JP3498808B2 - DNA polymerase gene - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA polymerase gene and a method for genetically engineering DNA polymerase.

[0002]

2. Description of the Related Art DNA polymerases generally used as reagents for genetic engineering research up to now include Escherichia coli-derived DNA polymerase, a Klenow fragment which is a modification thereof, Thermus aquaticus-derived DNA polymerase, and Bacillus caldotenax ( Ba
Cillus caldotenax) -derived DNA polymerase and the like.
These enzymes are used for DNA labeling, PCR, DNA base sequence determination, etc. depending on the properties of the enzymes.
In general, DNA polymerase has specificity due to its origin, and there is a method of utilization that takes advantage of its characteristics. Pyrodictium occultum (Pyrodictium occultum) is an extremely thermophilic archaea with an optimum growth temperature of about 105 ° C.
Since this DNA polymerase derived from bacteria is expected to be stable at high temperatures, it is expected to be useful as a reagent for genetic engineering research. However, since the bacterium has a very high growth temperature and is anaerobic, it is difficult to culture it in a large amount. Therefore, it is very difficult to directly collect a large amount of DNA polymerase from the culture of the bacterium. On the other hand, regarding the DNA polymerase gene derived from the bacterium, the Abstract of the American Society for Microbiology (Abstract of The
American Society for Microbiology), Volume 93, Volume 1
On page 97 (1993), it is described that the DNA polymerase gene was isolated from the bacterium. However,
There is no description sufficient to specify the gene such as a nucleotide sequence other than the existence of an open reading frame capable of encoding 803 amino acids, and a sufficient amino acid sequence and enzymatic chemical properties to specify the DNA polymerase are sufficient. There is no description. Further, other DNA polymerase genes of the bacterium are not described.

[0003]

The object of the present invention is to identify a novel DNA polymerase gene and use a transformant carrying a plasmid containing the gene to obtain a novel D gene.
It is to provide a method for genetically engineering NA polymerase.

[0004]

Means for Solving the Problems To summarize the present invention, the first invention of the present invention is the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or a part thereof , and the sequence of the sequence listing.
Amino acids represented by amino acid Nos. 36 to 914 of No. 1
It relates to a DNA polymerase gene encoding an amino acid sequence containing the sequence . The second invention of the present invention relates to a DNA polymerase gene capable of hybridizing to the DNA polymerase gene of the first invention under strict conditions. A third invention of the present invention relates to a method for producing a DNA polymerase,
Culturing a transformant carrying a plasmid containing the DNA polymerase gene of the first or second invention, and collecting the DNA polymerase encoded by the DNA polymerase gene of the first or second invention from the culture. Characterize. And the fourth invention of the present invention is the arrangement of the sequence listing.
The amino acid sequence represented by No. 1 or a part thereof
And the amino acid numbers 36 to 9 of SEQ ID NO: 1 in the sequence listing
Consisting of an amino acid sequence including the amino acid sequence shown in 14.
DNA polymerase.

As a result of diligent research, the present inventors have found two novel DNA polymerase genes from Pyrodictium occlutam and succeeded in cloning them. Furthermore, a transformant having these genes introduced therein was produced, and mass production of DNA polymerase was successful, thus completing the present invention.

The present invention will be described in detail below. The strain used in the present invention includes, for example, Pyrodictium
Occultum DSM2709 ^T strain [Deutsche Sammlung von Mikroorganismen (Deutsche Sammlung von Mikroorganismen
und Zellkulturen) Preserved strain of GmbH: DSM270
9 ^T ].

