Abstract:
The present invention provides a novel thermostable DNA polymerase I obtainable from  Rhodothermus obamensis , which possesses 3′-5′ exonuclease activity and has a half-life of about 35 minutes at 94° C. This polymerase also contains a tyrosine residue in the ribosome binding site which improves incorporation of dideoxyribonucleic acids. Also provided are isolated DNA and vectors encoding this polymerase, as well as its large fragment, and methods for producing recombinant enzyme using the same.

Description:
BACKGROUND OF INVENTION  
         [0001]    The present invention relates to a novel thermostable DNA polymerase I from  Rhodothermus obamensis , which possesses 3′-5′ exonuclease activity and has a preliminary estimated half-life of 35 minutes at 94° C., as well as methods for cloning and producing the large fragment of  R. obamensis  DNA polymerase I, as well as isolated DNA encoding this enzyme and vectors containing the same.  
           [0002]    DNA polymerases are important enzymes involved in chromosome replication and repair. These enzymes have also been employed in DNA diagnostics and analysis. In several of these applications, including PCR, thermocycle sequencing, and iso-thermal strand displacement amplification, DNA polymerases must maintain enzymatic activity at temperatures from 50° C.-95° C. One advantageous source for such polymerases is thermophiles. Here we describe a method for purifying, cloning and expressing  Rhodothermus obamensis  DNA polymerase I large fragment in  E. coli.    
           [0003]    [0003] E. coli  DNA polymerase I and T4 DNA polymerase were cloned, purified and characterized previously (Joyce C. M. and Derbyshire V.  Methods in Enzymology , 262:3-13, (1995); Nossal N. G. et al.  Methods in Enzymology , 262: 560-569, (1995)). These enzymes have a variety of uses in recombinant DNA technology including DNA labeling by nick translation, second-strand cDNA synthesis in cDNA cloning, and DNA sequencing.  
           [0004]    U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159 disclosed the use of DNA polymerases in a process for amplifying, detecting, and/or cloning nucleic acid sequences. This process, commonly referred to as polymerase chain reaction (PCR), involves the use of a polymerase, primers and nucleotide triphosphates and amplifying existing nucleic acid sequences.  
           [0005]    A number of thermostable DNA polymerases have been isolated and cloned from thermophilic eubacteria. The thermostable Bst DNA polymerase from  Bacillus stearothermophilus  and the Bca DNA polymerase from  Bacillus caldotenax  have been cloned and expressed in  E. coli  (Aliotta J. M. et al.  Genetic Analysis: Biomol. Engin , 12:185-195, (1996); Uemori, T. et al.  J. Biochem . 113:401-410, (1993)). These two DNA polymerases have been used in strand displacement amplification (Milla, M. A. et al.  Biotechniques , 24:392-395, (1998)).  
           [0006]    DNA polymerases have also been cloned from a number of Thermus species such as  T. aquaticus  (Lawyer, F. C., et al.  J. Biol. Chem . 264:6427-6437 (1989)).  T. thermophilus  (Asakura, K. et al.  J. Ferment. Bioeng ., 76:265-269, (1993), and  T. filiformis  (Jung, S. E. et al. GenBank Accession No. AF030320, (1997)). These characterized Thermus-DNA polymerases, belonging to the Family A DNA polymerases, exhibit 5′-3′ exonuclease activity while lacking 3′-5′ proof-reading exonuclease activity. For thermocycling sequencing, a Taq DNA polymerase variant called ThermoSequenase (F667Y) has been constructed that efficiently incorporates dideoxy terminators and dye-terminators (Tabor S. and Richardson C. C.,  Proc. Natl. Acad. Sci. USA , 92:6339-6343, (1995); Vander Horn P. B. et al.  Biotechniques , 22:758-765, (1996)). Although readable DNA sequence for one sequencing reaction has improved from 300 bp to about 600 bp, further technical improvements are needed to achieve 1000 or more bases of reliable sequence for each reaction. Such improvement most likely requires the introduction of new DNA polymerases such as thermostable T7-like DNA polymerases.  
           [0007]    Research was conducted on the isolation and purification of DNA polymerases from  Thermus aquaticus  (Chien, A. et al.  J. Bacteriol . 127:1550-1557, (1976)). The publication of Chien, A. et al. discloses the isolation and purification of a DNA polymerase with a temperature optimum of 80° C. from  T. aquaticus  YT1 strain. The Chien et al., purification procedure involves a four-step process. These steps include preparation of crude extract, DEAE-Sephadex chromatography, phosphocellulose chromatography and chromatography on DNA cellulose.  
           [0008]    U.S. Pat. No. 4,889,818 discloses a purified thermostable DNA polymerase from  T. aquaticus , Taq DNA polymerase, having a molecular weight of about 86,000 to 90,000 daltons prepared by a process substantially identical to the process of Kaledin with the addition of the substitution of a phosphocellulose chromatography step in lieu of chromatography on single-strand DNA-cellulose. In addition, European Patent Application 0258017 disclose Taq polymerase as the preferred enzyme for use in the PCR process discussed above. Research has indicated that while the Taq DNA polymerase has a 5′-3′ polymerase-dependent exonuclease function, Taq DNA polymerase does not possess a 3′-5′ proofreading exonuclease function (Lawyer, F. C., et al. J. Biol. Chem. 264:6427-6437 (1989)). As a result, Taq DNA polymerase is prone to base incorporation errors, making its use in certain applications undesirable. For example, attempting to clone an amplified gene is problematic since any one copy of the gene may contain an error due to a random misincorporation event. Depending on where in the replication cycle that error occurs (e.g., in an early replication cycle), the entire DNA amplified could contain the erroneously incorporated base, thus, giving rise to a mutated gene product.  
           [0009]    Accordingly, it would be desirable to clone and produce a thermostable DNA polymerase with 3′-5′ proof-reading exonuclease activity that may be used to improve the fidelity of DNA amplification reactions described above. It would also be desirable to clone a thermostable and processive DNA polymerase which efficiently incorporates dye terminators.  
         SUMMARY OF THE INVENTION  
         [0010]    In accordance with the present invention, there is provided a novel thermostable DNA polymerase I from  Rhodothermus obamensis , which possesses 3′-5′ exonuclease activity and has a preliminarily estimated half-life of 35 minutes at 94° C. This thermostable enzyme obtainable from  Rhodothermus obamensis , a thermophile isolated from a shallow marine hydrothermal vent in Tachibana Bay, Japan, has a molecular weight of about 104 kDa, and possesses a tyrosine residue in the ribosome binding domain which increases the incorporation rate of dideoxynucleotides.  
           [0011]    Also provided by the instant invention are methods for cloning and producing the large fragment of  R. obamensis  DNA polymerase I, as well as isolated DNA encoding this enzyme and vectors containing the same. The  Rhodothermus obamensis  DNA polymerase I large fragment has a molecular weight of about 71 kDa. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]    [0012]FIG. 1 is the nucleotide sequence (SEQ ID NO:1) and the predicted amino acid sequences (SEQ ID NO:2) of  R. obamensis  DNA polymerase I.  
         [0013]    [0013]FIG. 2 is the nucleotide sequence (SEQ ID NO:3) and the predicted amino acid sequences (SEQ ID NO:4) of  R. obamensis  DNA polymerase I large fragment.  
         [0014]    [0014]FIG. 3 is the SDS-PAGE gel showing the purification steps for recombinant  R. obamensis  DNA polymerase I large fragment. Lane 1 and 3, IPTG-induced cell extract after heat treatment; lane 2 and 4, non-induced cell extract after heat treatment; lane 5 and 7, protein size marker (7 to 212 kDa); lane 6, partially purified recombinant  R. obamensis  DNA polymerase I large fragment. Arrow I, indicating recombinant  R. obamensis  DNA polymerase I large fragment; arrow II indicating  E. coli  GroEL protein.  
