Val Arg Ser Lys Asp Gly Val Lys Pro Val Gin Leu Ala 820 825 830

Lys Val Thr Glu He Asp Ala Glu Lys Tyr Leu Glu Ala Leu Arg Ser 835 840 845

Thr Phe Glu Gin He Leu Arg Ala Phe Gly Val Ser Trp Asp Glu He 850 855 860

Ala Ala Thr Met Ser He Asp Ser Phe Phe Ser Tyr Pro Ser Lys Gly 865 870 875 880

Asn Ser

Claims

1. 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.

2. Nucleic acid according to Claim 1 wherein said nucleotide sequence is derived from bacteria of the ArcΛaeadomain.

3. Nucleic acid according to Claim 2 wherein said nucleotide sequence is derived from bacteria of the Sulfolobus genus.

4. Nucleic acid according to Claim 2 wherein said nucleotide sequence is derived from bacteria of the S. solfataricusspec±es .

5. Nucleic acid according to any of previous Claims wherein said polypeptide or fragments thereof have also a 3'-5' exonuclease activity.

6. Nucleic acid according to Claim 5 wherein said nucleotide sequence codes the polypeptide having the aminoacid sequence of SEQ ID N2 or fragments thereof.

7. Nucleic acid according to Claim 6 wherein said nucleotide sequence codes the polypeptide having the aminoacid sequence of SEQ ID N2 or fragments thereof, deleted or substituted for one or more aminoacids, so that said DNA polymerase activity is maintained.

8. 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.

9. Nucleic acid according to Claim 8 wherein said coding sequence lacks or is substituted of one or more nucleotides so that said DNA polymerase activity is maintained.

10. Nucleic acid able to hybridize at least at medium stringency to a nucleic acid according to any of previous Claims.

11. Nucleic acid according to Claim 10 complementary to nucleotide sequences from Claim 1 to 9.

12. Polypeptide with a thermostable and thermofilic DNA polymerase activity.

13. Polypeptide according to Claim 12 produced through recombinant DNA techniques by nucleic acids according to any of previous Claims from 1 to 11.

14. Polypeptide according to Claim 13 produced by the nucleotide sequence comprised in SEQ ID Nl.

15. Polypeptide according to Claim 14 having a sequence comprised in SEQ ID N2.

16. Recombinant cloning or expression vectors, having a plasmid or viral derivation, comprising nucleotide sequences accoprding to any of previous Claims from 1 to 11.

17. Recombinant vector according to Claim 16 being the plasmid pFCpolS (DSM N.7091).

18. Cells transformed with vectors according to Claims 16 or 17.