The DNA polymerase gene of the present invention can be obtained by the steps exemplified below. (1) Extract DNA from Pyrodictium occultum. (2) An oligonucleotide primer for gene amplification is prepared based on an amino acid sequence common to α-type DNA polymerase, and PCR is performed using the DNA obtained in (1) as a template. (3) cut the DNA obtained in (1) with an appropriate restriction enzyme,
On the other hand, Southern hybridization is performed using the amplified DNA fragment obtained in (2) as a probe. (4) The DNA of (1) is cleaved with the restriction enzyme for which an appropriate positive signal was obtained in (3), and a cassette matching each cleavage site is ligated with a DNA ligase. (5) PCR is performed using a common primer in the cassette and a primer attached to the DNA fragment used as the probe, and restriction enzyme mapping of the amplified DNA fragment is performed to limit the peripheral region containing the DNA polymerase gene. Create an enzyme map. (6) A restriction enzyme that yields a fragment containing the entire region encoding DNA polymerase based on the results of (5).
The NA is cleaved and ligated into the vector. (7) A vector to which a DNA fragment is ligated is introduced into a host bacterium, and a transformant containing the target DNA fragment is selected. (8) The transformant obtained in (7) is cultured, and the DNA polymerase activity of the cultured bacterial cell extract is confirmed.

The DNA derived from Pyrodictium occultum DSM2709, which is the above-mentioned DNA donor, is 100
Extract from the cultured bacterial cells anaerobically cultured at ° C. Known methods can be used for extraction, purification, restriction enzyme digestion, and the like. For details of the method, see 1982, Cold Spring Harbor Laboratory, T.S. T. Maniatis et al., Molecular cloning,
Laboratory Manual (Molecular Cloning, A
Laboratory Manual) pages 75-178.

As a method for selecting the desired DNA fragment, for example, the amino acid sequences of known DNA polymerases are compared with each other, and PCR is performed based on a region showing a common amino acid sequence.
Make a primer for. Since the archaea-derived DNA polymerases known so far are of α type,
For example, the amino acid sequences of known α-type DNA polymerases can be compared to prepare a mixed primer based on a common region. A primer for cloning an α-type DNA polymerase is disclosed in Japanese Patent Application Laid-Open No. 6-
The GC type, AT type, or intermediate type primers described in Japanese Patent No. 14780 can be used. These can be used properly according to the GC content of the target DNA.

Pyrodictium occultum is a product of Systematic and Applied Microbiology, Volume 4,
Since the GC content is as high as 62% as described in pp. 535-551 (1983), the present inventors have used the GC-type primers shown in SEQ ID NOS: 3 and 4 of the Sequence Listing to determine Pyrodycium ocultum. PCR was performed using the DNA as a template. As a result, they have found that a specific DNA fragment is amplified. Furthermore, when the base sequence of this amplified DNA fragment was determined, two types of sequences were found in which the deduced amino acid sequence had homology to known DNA polymerases. The nucleotide sequences of these two kinds of amplified DNA fragments are SEQ ID NO: 5 (referred to as sequence I) and SEQ ID NO: 6 in the sequence listing.
(Designated as Sequence II). This suggested that Pyrodictium occlutam had at least two types of DNA polymerases.

The genes encoding these DNA polymerases can be selected, for example, by carrying out hybridization using the amplified DNA fragment as a probe. For selection by hybridization, a known method, for example, the method described in the above-mentioned Molecular Cloning, Laboratory Manual, page 309 (1982) can be used. After analyzing which restriction enzyme fragment of the Pyrodictium occlutam DNA the target DNA polymerase gene is present by Southern hybridization, a cassette having an appropriate restriction enzyme site is ligated to each cleavage fragment, and PCR with a part of the reaction solution and a common primer in the cassette and a primer attached to the region in the probe
Then, a restriction enzyme map of the region containing the DNA polymerase gene can be obtained by performing restriction enzyme analysis on the obtained amplified fragment. From this result, the Pyrodictium occultum DNA is cleaved and integrated into a vector in order to clone a fragment containing the entire region encoding the DNA polymerase. Known plasmid vectors can be used, for example, pUC18, pUC19, pTV1.
18N, pTV119N and the like, but are not limited thereto. Also, as a means for incorporating, a known method can be used, and it may be incorporated by an enzymatic reaction using DNA ligase.

Next, the recombinant plasmid is introduced into host E. coli. As the host E. coli, both wild strains and mutant strains can be used as long as they have transformability.
It is desirable that the modified mutant strain is a modified wild strain (r ⁻ , m ⁺ ). As a means for introducing itself, a known method, for example, the above-mentioned molecular cloning, laboratory manual, page 250 (1982) can be used. Thus, a cloned DNA population can be prepared by introducing a target DNA fragment into a host and selecting a colony having characteristics of a plasmid vector, for example, ampicillin resistance in the case of pUC18.