         [0015]    [0015]FIG. 4 illustrates the thermostability of the recombinant  R. obamensis  DNA polymerase I large fragment at 94° C. The polymerase assay was carried out at 65° C. for 20 min after incubation of the DNA polymerase at 94° C. for 1 to 40 min. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0016]    [0016] Rhodothermus obamensis  was isolated from a shallow marine hydrothermal vent in Tachibana Bay, Japan. It can grow in the temperature range of 50 to 85° C. with optimal growth temperature at 80° C. The pH range for growth media is pH 5.5 to 9.0. It can be cultured in a marine broth with NaCl concentration of 1 to 5%. In a preferred embodiment, the type strain is  Rhodothermus obamensis  OKD7 (Sako Y. et al.  Int. J. Syst. Bactriol.  46:1099-1104, (1996)).  
         [0017]    Purification of  R. Obamensis  DNA Polymerase I  
         [0018]    The native or recombinant  R. obamensis  DNA polymerase can be purified by the following procedure:  
         [0019]    Cells are resuspended in a lysis buffer (50 mM Tris-HCl, pH 8, 1 mM EDTA, 5 mM DTT) and lysed by sonication. Pulverized ammonium sulfate is added slowly with gentle stirring to a final concentration of 30% (W/V), and the suspension is allowed to sit at 40° C. overnight. The ammonium sulfate precipitate is collected by centrifugation in a rotor at 12,000 rpm for 30 min. The supernatant is discarded. The pellet is resuspended in a buffer containing 50 mM Tris-HCl, pH 8, 10% glycerol, 1 mM EDTA, 5 mM DTT. The  R. obamensis  DNA polymerase I may be further purified by chromatography, for example:  
         [0020]    [0020] R. obamensis  DNA polymerase I may be purified by phosphocellulose chromatography (Whatman cellulose phosphate ion-exchange resin P11). Fractions may be assayed for thermostable DNA polymerase activity and peak fractions may be pooled and dialysed.  
         [0021]    [0021] R. obamensis  DNA polymerase I may be purified by DEAE chromatography (Whatman ion exchange cellulose DE52 resin). Fractions may then be assayed for thermostable DNA polymerase activity and peak fractions can be pooled and dialysed.  
         [0022]    [0022] R. obamensis  DNA polymerase I may be purified, as in a preferred embodiment, by DNA binding affinity column chromatography (Heparin sepharose or Heparin TSK). Fractions may be assayed for thermostable DNA polymerase activity, and peak fractions may be pooled and dialysed.  
         [0023]    [0023] R. obamensis  DNA polymerase I can be purified by Mono Q FPLC. Fractions may be assayed for thermostable DNA polymerase activity. Peak fractions may be pooled and dialysed.  
         [0024]    [0024] R. obamensis  DNA polymerase I may be further purified by Mono S FPLC. Fractions may then be assayed for thermostable DNA polymerase activity, and peak fractions can be pooled and dialysed in a storage buffer with 50% glycerol.  
         [0025]    Alternatively, recombinant  R. obamensis  DNA polymerase I may be purified by affinity purification via the use of a fusion protein. For example, fusion of  R. obamensis  DNA polymerase I to maltose binding protein, chitin binding protein, GST, or His tag. After the fusion protein is purified, the affinity tag may be removed by a protease or by controlled protein splicing/cleavage reaction. (U.S. Pat. Nos. 5,643,758 and 5,834,247.)  
         [0026]    Cloning of  R. Obamensis  DNA Polymerase I  
         [0027]    The method described herein by which the  R. obamensis  DNA polymerase I gene is cloned and its large fragment is expressed includes the following steps:  
         [0028]    1. The genomic DNA is purified from  R. obamensis  cells.  
         [0029]    2. Conserved regions in DNA polymerase I are found by nucleotide sequence comparison of Pol I type DNA polymerases from Eubacteria and especially thermophilic bacteria. Based on the conserved sequences, one set of degenerate primers is designed and an initial PCR is carried out using the degenerate primers to amplify part of the  R. obamensis  DNA polymerase I. A 609 bp DNA fragment in the DNA polymerase domain is amplified and sequenced.  
         [0030]    3. Single stranded DNA primers are designed based on the initial 609 bp sequence. Inverse PCR is used to amplify upstream and downstream DNA sequences.  R. obamensis  genomic DNA is digested with restriction enzymes with 4-6 bp recognition sequences, giving rise to reasonable size template DNA for inverse PCR reactions. The digested DNA is self-ligated at a low DNA concentration. The ligated circular DNA is used as templates for inverse PCR reaction using a set of primers that annealed to the left or right ends of the initial fragment. The inverse PCR products are purified in low-melting agarose gel and sequenced directly using primers. The newly derived DNA sequences are compared with sequences in GenBank using BlastX program. This step is repeated until the start codon was found upstream and stop codon was found downstream. The entire DNA polymerase gene is found to be 2772 bp long, encoding a protein with predicted molecular weight of 104.7 kDa.  
         [0031]    4. The 3′-5′ exonuclease domain is compared with that of  E. coli  DNA polymerase I. It is found that  R. obamensis  DNA polymerase I contains three conserved motifs of 3′-5′ exonuclease. The three conserved motifs have the following amino acid sequence: motif I, DTE, motif II, NLKYD, motif III, YACED. It is concluded that  R. obamensis  DNA polymerase I may contain 3′-5′ exonuclease proofreading activity. In addition,  R. obamensis  DNA polymerase I contains a Tyr residue (Y761) in the ribose binding region ( E. coli  O helix homolog). It&#39;s known that Tyr residue at this position increases the incorporation rate for dideoxynucleotides.  
         [0032]    5. To overexpress the large fragment of  R. obamensis  DNA polymerase I, 888-bp DNA encoding N-terminus 5′-3′ exonuclease domain is deleted by PCR. The deletion variant lacking 5′-3′ exonuclease region is 1884 bp long, encoding the 628-aa DNA polymerase I large fragment with predicted molecular weight of 71.3 kDa. This  R. obamensis  DNA polymerase I large fragment is similar to  E. coli  Klenow fragment, but it contains 28 extra amino acid residues at the N-terminus. The DNA coding for the large fragment is amplified by PCR, digested with NdeI and BamHI and cloned into a T7 expression vector pAII17. One clone #7 is further characterized.  
         [0033]    6 . E. coli  cells ER2566 [pAII17-Rob polI large fragment] is cultured to late log phase and induced by addition of IPTG ( R. obamensis  is abbreviated as Rob). Cell extract is prepared and heated at 65° C. for 30 min. Heat-denatured  E. coli  proteins were removed by centrifugation and the supernatant is assayed at 65° C. for DNA polymerase activity on activated calf thymus DNA. It is found that the large fragment has thermostable DNA polymerase activity.  
         [0034]    7 . R. obamensis  DNA polymerase I large fragment is purified by chromatography through Heparin-Sepharose column. The large fragment is partially purified. Another protein of 60 kDa is copurified with  R. obamensis  DNA polymerase I large fragment. To determine if this 60 kDa protein is a protease degradation product, the N-terminus of the 60 kDa protein is sequenced. The first 15 residues are compared with known proteins in protein data base. It has 100% identity to  E. coli  GroEL protein.  
         [0035]    8. To determine the half-life of the partially purified large fragment, the protein is heated at 94° C. for 1 to 40 min. Samples are taken and assayed for remaining DNA polymerase activity. It is found that  R. obamensis  DNA polymerase I large fragment has an half-life of 35 min at 94° C.  
         [0036]    The following Examples are given to illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that these Examples are illustrative, and that the invention is not to be considered as restricted thereto as indicated in the appended claims.  
         [0037]    The references cited above and below are herein incorporated by reference.  
       EXAMPLE I  
     Cloning of  R. obamensis  DNA Polymerase I Gene  
       [0038]    [0038] Rhodothermus obamensis  (JCM 9785, Japan Collection of Microorganisms, Wako-shi, Saitama, Japan) was cultured in Bacto marine broth at 70° C. overnight. Cells from one liter of culture were collected by centrifugation. Genomic DNA was prepared from the cell pellet by the standard procedure. A set of degenerate primers were designed based on the conserved amino acid sequence in the DNA polymerase domain. The primers have the following sequences:  
                                                                 (SEQ ID NO:5)                5′-TCCGA(C/T)CCCAACCT(G/C)CAGAACATCCC-3′   138-151                        (SEQ ID NO:6)                5′-AGGA(G/C) (G/C)AGCTCGTCGTG(G/C)ACCTG-3′   138-152              
 