Next, a clone having the desired fragment is selected from the above population. The selection method may be colony hybridization or plaque hybridization depending on the type of vector, and the method itself is known.

The present inventors have cloned a DNA fragment of about 4.2 kb using the amplified DNA fragment having the sequence I (probe I) as a probe by the above method. A part of the nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing. Also, the sequence II
A DNA fragment of about 3.1 kb was cloned by using the amplified DNA fragment (probe II) having the following as a probe. A part of the nucleotide sequence is shown in SEQ ID NO: 8 in the sequence listing. Furthermore, the present inventors cloned about 3.1 using probe II.
A plasmid having the kb fragment incorporated into pTV119N was prepared and named pPO500-II. Plasmid pPO
Escherichia coli having 500-II was cultured to obtain a crude extract of bacterial cells. The extract was treated with a sufficient amount of DNA even after treatment at 90 ° C for 20 minutes.
E. coli crude extract having a polymerase activity and having only an expression vector does not have such activity.
It was concluded that the thermostable DNA polymerase production information was present on pPO500-II and that the gene having the information was expressed in E. coli. Escherichia coli JM109 transformed with pPO500-II was Escherichia coli JM10.
It is named and displayed as 9 / pPO500-II and has been deposited as FERM P-13659 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

The present inventors also obtained a fragment of about 4.2 kb cloned with probe I into pTV118N.
Was constructed and was named pPO100-I. Escherichia coli containing the plasmid pPO100-I was cultured to obtain a crude cell extract. The extract is 70 ° C 20
After treatment with sufficient amount of DNA polymerase activity,
It was concluded that there is thermostable DNA polymerase production information on PO100-I, and the gene having this information is expressed in E. coli. E. coli JM109 transformed with pPO100-I is Escherichia coli JM109.
It is named and displayed as / pPO100-I and has been deposited as FERM P-13660 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

Further, if the obtained DNA polymerase gene is used as a probe for hybridization under strict conditions, another DNA polymerase gene having a slightly different nucleotide sequence but substantially the same can be obtained.

The strict conditions are, for example,
It is as follows. That is, a DNA-immobilized nylon membrane was treated with 6 × SSC (1 × SSC is prepared by dissolving 8.76 g of sodium chloride and 4.41 g of sodium citrate in 1 liter of water), 1% sodium lauryl sulfate, 100 μg / ml. Hybridization with a probe for 20 hours at 65 ° C. in a solution containing salmon sperm DNA, 5 × Denhardt's (containing 0.1% bovine serum albumin, polyvinylpyrrolidone, and ficoll). .

As a method for purifying these thermostable DNA polymerases, for example, ultrasonic treatment, heat treatment, phenyl sepharose column chromatography, heparin sepharose column chromatography, mono (Mono) Q (Pharmacia Co.), mono Each treatment of S (Pharmacia) can be performed to purify the DNA polymerase until it becomes a substantially single band. p
The thermostable DNA polymerase encoded by the DNA polymerase gene on PO500-II is a polypeptide showing a molecular weight of about 90,000 daltons by SDS-PAGE, and
It had NA synthesis activity and 5 '→ 3', 3 '→ 5' exonuclease activity. The thermostable DNA polymerase encoded by the DNA polymerase gene on pPO100-I is a polypeptide showing a molecular weight of about 950,000 daltons by SDS-PAGE, and has a DNA synthesis activity and 5 '→ 3', 3 '. → It had a 5'exonuclease activity. These thermostable DNA polymerases are useful as reagents for genetic engineering research.

The discovery of two types of DNA polymerases from archaea by the present invention also provides the following interesting findings. In other words, it has been proposed that multiple DNA polymerases act on DNA replication in eukaryotic systems, suggesting that leading strand synthesis and lagging strand synthesis may be performed by different DNA polymerases. Has been done. On the other hand, in prokaryotes, analysis is progressing in the Escherichia coli system, and the replication enzyme D
NA polymerase III is known. This enzyme has 10
In a complex of different kinds of proteins, asymmetry appears depending on the combination when the complex becomes a dimer, which explains the mechanical difference between the synthesis of the leading chain and the lagging chain. However, although it is known that archaea have a DNA polymerase having a structure similar to that of DNA polymerase α possessed by eukaryotic cells, the mechanism of DNA replication is unknown at all. This time, the present inventors have confirmed that two types of D that are structurally in the same family as α-type.
The NA polymerase gene was found. It is considered that eukaryotic cells evolved due to symbiosis of archaea with eubacteria. Probably the two types of α-type DNA polymerase genes found by the present inventors are the eukaryotic DNA polymerases α, δ, It is considered to correspond to two types of DNA polymerases in ε. The isolation of two types of DNA polymerase genes that are thought to be involved in DNA replication from hyperthermophilic archaea provides clues to elucidate the mechanism of DNA replication closer to the primitive of organisms. Elucidation of the DNA replication mechanism of archaea can be useful for studying DNA replication of higher organisms.

[0020]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

Example 1 (1) Preparation of Pyrodictium occultum chromosomal DNA Pyrodycium occultum DSM2709 ^T was anaerobically cultured under the conditions specified by DSM. 150
750 μl of 25% sucrose,
Suspend in 0.05M Tris-hydrochloric acid (pH 8.0) and
50 μl of 0.5 M EDTA and 75 ml of lysozyme solution (10 mg / ml) were added and left at 20 ° C. for 1 hour. Further, 6 ml of SET solution (20 mM Tris-hydrochloric acid pH 8.0, 1 mM EDTA, 150 mM NaC
l) was added, followed by 375 μl of 10% SDS and 75 μl
Proteinase K solution (20 mg / ml) of
It was left at 37 ° C. for 1 hour. Phenol extraction and chloroform extraction were followed by ethanol precipitation to recover long-chain DNA. About 7 μg of DNA was obtained by the above operation.

(2) Amplification of specific DNA by PCR Two kinds of oligonucleotides (GC type primers 1 and 2) shown in SEQ ID NOS: 3 and 4 of the sequence listing were synthesized. 100 pmol of each of these primers and Example 1-
1 ng of chromosomal DNA prepared in (1) was used for a total amount of 10
0 μl system at 94 ° C for 1 minute, 45 ° C for 2 minutes, 72 ° C for 2 minutes
PCR was performed for 50 cycles under the condition of minutes. 5μ of reaction solution
As a result of taking 1 and analyzing by agarose gel electrophoresis, a DNA fragment of about 400 bp was specifically amplified. This DNA fragment was incorporated into pUC118 vector cleaved with SmaI to determine the nucleotide sequence. As a result, two types of sequences were found. The sequences are shown in SEQ ID NO: 5 (sequence I) and SEQ ID NO: 6 (sequence II) in the sequence listing. All of these sequences had homology to the sequences of known DNA polymerases.

(3) Search for DNA polymerase gene by genomic Southern method BamHI and Eco were prepared using 0.15 μg of the chromosomal DNA prepared in Example 1- (1) per restriction enzyme.
It was digested with 5 kinds of restriction enzymes RI, Hind III, PstI, and XbaI and subjected to agarose gel electrophoresis. Then, the DNA on the agarose gel was transferred to a nylon membrane, and Southern hybridization was performed using probe II. The label of the probe was radioactively labeled by the random priming method. Hybridization conditions are 5
X SSC, 0.1% SDS, 5 x Denharz solution, 100
Performed in μg / ml calf thymus DNA for 5 hours at 65 ° C. Two
After washing in × SSC, 0.1% SDS at 5.5 ° C. for 1 hour, it was exposed to an imaging plate (Fuji Film Co., Ltd.) and image data was obtained with an image analyzer BAS-2000 (Fuji Film Co., Ltd.). As a result, BamH
2.7 kb, 20 kb, 4.4 kb, and 6.6 k for I, EcoRI, Hind III, PstI, and XbaI, respectively.
A positive signal was detected at a position of b and 9.4 kb.