         [0039]    (G/C) indicates degenerate position, G or C.  
         [0040]    Primers 138-151 and 138-152 were used to amplify a portion of  R. obamensis  DNA polymerase I in PCR under the following condition: 95° C. for 30 sec, 50° C. for 1 min, 72° C. for 1 min, 35 cycles, 2.5 units of Taq plus Vent® DNA polymerase (50:1 ratio). A ˜600 bp PCR product was found. The PCR product was gel-purified in low-melting agarose gel and sequenced directly by thermocycling sequencing using primer 138-151 which generated a 609 bp DNA fragment. When this DNA fragment was translated into amino acid sequence and compared to known proteins in GenBank, it was found that it has 50% aa sequence identity to  E. coli  DNA polymerase I (pol I) and 54% aa sequence identity to Taq DNA polymerase.  
         [0041]    Two primers were synthesized based on the known 609 bp DNA sequence. They have the following sequences:  
                                   5′-CGCAGGGCGTTTGTGCCGCGG-3′   202-154   (SEQ ID NO:7)                   5′-GTCTCCCGCCCCATCTCGGTG-3′   202-155   (SEQ ID NO:8)          
 
         [0042]    [0042] R. obamensis  genomic DNA was digested individually with the following restriction enzymes: AvaI, BsaAI, BsaHI, BstNI, EagI, HaeII, HhaI, HincII, MspI, NcoI, NspI, SacII, Sau3AI, TaqI, TseI, Tsp45I, BanI, or AluI. After restriction digestion, the DNA was purified by phenol-CHCl 3  extraction and ethanol precipitation. The digested DNA was self-ligated at a low DNA concentration (2 ug/ml). T4 DNA ligase was inactivated by heating at 65° C. for 30 min and the DNA was precipitated and resuspended in TE buffer. The self-ligated genomic DNA was used in inverse PCR to amplify the remaining portion of the DNA polymerase I gene. The following condition was used in inverse PCR: 95° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 2 min, 30 cycles. Inverse PCR products were found in BsaHI, HaelI, NcoI, and NspI digested and self-ligated DNA templates. The NcoI inverse PCR fragment was the largest, giving rise to about 1950 bp of new DNA sequence (2550 bp−600 bp=˜1950 bp). This fragment was gel-purified in low-melting agarose gel and sequenced directly using primers 202-154 and 202-155. Four new primers were made to finish sequencing the NcoI fragment.  
         [0043]    Two new inverse PCR primers were made to amplify the DNA beyond the NcoI site. The two primers have the following sequences:  
                                   5′-GCCGGCCGCTTGTCAACTCGA-3′   205-7   (SEQ ID NO:9)                   5′-TGATGAACACGTATTGCGCCC-3′   205-8   (SEQ ID NO:10)          
 
         [0044]    [0044] R. obamensis  genomic DNA was digested with restriction enzymes AvaI, BsaHI, BstNI, SacII, Sau3AI, TaqI, TseI, Tsp45I, BanI, AluI and self-ligated as described above. The ligated genomic DNA was used in inverse PCR. Inverse PCR condition was 95° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 2 min, 35 cycles. Inverse PCR products were found in Sau3AI, TaqI, and TseI digested and self-ligated DNA. The inverse PCR products were gel-purified and sequenced which gave rise to 27 bp of new DNA sequence. A start codon was found in the newly derived sequence.  
         [0045]    To amplify the C-terminus coding region of  R. obamensis  DNA polymerase I, two inverse PCR primers were made:  
                                                                         (SEQ ID NO:11)                    5′-GAAGCGGGAAGGCTACCGGGCCAA-3′   204-7                            (SEQ ID NO:12)                    5′-AGTCGGTGGTAGATGTGCACCATG-3′   204-8              
 
         [0046]    Inverse PCR condition was 95° C. for 30 sec, 55° C. for 30 sec. and 72° C. for 2 min, 35 cycles. Inverse PCR products were found in HaeII, NspI, Sau3AI, and Tsp45I digested and self-ligated templates. The inverse PCR products were gel-purified and sequenced which gave rise to the C-terminus coding region. The entire  R. obamensis  DNA polymerase gene is 2772 bp long, encoding a protein with predicted molecular weight of 104.7 kDa (FIG. 1). Unlike Taq DNA polymerase,  R. obamensis  DNA polymerase I contains three conserved 3′-5′ exonuclease motifs. The three conserved motifs have the following amino acid sequence:  
         [0047]    motif I, DTE  
         [0048]    motif II, NLKYD  
         [0049]    motif III, YACED.  
         [0050]    It is concluded that  R. obamensis  DNA polymerase I may contain 3′-5′ exonuclease proofreading activity. In addition,  R. obamensis  DNA polymerase I contains a Tyr residue (Y761) in the ribose binding region ( E. coli  O helix homolog). It&#39;s known that Tyr residue at this position increases the incorporation rate for dideoxynucleotides. Pol I-like DNA polymerases that have a Tyr residue at the ribose selectivity site include DNA polymerases from phage T7 and T3, yeast mitochondria,  Mycobacterium tuberculosis, Mycobacterium leprae, Rhodothermus obamensis , and Rhodothermus sp. ‘ITI518’.  
       EXAMPLE II  
     Expression of  R. obamensis DNA Polymerase I Large Fragment    
       [0051]    To construct a large fragment of  R. obamensis  DNA polymerase I, 888-bp DNA encoding N-terminus 5′-3′ exonuclease domain was deleted. The deletion variant lacking 5′-3′ exonuclease region is 1884 bp long, encoding 628-aa DNA polymerase I large fragment with predicted molecular weight of 71.3 kDa. This  R. obamensis  DNA polymerase I large fragment is similar to  E. coli  Klenow fragment, but it contains 28 extra amino acid residues at the N-terminus (FIG. 2). The DNA coding for the large fragment was amplified by PCR under the PCR condition of 95° C. for 30 sec, 55° C. for 30 sec. and 72° C. for 2 min, 20 cycles, 2 units of Vent® DNA polymerase. The PCR primers have the following sequence:  
                                       5′-CTGGCCGGC CATATG AACGGCGAAGCCGCCTTGGATGAG-3′   204-146.   ( CATATG =  Nde I site).   (SEQ ID NO:13)                   5′-GTT GGATCC GCTTCAGTGGGCATCCAGCCAGTTGTC-3′   204-147.   ( GGATCC =  Bam HI site).   (SEQ ID NO:14)          
 
         [0052]    The amplified PCR product was digested with NdeI and BamHI and inserted into a T7 expression vector pAII17 precut with NdeI and BamHI. The ligated DNA was used to transform  E. coli  competent cell ER2566. Eighteen Amp R  transformants were screened for insert. Six plasmids contained the correct size insert (#2, #5, #6, #7, #12, and #14). To test DNA polymerase activity in all six isolates,  E. coli  cells ER2566 [pAII17-Rob-polI-large fragment] were cultured to late log phase and induced by addition of IPTG to 0.5 mM concentration ( R. obamensis  is abbreviated as Rob). Cell extract was prepared by sonication and centrifugation. The cleared lysate was heated at 65° C. for 30 min. Heat-denatured  E. coli  proteins were removed by centrifugation and the supernatant was analyzed on an SDS-PAGE gel (FIG. 3, lanes 1-4) and was assayed at 65° C. for DNA polymerase activity on activated calf thymus DNA. The DNA polymerase activity was performed in a total of 50 ul volume at 65° C. It contains 20 ul of cell extract, 5 ul (10 ug) of activated calf thymus DNA, 1 ul of dNTP (5.4 mM), 5 ul of 10× thermopol buffer, 1 ul of [ 3 H]TTP, 18 ul of sdH 2 O. The components of 1× Thermopol buffer are 10 mM KCl, 20 mM Tris-HCl, pH 8.8, 10 mM (NH 4 ) 2 SO 4 471 , 2 mM MgSO 4 , 0.1% Triton X-100. Following incubation at 65° C. for 20-30 min, the entire volume was spotted on to DE81 membrane discs and dried under a heating lamp for 30 min. The membranes were washed 2× in 500 ml of 10% TCA. The acid-insoluble [ 3 H]TMP incorporated DNA was counted in scintillation counting solution. It was found that isolates #2, #5, #7, #12, and #14 have thermostable DNA polymerase activity. #7 and #12 displayed highest activity. #7 was chosen to be further characterized. Two liters of cells of #7 clone were induced with IPTG and cell extract was prepared by sonication and centrifugation. The cell extract was heated at 65° C. for 30 min and the denatured  E. coli  proteins were removed by centrifugation.  R. obamensis  DNA polymerase I large fragment was purified by chromatography through Heparin-Sepharose column.  R. obamensis  DNA polymerase I large fragment was eluted with 50 mM to 1 M NaCl gradient. Fractions 19 and 20 contained the most DNA polymerase activity. Proteins from fractions 15 to 20 were analyzed on an SDS-PAG gel. Two major proteins were found, one with expected size of 71 kDa. Another protein of 60 kDa is copurified with  R. obamensis  DNA polymerase I large fragment (FIG. 3, lane 6). To determine if this 60 kDa protein was a protease degradation product, the N-terminus of the 60 kDa protein was sequenced. The first 15 residues (AAKDVKFGNDARVKM (SEQ ID NO:15)) are compared with protein data base. It has 100% identity to  E. coli  GroEL protein. It was concluded that the 60 kDa protein is not a protease degradation product. Since  R. obamensis  DNA polymerase I large fragment is a foreign protein to  E. coli , perhaps it needs more GroEL protein to help it to fold correctly.  
         [0053]    To increase stability of the T7 expression clone, ER2566[pLysS] was transformed with the plasmid carrying Rob polI large fragment. The final expression strain is ER2566[pAII17-Rob polI large fragment, pLysS], Amp R  and Cm R .  
         [0054]    A sample of the  E. coli  containing ER2566[pAII17-Rob polI large fragment, pLysS], (NEB#1186) has been deposited under the terms and conditions of the Budapest Treaty with the American Type Culture Collection on Mar. ______, 1999 and received ATCC Accession No. ______.  
         [0055]    To determine the half-life of the partially purified large fragment, the protein is heated at 94° C. for 1 to 40 min. Samples are taken and assayed for remaining DNA polymerase activity. DNA polymerase assay was about the same as described above except that 5 ul of the heat-treated large fragment was used in the assay. The time of heat treatment was plotted against the percentage of remaining DNA polymerase activity. It was found that  R. obamensis  DNA polymerase I large fragment has an half-life of 35 min at 94° C. (FIG. 4).  
         [0056]    During the course of this work, the DNA polymerase I gene was cloned from Rhodothermus sp. ‘ITI518’ and was released in GenBank on Jan. 1, 1999 (Blondal et al., GenBank Accession No. AF028719).  Rhodothermus obamensis  and Rhodothermus sp. ‘ITI518’ DNA polymerase I share 98% amino acid sequence identity. However, the thermostability of  Rhodothermus obamensis  and Rhodothermus sp. ‘ITI518’ DNA polymerase I large fragments are different. It was reported that the half-life of Rhodothermus sp. ‘ITI518’ DNA polymerase I large fragment at 90° C. is about 10 min (Blondal, T. et al. International Conference: Thermophile 98, Abstract, page G-P20).  R. obamensis  DNA polymerase I large fragment is more thermostable. It has an half-life of 35 min at 94° C. There are two possible explanations. One possibility is that  R. obamensis  DNA polymerase I large fragment has a different N-terminus than Rhodothermus sp. ‘ITI518’ DNA polymerase I large fragment (due to different aa deletion in the 5′-3′ exonuclease region). It&#39;s known that N-terminus deletion of 5′-3′ exonuclease domain can increase thermostability of DNA polymerases. The second possibility is that  R. obamensis  DNA polymerase I large fragment fortuitously copurified with  E. coli  protein GroEL, which is a chaperon for protein folding. The inclusion of GroEL protein in the polymerase assay may increase the thermostability of  R. obamensis  DNA polymerase I large fragment at 94° C.  
       EXAMPLE III  
     Expression of  R. obamensis  DNA Polymerase I and its Large Fragment in any Expression Host  
       [0057]    [0057] R. obamensis  DNA polymerase I gene or its deletion derivative can be amplified by PCR using primers. The deletion can be in the 5′-3′ or 3′-5′ exonuclease domains. Alternatively, the active site residues of 5′-3′ or 3′-5′ exonuclease domains can mutagenized without affecting the DNA polymerase domain. Restriction sites can be engineered in the PCR primers to aid the cloning of the PCR products into appropriate cloning vectors. PCR conditions can be 90-95° C. for 30 sec, 50-65° C. for 30 sec. and 72° C. for 1-3 min, 20-30 cycles, 1-5 units of Vent® DNA polymerase or any proofreading DNA polymerase. PCR products can be digested with appropriate restriction enzymes. After ligation of PCR products to vectors, the ligated DNA can be used to transform expression host by transformation or electroporation. Plasmid mini-preparations can be made to screen inserts. Once the correct inserts are found, cells can be induced to produce the desired proteins. Cell extract can be prepared by lysozyme treatment or sonication and centrifugation. The cleared lysate can be heated at 65-85° C. for 30-60 min. Heat-denatured  E. coli  proteins can be removed by centrifugation and the supernatant can be analyzed on an SDS-PAG gel. The lysate can be assayed at 65-85° C. for DNA polymerase activity on activated calf thymus DNA or single-stranded DNA with a primer. The DNA polymerase activity can be , performed in a total of 50-100 ul volume at 65-85° C. It contains 1-20 ul of cell extract, 5 ul (10 ug) of activated calf thymus DNA, 1 ul of dNTP (5.4 mM), 5 ul of 10× thermopol buffer or any DNA polymerase buffer, 1 ul of [ 3 H]TTP, 18 ul of sdH 2 O. The components of 1× Thermopol buffer are 10 mM KCl, 20 mM Tris-HCl, pH 8.8, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100. Following incubation at 65-85° C. for 10-30 min, the entire volume can be spotted on to DE81 membrane discs and dried. The membranes can be washed 1-2× in 500 ml of 10% TCA. The acid-insoluble [ 3 H]TMP incorporated DNA can be counted in scintillation counting solution.  R. obamensis  DNA polymerase I and its large fragments can be purified by chromatography through affinity column, cation/anion exchange columns, or gel filtration columns.  
         [0058]    To determine the half-life of the partially purified large fragment, the protein can be heated at 94° C. for 1 to 60 min. Samples can be taken and assayed for remaining DNA polymerase activity. The time course can be plotted against the percentage of remaining DNA polymerase activity. Heat shock proteins such as GroEL chaperon can be added to the polymerase reaction to increase the thermostability of DNA polymerase.  
     
       
       
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             Rhodothermus obamensis  
             
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            1 

atg cag cgc ctg tac ctg atc gat gcc atg gcg ctg gcc tat cgg gcg       48 
Met Gln Arg Leu Tyr Leu Ile Asp Ala Met Ala Leu Ala Tyr Arg Ala 
  1               5                  10                  15 

caa tac gtg ttc atc agc cgg ccg ctt gtc aac tcg aag gga cag aac       96 
Gln Tyr Val Phe Ile Ser Arg Pro Leu Val Asn Ser Lys Gly Gln Asn 
             20                  25                  30 

acc tcg gcc gcc tac ggt ttt acg acc tcc ctt ctg aag ctg atc gaa      144 
Thr Ser Ala Ala Tyr Gly Phe Thr Thr Ser Leu Leu Lys Leu Ile Glu 
         35                  40                  45 

gaa cac ggc atg gac tac atg gcc gtg gtc ttc gac gcc ggc ggg gag      192 
Glu His Gly Met Asp Tyr Met Ala Val Val Phe Asp Ala Gly Gly Glu 
     50                  55                  60 

gag ggc acg ttt cgc gaa gcg atc tat gag gaa tac aag gcg cat cgg      240 
Glu Gly Thr Phe Arg Glu Ala Ile Tyr Glu Glu Tyr Lys Ala His Arg 
 65                  70                  75                  80 

gag ccg ccg ccg gaa gat ctg ctg gcc aac ctg ccc tgg atc aag gag      288 
Glu Pro Pro Pro Glu Asp Leu Leu Ala Asn Leu Pro Trp Ile Lys Glu 
                 85                  90                  95 

atc gtc cgg gcg ctg gac att ccc gtc atc gag gag ccg ggc gtc gag      336 
Ile Val Arg Ala Leu Asp Ile Pro Val Ile Glu Glu Pro Gly Val Glu 
            100                 105                 110 

gcc gac gac gtg atc gga acg ctg gcc cgt cgg gcc gag gcg cac ggc      384 
Ala Asp Asp Val Ile Gly Thr Leu Ala Arg Arg Ala Glu Ala His Gly 
        115                 120                 125 

atc gac gtg gtg atc gtc tca ccc gac aag gac ttt ctg cag ctg ctg      432 
Ile Asp Val Val Ile Val Ser Pro Asp Lys Asp Phe Leu Gln Leu Leu 
    130                 135                 140 

agc ccg cac gtt tcc atc tac aaa ccg gcg cgg cgc ggc gaa acc ttc      480 
Ser Pro His Val Ser Ile Tyr Lys Pro Ala Arg Arg Gly Glu Thr Phe 
145                 150                 155                 160 

gac ctg atc acc atc gag act ttc cgg gag acc tac ggc ctg gag ccg      528 
Asp Leu Ile Thr Ile Glu Thr Phe Arg Glu Thr Tyr Gly Leu Glu Pro 
                165                 170                 175 

cac cag ttc atc gac gtg ctg gct ctc atg ggc gat ccg agc gac aat      576 
His Gln Phe Ile Asp Val Leu Ala Leu Met Gly Asp Pro Ser Asp Asn 
            180                 185                 190 

gtg ccg ggc gtg ccg ggc atc ggc gaa aag acc gcc gtg cag ctc atc      624 
Val Pro Gly Val Pro Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Ile 
        195                 200                 205 

caa cag tac ggc tcg gtg gaa aac ctg ctg gcc cat gcc gag gag gtg      672 
Gln Gln Tyr Gly Ser Val Glu Asn Leu Leu Ala His Ala Glu Glu Val 
    210                 215                 220 

aaa ggg aag cgg gcc cgc gag ggg ctc ctg aac cac cgc gag gaa gcg      720 
Lys Gly Lys Arg Ala Arg Glu Gly Leu Leu Asn His Arg Glu Glu Ala 
225                 230                 235                 240 

ctc ctc tcg aag cgg ctg gtg acg atc cgg acc gat gtg ccg ttg cgc      768 
Leu Leu Ser Lys Arg Leu Val Thr Ile Arg Thr Asp Val Pro Leu Arg 
                245                 250                 255 

att cgc tgg gag gcg ttc cat cgc gcc cgg ccc gat ctg ccg cgc ctg      816 
Ile Arg Trp Glu Ala Phe His Arg Ala Arg Pro Asp Leu Pro Arg Leu 
            260                 265                 270 

ctg cag atc ttt cag gag ctg gaa ttc gac tcg ctg gtg cgg cgc atc      864 
Leu Gln Ile Phe Gln Glu Leu Glu Phe Asp Ser Leu Val Arg Arg Ile 
        275                 280                 285 

cgg gaa ggc gga ctg gcc ggc att gtg aac ggc gaa gcc gcc ttg gat      912 
Arg Glu Gly Gly Leu Ala Gly Ile Val Asn Gly Glu Ala Ala Leu Asp 
    290                 295                 300 