(4) Construction of restriction enzyme map of region containing DNA polymerase gene 0.3 μg of chromosomal DNA prepared in Example 1- (1) was added to BamHI, EcoRI, HindIII or PstI.
After cleaving at 50 ng, 50 ng of each cassette (Takara Shuzo Co., Ltd.) having each cleavage site was ligated using DNA ligase. DNA was recovered from this reaction solution by ethanol precipitation, and a portion of this was used for PCR. In order to perform nested PCR, specific primers II-S1 (SEQ ID NO: 11), II- in the same direction as GC type primers 1, 2 (SEQ ID NO: 3, 4) were previously prepared in the sequence of probe II.
S2 (SEQ ID NO: 12) was synthesized. The first PCR was performed using a combination of a common primer represented by SEQ ID NO: 9 (cassette primer C1, Takara Shuzo) attached to the sequence in the cassette and a primer represented by SEQ ID NO: 3 or 4. . Next, using 1 ml of the PCR solution as a template, a cassette primer C2 (Takara Shuzo) represented by SEQ ID NO: 10 in the sequence listing and a primer II-S1 or II-S.
PCR was performed on 2 combinations. The amplified DNA fragments were BamHI, HindIII, EcoRI, PstI, K
A restriction enzyme map was prepared by cutting with a restriction enzyme such as pnI, XbaI, SmaI, and SalI. The restriction enzyme map is shown in FIG. From this restriction map and the positions of the primers used for PCR, it was estimated that the entire region encoding DNA polymerase was contained in the approximately 3.1 kb fragment obtained by double cleavage of PstI-KpnI.

(5) D containing a DNA polymerase gene
Cloning of NA fragment 4 μg of chromosomal DNA prepared in Example 1- (1) was added to Ps.
It was cleaved with tI and KpnI, and the DNA around 3.1 kb was recovered from the agarose gel. Recovery is Supple (SUPRE
C) -01 (Takara Shuzo) was used. pTV
119N (Takara Shuzo) was cleaved with PstI and KpnI to prepare a fragment, which was mixed with the recovered fragment and ligated with DNA ligase. Next, a target clone was selected by colony hybridization using probe II as a probe from the transformant population obtained by introducing into E. coli JM109 strain. A plasmid was recovered from the selected transformant, it was confirmed that the desired PstI-KpnI fragment had been introduced, and the plasmid was named pPO500-II. PPO500-II was introduced into E. coli JM109 again to prepare Escherichia coli JM109 / pPO50.
0-II (FERM P-13659) was obtained. Furthermore, p
PstI-K cloned in PO500-II
The base sequence of the pnI fragment was determined. The base sequence is shown in SEQ ID NO: 8 in the sequence listing. As a result, an open reading frame (ORF) consisting of 803 amino acids was recognized. The amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.

(6) Culture of transformant and preparation of crude extract Escherichia coli JM109 / pPO500-II (F
ERM P-13659) with 100 μg of ampicillin
The cells were inoculated into 5 ml of L medium existing at a concentration of / ml and cultured at 37 ° C. When the turbidity of the culture broth was 0.6 (A ₆₆₀ ), isopropyl-β-D-thiogalactoside (IPTG), which was an inducer, was added and the culture was further continued for 15 hours. After collecting the cells, the cells were suspended in 150 μl of 25% sucrose, 50 mM Tris-hydrochloric acid (pH 7.6), 10 mM NaCl, 1 mM 2-mercaptoethanol, 2 μM phenylmethanesulfonyl fluoride (PMSF), and lysozyme was suspended in 0.
Add at a concentration of 5 mg / ml and let stand at 0 ° C for 30 minutes, then 3
It was transferred to 7 ° C. and left still for 30 minutes. After freeze-thawing once, the supernatant was obtained by centrifugation (14000 rpm, 10 minutes). The obtained supernatant is heat-treated at 100 ° C. for 15 minutes and then centrifuged again (14000 rpm, 10 minutes).
Then, the supernatant was recovered and used as a crude extract.