gag gcg ctt gaa gcg gag acc gag ccg gag ttc gat ttc ggg cca tac      960 
Glu Ala Leu Glu Ala Glu Thr Glu Pro Glu Phe Asp Phe Gly Pro Tyr 
305                 310                 315                 320 

gag ccg ctg cag gtg tac gat ccg gaa aag gcg gac tac cgg atc gtc     1008 
Glu Pro Leu Gln Val Tyr Asp Pro Glu Lys Ala Asp Tyr Arg Ile Val 
                325                 330                 335 

cgc aac cgc cag cag ctc gac gaa ctc gtg gcg cat ctg gac gga ttc     1056 
Arg Asn Arg Gln Gln Leu Asp Glu Leu Val Ala His Leu Asp Gly Phe 
            340                 345                 350 

gaa cgg ctg gcc atc gac acg gag acg act tcg acc gag gcc atg tgg     1104 
Glu Arg Leu Ala Ile Asp Thr Glu Thr Thr Ser Thr Glu Ala Met Trp 
        355                 360                 365 

gcc tcg ctg gtg ggc att gcc ttt tcc tgg gag aaa ggc cag ggc tac     1152 
Ala Ser Leu Val Gly Ile Ala Phe Ser Trp Glu Lys Gly Gln Gly Tyr 
    370                 375                 380 

tac gtg ccc acg ccg ctg ccg gac ggc acg ccg acc gag acg gtg ctc     1200 
Tyr Val Pro Thr Pro Leu Pro Asp Gly Thr Pro Thr Glu Thr Val Leu 
385                 390                 395                 400 

gag cga ctg gcg ccg atc ctc cga cgg gcg cag cgc aaa gtc ggt cag     1248 
Glu Arg Leu Ala Pro Ile Leu Arg Arg Ala Gln Arg Lys Val Gly Gln 
                405                 410                 415 

aac ctg aag tac gat ctg gtg gtg ctg gcg cgg cac ggc gtc caa gtc     1296 
Asn Leu Lys Tyr Asp Leu Val Val Leu Ala Arg His Gly Val Gln Val 
            420                 425                 430 

ccg ccc ccg tac ttc gac acg atg gtg gcg cac tac ctg att gcg ccc     1344 
Pro Pro Pro Tyr Phe Asp Thr Met Val Ala His Tyr Leu Ile Ala Pro 
        435                 440                 445 

gag gaa ccg cat aac ctg gac gtg ctg gcc cgc cag tac ctt cgc tac     1392 
Glu Glu Pro His Asn Leu Asp Val Leu Ala Arg Gln Tyr Leu Arg Tyr 
    450                 455                 460 

cag atg gtt tcc atc acg gaa ctg atc ggc tcg ggt cgc gac cag aag     1440 
Gln Met Val Ser Ile Thr Glu Leu Ile Gly Ser Gly Arg Asp Gln Lys 
465                 470                 475                 480 

tcc atg cgc gac gtg tcg atc gac gag gtg ggg ccc tat gcc tgt gaa     1488 
Ser Met Arg Asp Val Ser Ile Asp Glu Val Gly Pro Tyr Ala Cys Glu 
                485                 490                 495 

gac acg gac att gcg ctg caa ctg gcc gat gtg ctg gcc gcc gag ttg     1536 
Asp Thr Asp Ile Ala Leu Gln Leu Ala Asp Val Leu Ala Ala Glu Leu 
            500                 505                 510 

gac cga cac gga ctc cgg cat atc gcc gag gag atg gag ttc ccg ctc     1584 
Asp Arg His Gly Leu Arg His Ile Ala Glu Glu Met Glu Phe Pro Leu 
        515                 520                 525 

atc gag gtg ctg gcc gat atg gag cgg acg ggc atc tgc atc gat cgc     1632 
Ile Glu Val Leu Ala Asp Met Glu Arg Thr Gly Ile Cys Ile Asp Arg 
    530                 535                 540 

gcg gtg ctt cgg gaa atc ggt aag caa ctc gaa gcg gag ctt cac gaa     1680 
Ala Val Leu Arg Glu Ile Gly Lys Gln Leu Glu Ala Glu Leu His Glu 
545                 550                 555                 560 

ctg gag gtg aag atc tat gag gtg gcc ggc gtc gaa ttc aac atc ggc     1728 
Leu Glu Val Lys Ile Tyr Glu Val Ala Gly Val Glu Phe Asn Ile Gly 
                565                 570                 575 

tcg ccg cag caa ctg gcg gac gtc ttg ttc aag aag ctc ggg ttg aag     1776 
Ser Pro Gln Gln Leu Ala Asp Val Leu Phe Lys Lys Leu Gly Leu Lys 
            580                 585                 590 

ccg cgg gcg cgc acc agc acc ggc cgg cct tcc acc aaa gag agc gtg     1824 
Pro Arg Ala Arg Thr Ser Thr Gly Arg Pro Ser Thr Lys Glu Ser Val 
        595                 600                 605 

ctg cag gag ctg gcc acg cag cac ccg ctc ccc ggc ctg atc ctg gac     1872 
Leu Gln Glu Leu Ala Thr Gln His Pro Leu Pro Gly Leu Ile Leu Asp 
    610                 615                 620 

tgg cga cac ctg gcc aag ctc aaa agc acc tac gtg gac ggc ctc gag     1920 
Trp Arg His Leu Ala Lys Leu Lys Ser Thr Tyr Val Asp Gly Leu Glu 
625                 630                 635                 640 

ccg ctc atc cat ccg gag acc ggc cgc atc cac acc acg ttc aac cag     1968 
Pro Leu Ile His Pro Glu Thr Gly Arg Ile His Thr Thr Phe Asn Gln 
                645                 650                 655 

acg gtg acg gct acc ggg cgg ctt tcc tcg agc aac ccg aac ctg cag     2016 
Thr Val Thr Ala Thr Gly Arg Leu Ser Ser Ser Asn Pro Asn Leu Gln 
            660                 665                 670 

aac atc ccg gtt cgc acc gag atg ggg cgg gag atc cgc agg gcg ttt     2064 
Asn Ile Pro Val Arg Thr Glu Met Gly Arg Glu Ile Arg Arg Ala Phe 
        675                 680                 685 

gtg ccg cgg ccg ggc tgg aag ctg ctc tcg gcc gac tac gtc cag atc     2112 
Val Pro Arg Pro Gly Trp Lys Leu Leu Ser Ala Asp Tyr Val Gln Ile 
    690                 695                 700 

gaa ctt cgc att ctg gcc gcg ctg agc ggc gac gag gcg ctt cgc cgg     2160 
Glu Leu Arg Ile Leu Ala Ala Leu Ser Gly Asp Glu Ala Leu Arg Arg 
705                 710                 715                 720 

gcc ttt ctg gag gga cag gac atc cat acg gcc acg gca gcc cgc gtc     2208 
Ala Phe Leu Glu Gly Gln Asp Ile His Thr Ala Thr Ala Ala Arg Val 
                725                 730                 735 

ttc aag gtg ccg ccc gag cag gtg acg ccc gag cag cgc cgc cgc gcc     2256 
Phe Lys Val Pro Pro Glu Gln Val Thr Pro Glu Gln Arg Arg Arg Ala 
            740                 745                 750 

aag atg gtc aac tac ggc att ccc tac ggg att tcg gcc tgg ggg ctg     2304 
Lys Met Val Asn Tyr Gly Ile Pro Tyr Gly Ile Ser Ala Trp Gly Leu 
        755                 760                 765 

gcg cag cgg ctt cgc tgc tcc acg cgc gag gcg cag gag ctt atc gaa     2352 
Ala Gln Arg Leu Arg Cys Ser Thr Arg Glu Ala Gln Glu Leu Ile Glu 
    770                 775                 780 

gaa tat cag cgg gcc ttt ccg ggc gtg acg cgc tac ctg cac cgc gtc     2400 
Glu Tyr Gln Arg Ala Phe Pro Gly Val Thr Arg Tyr Leu His Arg Val 
785                 790                 795                 800 

gtc gaa gag gcc cgc cag aag ggc tac gtc gag acg ctg ctg ggc cgc     2448 
Val Glu Glu Ala Arg Gln Lys Gly Tyr Val Glu Thr Leu Leu Gly Arg 
                805                 810                 815 

cgc cgc tac gta ccg aac atc aac tcc cgc aac cgg gcc gag cgc tcg     2496 
Arg Arg Tyr Val Pro Asn Ile Asn Ser Arg Asn Arg Ala Glu Arg Ser 
            820                 825                 830 

atg gcc gaa cgc atc gcc gtg aac atg ccc atc cag ggc acg cag gcc     2544 
Met Ala Glu Arg Ile Ala Val Asn Met Pro Ile Gln Gly Thr Gln Ala 
        835                 840                 845 

gac atg atc aag ctg gcc atg gtg cac atc tac cac cga ctg aag cgg     2592 
Asp Met Ile Lys Leu Ala Met Val His Ile Tyr His Arg Leu Lys Arg 
    850                 855                 860 

gaa ggc tac cgg gcc aag atg ctg ctc cag gtg cac gac gag ctg gtc     2640 
Glu Gly Tyr Arg Ala Lys Met Leu Leu Gln Val His Asp Glu Leu Val 
865                 870                 875                 880 

ttc gag atg ccc ccc gaa gag gtg gag ccc gtg cgc caa ctg gtc gag     2688 
Phe Glu Met Pro Pro Glu Glu Val Glu Pro Val Arg Gln Leu Val Glu 
                885                 890                 895 

cag gag atg aag cag gcc ctg ccg ctg gaa ggt gtg ccc atc gag gtg     2736 
Gln Glu Met Lys Gln Ala Leu Pro Leu Glu Gly Val Pro Ile Glu Val 
            900                 905                 910 

gac atc ggc gtc ggc gac aac tgg ctg gat gcc cac tga                 2775 
Asp Ile Gly Val Gly Asp Asn Trp Leu Asp Ala His 
        915                 920 

 
           
             2  
             924  
             PRT  
             Rhodothermus obamensis  
           
            2 

Met Gln Arg Leu Tyr Leu Ile Asp Ala Met Ala Leu Ala Tyr Arg Ala 
  1               5                  10                  15 

Gln Tyr Val Phe Ile Ser Arg Pro Leu Val Asn Ser Lys Gly Gln Asn 
             20                  25                  30 

Thr Ser Ala Ala Tyr Gly Phe Thr Thr Ser Leu Leu Lys Leu Ile Glu 
         35                  40                  45 

Glu His Gly Met Asp Tyr Met Ala Val Val Phe Asp Ala Gly Gly Glu 
     50                  55                  60 

Glu Gly Thr Phe Arg Glu Ala Ile Tyr Glu Glu Tyr Lys Ala His Arg 
 65                  70                  75                  80 

Glu Pro Pro Pro Glu Asp Leu Leu Ala Asn Leu Pro Trp Ile Lys Glu 
                 85                  90                  95 

Ile Val Arg Ala Leu Asp Ile Pro Val Ile Glu Glu Pro Gly Val Glu 
            100                 105                 110 

Ala Asp Asp Val Ile Gly Thr Leu Ala Arg Arg Ala Glu Ala His Gly 
        115                 120                 125 

Ile Asp Val Val Ile Val Ser Pro Asp Lys Asp Phe Leu Gln Leu Leu 
    130                 135                 140 

Ser Pro His Val Ser Ile Tyr Lys Pro Ala Arg Arg Gly Glu Thr Phe 
145                 150                 155                 160 

Asp Leu Ile Thr Ile Glu Thr Phe Arg Glu Thr Tyr Gly Leu Glu Pro 
                165                 170                 175 

His Gln Phe Ile Asp Val Leu Ala Leu Met Gly Asp Pro Ser Asp Asn 
            180                 185                 190 

Val Pro Gly Val Pro Gly Ile Gly Glu Lys Thr Ala Val Gln Leu Ile 
        195                 200                 205 

Gln Gln Tyr Gly Ser Val Glu Asn Leu Leu Ala His Ala Glu Glu Val 
    210                 215                 220 

Lys Gly Lys Arg Ala Arg Glu Gly Leu Leu Asn His Arg Glu Glu Ala 
225                 230                 235                 240 

Leu Leu Ser Lys Arg Leu Val Thr Ile Arg Thr Asp Val Pro Leu Arg 
                245                 250                 255 

Ile Arg Trp Glu Ala Phe His Arg Ala Arg Pro Asp Leu Pro Arg Leu 
            260                 265                 270 

Leu Gln Ile Phe Gln Glu Leu Glu Phe Asp Ser Leu Val Arg Arg Ile 
        275                 280                 285 

Arg Glu Gly Gly Leu Ala Gly Ile Val Asn Gly Glu Ala Ala Leu Asp 
    290                 295                 300 

Glu Ala Leu Glu Ala Glu Thr Glu Pro Glu Phe Asp Phe Gly Pro Tyr 
305                 310                 315                 320 

Glu Pro Leu Gln Val Tyr Asp Pro Glu Lys Ala Asp Tyr Arg Ile Val 
                325                 330                 335 

Arg Asn Arg Gln Gln Leu Asp Glu Leu Val Ala His Leu Asp Gly Phe 
            340                 345                 350 

Glu Arg Leu Ala Ile Asp Thr Glu Thr Thr Ser Thr Glu Ala Met Trp 
        355                 360                 365 

Ala Ser Leu Val Gly Ile Ala Phe Ser Trp Glu Lys Gly Gln Gly Tyr 
    370                 375                 380 

Tyr Val Pro Thr Pro Leu Pro Asp Gly Thr Pro Thr Glu Thr Val Leu 
385                 390                 395                 400 

Glu Arg Leu Ala Pro Ile Leu Arg Arg Ala Gln Arg Lys Val Gly Gln 
                405                 410                 415 

Asn Leu Lys Tyr Asp Leu Val Val Leu Ala Arg His Gly Val Gln Val 
            420                 425                 430 

Pro Pro Pro Tyr Phe Asp Thr Met Val Ala His Tyr Leu Ile Ala Pro 
        435                 440                 445 

Glu Glu Pro His Asn Leu Asp Val Leu Ala Arg Gln Tyr Leu Arg Tyr 
    450                 455                 460 

Gln Met Val Ser Ile Thr Glu Leu Ile Gly Ser Gly Arg Asp Gln Lys 
465                 470                 475                 480 

Ser Met Arg Asp Val Ser Ile Asp Glu Val Gly Pro Tyr Ala Cys Glu 
                485                 490                 495 

Asp Thr Asp Ile Ala Leu Gln Leu Ala Asp Val Leu Ala Ala Glu Leu 
            500                 505                 510 

Asp Arg His Gly Leu Arg His Ile Ala Glu Glu Met Glu Phe Pro Leu 
        515                 520                 525 

Ile Glu Val Leu Ala Asp Met Glu Arg Thr Gly Ile Cys Ile Asp Arg 
    530                 535                 540 

Ala Val Leu Arg Glu Ile Gly Lys Gln Leu Glu Ala Glu Leu His Glu 
545                 550                 555                 560 

Leu Glu Val Lys Ile Tyr Glu Val Ala Gly Val Glu Phe Asn Ile Gly 
                565                 570                 575 

Ser Pro Gln Gln Leu Ala Asp Val Leu Phe Lys Lys Leu Gly Leu Lys 
            580                 585                 590 

Pro Arg Ala Arg Thr Ser Thr Gly Arg Pro Ser Thr Lys Glu Ser Val 
        595                 600                 605 

Leu Gln Glu Leu Ala Thr Gln His Pro Leu Pro Gly Leu Ile Leu Asp 
    610                 615                 620 

Trp Arg His Leu Ala Lys Leu Lys Ser Thr Tyr Val Asp Gly Leu Glu 
625                 630                 635                 640 

Pro Leu Ile His Pro Glu Thr Gly Arg Ile His Thr Thr Phe Asn Gln 
                645                 650                 655 

Thr Val Thr Ala Thr Gly Arg Leu Ser Ser Ser Asn Pro Asn Leu Gln 
            660                 665                 670 

Asn Ile Pro Val Arg Thr Glu Met Gly Arg Glu Ile Arg Arg Ala Phe 
        675                 680                 685 

Val Pro Arg Pro Gly Trp Lys Leu Leu Ser Ala Asp Tyr Val Gln Ile 
    690                 695                 700 

Glu Leu Arg Ile Leu Ala Ala Leu Ser Gly Asp Glu Ala Leu Arg Arg 
705                 710                 715                 720 

Ala Phe Leu Glu Gly Gln Asp Ile His Thr Ala Thr Ala Ala Arg Val 
                725                 730                 735 

Phe Lys Val Pro Pro Glu Gln Val Thr Pro Glu Gln Arg Arg Arg Ala 
            740                 745                 750 

Lys Met Val Asn Tyr Gly Ile Pro Tyr Gly Ile Ser Ala Trp Gly Leu 
        755                 760                 765 

Ala Gln Arg Leu Arg Cys Ser Thr Arg Glu Ala Gln Glu Leu Ile Glu 
    770                 775                 780 

Glu Tyr Gln Arg Ala Phe Pro Gly Val Thr Arg Tyr Leu His Arg Val 
785                 790                 795                 800 

Val Glu Glu Ala Arg Gln Lys Gly Tyr Val Glu Thr Leu Leu Gly Arg 
                805                 810                 815 

Arg Arg Tyr Val Pro Asn Ile Asn Ser Arg Asn Arg Ala Glu Arg Ser 
            820                 825                 830 

Met Ala Glu Arg Ile Ala Val Asn Met Pro Ile Gln Gly Thr Gln Ala 
        835                 840                 845 

Asp Met Ile Lys Leu Ala Met Val His Ile Tyr His Arg Leu Lys Arg 
    850                 855                 860 

Glu Gly Tyr Arg Ala Lys Met Leu Leu Gln Val His Asp Glu Leu Val 
865                 870                 875                 880 

Phe Glu Met Pro Pro Glu Glu Val Glu Pro Val Arg Gln Leu Val Glu 
                885                 890                 895 

Gln Glu Met Lys Gln Ala Leu Pro Leu Glu Gly Val Pro Ile Glu Val 
            900                 905                 910 

Asp Ile Gly Val Gly Asp Asn Trp Leu Asp Ala His 
        915                 920 

 
           
             3  
             1887  
             DNA  
             Rhodothermus obamensis  
             
               CDS  
               (1)..(1884)  
             
           
            3 

atg aac ggc gaa gcc gcc ttg gat gag gcg ctt gaa gcg gag acc gag       48 
Met Asn Gly Glu Ala Ala Leu Asp Glu Ala Leu Glu Ala Glu Thr Glu 
  1               5                  10                  15 

ccg gag ttc gat ttc ggg cca tac gag ccg ctg cag gtg tac gat ccg       96 
Pro Glu Phe Asp Phe Gly Pro Tyr Glu Pro Leu Gln Val Tyr Asp Pro 
             20                  25                  30 

gaa aag gcg gac tac cgg atc gtc cgc aac cgc cag cag ctc gac gaa      144 
Glu Lys Ala Asp Tyr Arg Ile Val Arg Asn Arg Gln Gln Leu Asp Glu 
         35                  40                  45 

ctc gtg gcg cat ctg gac gga ttc gaa cgg ctg gcc atc gac acg gag      192 
Leu Val Ala His Leu Asp Gly Phe Glu Arg Leu Ala Ile Asp Thr Glu 
     50                  55                  60 

acg act tcg acc gag gcc atg tgg gcc tcg ctg gtg ggc att gcc ttt      240 
Thr Thr Ser Thr Glu Ala Met Trp Ala Ser Leu Val Gly Ile Ala Phe 
 65                  70                  75                  80 

tcc tgg gag aaa ggc cag ggc tac tac gtg ccc acg ccg ctg ccg gac      288 
Ser Trp Glu Lys Gly Gln Gly Tyr Tyr Val Pro Thr Pro Leu Pro Asp 
                 85                  90                  95 

ggc acg ccg acc gag acg gtg ctc gag cga ctg gcg ccg atc ctc cga      336 
Gly Thr Pro Thr Glu Thr Val Leu Glu Arg Leu Ala Pro Ile Leu Arg 
            100                 105                 110 

cgg gcg cag cgc aaa gtc ggt cag aac ctg aag tac gat ctg gtg gtg      384 
Arg Ala Gln Arg Lys Val Gly Gln Asn Leu Lys Tyr Asp Leu Val Val 
        115                 120                 125 

ctg gcg cgg cac ggc gtc caa gtc ccg ccc ccg tac ttc gac acg atg      432 
Leu Ala Arg His Gly Val Gln Val Pro Pro Pro Tyr Phe Asp Thr Met 
    130                 135                 140 

gtg gcg cac tac ctg att gcg ccc gag gaa ccg cat aac ctg gac gtg      480 
Val Ala His Tyr Leu Ile Ala Pro Glu Glu Pro His Asn Leu Asp Val 
145                 150                 155                 160 

ctg gcc cgc cag tac ctt cgc tac cag atg gtt tcc atc acg gaa ctg      528 
Leu Ala Arg Gln Tyr Leu Arg Tyr Gln Met Val Ser Ile Thr Glu Leu 
                165                 170                 175 

atc ggc tcg ggt cgc gac cag aag tcc atg cgc gac gtg tcg atc gac      576 
Ile Gly Ser Gly Arg Asp Gln Lys Ser Met Arg Asp Val Ser Ile Asp 
            180                 185                 190 

gag gtg ggg ccc tat gcc tgt gaa gac acg gac att gcg ctg caa ctg      624 
Glu Val Gly Pro Tyr Ala Cys Glu Asp Thr Asp Ile Ala Leu Gln Leu 
        195                 200                 205 

gcc gat gtg ctg gcc gcc gag ttg gac cga cac gga ctc cgg cat atc      672 
Ala Asp Val Leu Ala Ala Glu Leu Asp Arg His Gly Leu Arg His Ile 
    210                 215                 220 

gcc gag gag atg gag ttc ccg ctc atc gag gtg ctg gcc gat atg gag      720 
Ala Glu Glu Met Glu Phe Pro Leu Ile Glu Val Leu Ala Asp Met Glu 
225                 230                 235                 240 

cgg acg ggc atc tgc atc gat cgc gcg gtg ctt cgg gaa atc ggt aag      768 
Arg Thr Gly Ile Cys Ile Asp Arg Ala Val Leu Arg Glu Ile Gly Lys 
                245                 250                 255 

caa ctc gaa gcg gag ctt cac gaa ctg gag gtg aag atc tat gag gtg      816 
Gln Leu Glu Ala Glu Leu His Glu Leu Glu Val Lys Ile Tyr Glu Val 
            260                 265                 270 

gcc ggc gtc gaa ttc aac atc ggc tcg ccg cag caa ctg gcg gac gtc      864 
Ala Gly Val Glu Phe Asn Ile Gly Ser Pro Gln Gln Leu Ala Asp Val 
        275                 280                 285 

ttg ttc aag aag ctc ggg ttg aag ccg cgg gcg cgc acc agc acc ggc      912 
Leu Phe Lys Lys Leu Gly Leu Lys Pro Arg Ala Arg Thr Ser Thr Gly 
    290                 295                 300 

cgg cct tcc acc aaa gag agc gtg ctg cag gag ctg gcc acg cag cac      960 
Arg Pro Ser Thr Lys Glu Ser Val Leu Gln Glu Leu Ala Thr Gln His 
305                 310                 315                 320 

ccg ctc ccc ggc ctg atc ctg gac tgg cga cac ctg gcc aag ctc aaa     1008 
Pro Leu Pro Gly Leu Ile Leu Asp Trp Arg His Leu Ala Lys Leu Lys 
                325                 330                 335 

agc acc tac gtg gac ggc ctc gag ccg ctc atc cat ccg gag acc ggc     1056 
Ser Thr Tyr Val Asp Gly Leu Glu Pro Leu Ile His Pro Glu Thr Gly 
            340                 345                 350 

cgc atc cac acc acg ttc aac cag acg gtg acg gct acc ggg cgg ctt     1104 
Arg Ile His Thr Thr Phe Asn Gln Thr Val Thr Ala Thr Gly Arg Leu 
        355                 360                 365 

tcc tcg agc aac ccg aac ctg cag aac atc ccg gtt cgc acc gag atg     1152 
Ser Ser Ser Asn Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Glu Met 
    370                 375                 380 

ggg cgg gag atc cgc agg gcg ttt gtg ccg cgg ccg ggc tgg aag ctg     1200 
Gly Arg Glu Ile Arg Arg Ala Phe Val Pro Arg Pro Gly Trp Lys Leu 
385                 390                 395                 400 

ctc tcg gcc gac tac gtc cag atc gaa ctt cgc att ctg gcc gcg ctg     1248 
Leu Ser Ala Asp Tyr Val Gln Ile Glu Leu Arg Ile Leu Ala Ala Leu 
                405                 410                 415 

agc ggc gac gag gcg ctt cgc cgg gcc ttt ctg gag gga cag gac atc     1296 
Ser Gly Asp Glu Ala Leu Arg Arg Ala Phe Leu Glu Gly Gln Asp Ile 
            420                 425                 430 

cat acg gcc acg gca gcc cgc gtc ttc aag gtg ccg ccc gag cag gtg     1344 
His Thr Ala Thr Ala Ala Arg Val Phe Lys Val Pro Pro Glu Gln Val 
        435                 440                 445 

acg ccc gag cag cgc cgc cgc gcc aag atg gtc aac tac ggc att ccc     1392 
Thr Pro Glu Gln Arg Arg Arg Ala Lys Met Val Asn Tyr Gly Ile Pro 
    450                 455                 460 

tac ggg att tcg gcc tgg ggg ctg gcg cag cgg ctt cgc tgc tcc acg     1440 
Tyr Gly Ile Ser Ala Trp Gly Leu Ala Gln Arg Leu Arg Cys Ser Thr 
465                 470                 475                 480 

cgc gag gcg cag gag ctt atc gaa gaa tat cag cgg gcc ttt ccg ggc     1488 
Arg Glu Ala Gln Glu Leu Ile Glu Glu Tyr Gln Arg Ala Phe Pro Gly 
                485                 490                 495 

gtg acg cgc tac ctg cac cgc gtc gtc gaa gag gcc cgc cag aag ggc     1536 
Val Thr Arg Tyr Leu His Arg Val Val Glu Glu Ala Arg Gln Lys Gly 
            500                 505                 510 

tac gtc gag acg ctg ctg ggc cgc cgc cgc tac gta ccg aac atc aac     1584 
Tyr Val Glu Thr Leu Leu Gly Arg Arg Arg Tyr Val Pro Asn Ile Asn 
        515                 520                 525 

tcc cgc aac cgg gcc gag cgc tcg atg gcc gaa cgc atc gcc gtg aac     1632 
Ser Arg Asn Arg Ala Glu Arg Ser Met Ala Glu Arg Ile Ala Val Asn 
    530                 535                 540 

atg ccc atc cag ggc acg cag gcc gac atg atc aag ctg gcc atg gtg     1680 
Met Pro Ile Gln Gly Thr Gln Ala Asp Met Ile Lys Leu Ala Met Val 
545                 550                 555                 560 

cac atc tac cac cga ctg aag cgg gaa ggc tac cgg gcc aag atg ctg     1728 
His Ile Tyr His Arg Leu Lys Arg Glu Gly Tyr Arg Ala Lys Met Leu 
                565                 570                 575 

ctc cag gtg cac gac gag ctg gtc ttc gag atg ccc ccc gaa gag gtg     1776 
Leu Gln Val His Asp Glu Leu Val Phe Glu Met Pro Pro Glu Glu Val 
            580                 585                 590 

gag ccc gtg cgc caa ctg gtc gag cag gag atg aag cag gcc ctg ccg     1824 
Glu Pro Val Arg Gln Leu Val Glu Gln Glu Met Lys Gln Ala Leu Pro 
        595                 600                 605 

ctg gaa ggt gtg ccc atc gag gtg gac atc ggc gtc ggc gac aac tgg     1872 
Leu Glu Gly Val Pro Ile Glu Val Asp Ile Gly Val Gly Asp Asn Trp 
    610                 615                 620 

ctg gat gcc cac tga                                                 1887 
Leu Asp Ala His 
625 

 
           
             4  
             628  
             PRT  
             Rhodothermus obamensis  
           
            4 

Met Asn Gly Glu Ala Ala Leu Asp Glu Ala Leu Glu Ala Glu Thr Glu 
  1               5                  10                  15 

Pro Glu Phe Asp Phe Gly Pro Tyr Glu Pro Leu Gln Val Tyr Asp Pro 
             20                  25                  30 

Glu Lys Ala Asp Tyr Arg Ile Val Arg Asn Arg Gln Gln Leu Asp Glu 
         35                  40                  45 

Leu Val Ala His Leu Asp Gly Phe Glu Arg Leu Ala Ile Asp Thr Glu 
     50                  55                  60 

Thr Thr Ser Thr Glu Ala Met Trp Ala Ser Leu Val Gly Ile Ala Phe 
 65                  70                  75                  80 

Ser Trp Glu Lys Gly Gln Gly Tyr Tyr Val Pro Thr Pro Leu Pro Asp 
                 85                  90                  95 

Gly Thr Pro Thr Glu Thr Val Leu Glu Arg Leu Ala Pro Ile Leu Arg 
            100                 105                 110 

Arg Ala Gln Arg Lys Val Gly Gln Asn Leu Lys Tyr Asp Leu Val Val 
        115                 120                 125 

Leu Ala Arg His Gly Val Gln Val Pro Pro Pro Tyr Phe Asp Thr Met 
    130                 135                 140 

Val Ala His Tyr Leu Ile Ala Pro Glu Glu Pro His Asn Leu Asp Val 
145                 150                 155                 160 

Leu Ala Arg Gln Tyr Leu Arg Tyr Gln Met Val Ser Ile Thr Glu Leu 
                165                 170                 175 

Ile Gly Ser Gly Arg Asp Gln Lys Ser Met Arg Asp Val Ser Ile Asp 
            180                 185                 190 

Glu Val Gly Pro Tyr Ala Cys Glu Asp Thr Asp Ile Ala Leu Gln Leu 
        195                 200                 205 

Ala Asp Val Leu Ala Ala Glu Leu Asp Arg His Gly Leu Arg His Ile 
    210                 215                 220 

Ala Glu Glu Met Glu Phe Pro Leu Ile Glu Val Leu Ala Asp Met Glu 
225                 230                 235                 240 

Arg Thr Gly Ile Cys Ile Asp Arg Ala Val Leu Arg Glu Ile Gly Lys 
                245                 250                 255 

Gln Leu Glu Ala Glu Leu His Glu Leu Glu Val Lys Ile Tyr Glu Val 
            260                 265                 270 

Ala Gly Val Glu Phe Asn Ile Gly Ser Pro Gln Gln Leu Ala Asp Val 
        275                 280                 285 

Leu Phe Lys Lys Leu Gly Leu Lys Pro Arg Ala Arg Thr Ser Thr Gly 
    290                 295                 300 

Arg Pro Ser Thr Lys Glu Ser Val Leu Gln Glu Leu Ala Thr Gln His 
305                 310                 315                 320 

Pro Leu Pro Gly Leu Ile Leu Asp Trp Arg His Leu Ala Lys Leu Lys 
                325                 330                 335 

Ser Thr Tyr Val Asp Gly Leu Glu Pro Leu Ile His Pro Glu Thr Gly 
            340                 345                 350 

Arg Ile His Thr Thr Phe Asn Gln Thr Val Thr Ala Thr Gly Arg Leu 
        355                 360                 365 

Ser Ser Ser Asn Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Glu Met 
    370                 375                 380 

Gly Arg Glu Ile Arg Arg Ala Phe Val Pro Arg Pro Gly Trp Lys Leu 
385                 390                 395                 400 

Leu Ser Ala Asp Tyr Val Gln Ile Glu Leu Arg Ile Leu Ala Ala Leu 
                405                 410                 415 

Ser Gly Asp Glu Ala Leu Arg Arg Ala Phe Leu Glu Gly Gln Asp Ile 
            420                 425                 430 

His Thr Ala Thr Ala Ala Arg Val Phe Lys Val Pro Pro Glu Gln Val 
        435                 440                 445 

Thr Pro Glu Gln Arg Arg Arg Ala Lys Met Val Asn Tyr Gly Ile Pro 
    450                 455                 460 

Tyr Gly Ile Ser Ala Trp Gly Leu Ala Gln Arg Leu Arg Cys Ser Thr 
465                 470                 475                 480 

Arg Glu Ala Gln Glu Leu Ile Glu Glu Tyr Gln Arg Ala Phe Pro Gly 
                485                 490                 495 

Val Thr Arg Tyr Leu His Arg Val Val Glu Glu Ala Arg Gln Lys Gly 
            500                 505                 510 

Tyr Val Glu Thr Leu Leu Gly Arg Arg Arg Tyr Val Pro Asn Ile Asn 
        515                 520                 525 

Ser Arg Asn Arg Ala Glu Arg Ser Met Ala Glu Arg Ile Ala Val Asn 
    530                 535                 540 

Met Pro Ile Gln Gly Thr Gln Ala Asp Met Ile Lys Leu Ala Met Val 
545                 550                 555                 560 

His Ile Tyr His Arg Leu Lys Arg Glu Gly Tyr Arg Ala Lys Met Leu 
                565                 570                 575 

Leu Gln Val His Asp Glu Leu Val Phe Glu Met Pro Pro Glu Glu Val 
            580                 585                 590 

Glu Pro Val Arg Gln Leu Val Glu Gln Glu Met Lys Gln Ala Leu Pro 
        595                 600                 605 

Leu Glu Gly Val Pro Ile Glu Val Asp Ile Gly Val Gly Asp Asn Trp 
    610                 615                 620 

Leu Asp Ala His 
625 

 
           
             5  
             26  
             DNA  
             synthetic  
           
            5 

tccgayccca acctscagaa catccc                                          26 

 
           
             6  
             23  
             DNA  
             Synthetic  
           
            6 

aggassagct cgtcgtgsac ctg                                             23 

 
           
             7  
             21  
             DNA  
             synthetic  
           
            7 

cgcagggcgt ttgtgccgcg g                                               21 

 
           
             8  
             21  
             DNA  
             synthetic  
           
            8 

gtctcccgcc ccatctcggt g                                               21 

 
           
             9  
             21  
             DNA  
             synthetic  
           
            9 

gccggccgct tgtcaactcg a                                               21 

 
           
             10  
             21  
             DNA  
             synthetic  
           
            10 

tgatgaacac gtattgcgcc c                                               21 

 
           
             11  
             24  
             DNA  
             Synthetic  
           
            11 

gaagcgggaa ggctaccggg ccaa                                            24 

 
           
             12  
             24  
             DNA  
             Synthetic  
           
            12 

agtcggtggt agatgtgcac catg                                            24 

 
           
             13  
             39  
             DNA  
             Synthetic  
           
            13 

ctggccggcc atatgaacgg cgaagccgcc ttggatgag                            39 

 
           
             14  
             36  
             DNA  
             Synthetic  
           
            14 

gttggatccg cttcagtggg catccagcca gttgtc                               36 

 
           
             15  
             15  
             PRT  
             Escherichia coli  
           
            15 

Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met 
  1               5                  10                  15