(7) Measurement of DNA polymerase activity As a reaction solution, 20 mM Tris-hydrochloric acid (pH 6.3,
75 ° C.), 2.5 mM magnesium chloride, 1 mM 2-
Mercaptoethanol, 2μM activated DNA, 33μ
M dATP, dCTP, dGTP, TTP, 60 nM
[ ³ H] TTP was prepared, an appropriate amount of crude extract was added to 150 μl of this solution, and the mixture was reacted at 75 ° C. for 5 minutes.
50 mM pyrophosphate, 10% trichloroacetic acid (TC
The reaction was stopped by adding 1 ml of A). After standing still in ice for 5 minutes, the whole amount was transferred onto a glass filter and suction filtered. After washing several times with 10% TCA, the mixture was replaced with 70% ethanol, the filter was dried, and the radioactivity on the filter was measured with a liquid scintillation counter. 1
3.0 U of DNA polymerase was obtained from the culture medium of ml.

(8) Production of Thermostable DNA Polymerase by Escherichia coli Introduced with Plasmid pPO500-II 4.2 g of bacterial cells obtained from 3 liters of E. coli JM109 / pPO500-II culture medium was buffered (150 mM Tris-hydrochloric acid pH 7). .6, 2 mM EDTA, 2.4 mM
PMSF) was suspended in 40 ml and crushed by ultrasonic treatment. Ammonium sulfate was added to the final concentration of 0.2 M and the mixture was centrifuged (12,000 rpm, 10 minutes), and the supernatant was subjected to heat treatment at 90 ° C. for 10 minutes to obtain a crude extract. This A ₂₈₀ is measured and 1000 (A ₂₈₀ ) is 5
% Polyethyleneimine (PEI) solution (pH 7.6)
After adding 0.5 ml and stirring at 4 ° C for 1 hour, centrifugation (12,000 rpm, 20 minutes) was performed to remove nucleic acids,
The supernatant was added to a buffer solution (50 mM Tris-hydrochloric acid pH 7.6,
Phenyl Sepharose column (6FFHig) equilibrated with 2 mM EDTA, 0.2 M ammonium sulfate)
h sub (Pharmacia)). Buffer solution 50 mM Tris-hydrochloric acid pH 7.6, 2 mM EDT
A, buffer solution (50 mM Tris-hydrochloric acid pH 7.6, 2 m
M EDTA, 20% ethylene glycol) were sequentially washed, and then eluted with a linear concentration gradient of 0 M to 4 M urea to fractionate, and the DNA active polymerase activity was examined according to Example 1- (7). Next, the active fractions were collected and collected in buffer [50
mM Tris-hydrochloric acid pH 7.6, 100 mM KCl,
0.1 mM EDTA, 0.2% Tween 2
[0] was added to 1 ml of a high trap heparin column (Pharmacia) and eluted with a linear concentration gradient of 150 mM KCl and 150 mM to 650 mM KCl to collect active fractions. The active fraction is a buffer solution (50 m
M Tris-hydrochloric acid pH 7.6, 0.1 mM EDTA,
It was dialyzed against 0.2% Tween 20) and subjected to Mono-Q (5 ml) equilibrated with the same buffer, and the unadsorbed fraction was added to Mono-S (5 ml) equilibrated with the same buffer to give 0 mM to 5 mM.
Active fractions were collected by elution with a linear gradient of 00 mM NaCl. 2200U enzyme preparation (PocDN) from Mono S fraction
A polymerase II) was obtained and subjected to SDS-PAGE analysis to give a single band with a molecular weight of about 90,000 daltons.

(9) Measurement of 5 '→ 3' exonuclease activity pUC119 was cleaved with SspI and electrophoresed on an agarose gel to recover a 386 bp fragment using Suplex-01 (Takara Shuzo). The 5'end of this fragment was [γ- ³²
P] ATP and a megalabel kit (Takara Shuzo) were used for radiolabeling, and gel filtration was carried out using a NICK column (G50) (Pharmacia) to remove free [γ- ³² P] ATP to obtain a substrate. 1 ng of this substrate and 0.05 U of PocDNA polymerase II obtained in Example 1- (8) were added to 20 mM.
Tris-hydrochloric acid (pH 6.3 or pH 7.7) 2.5 m
75 ° C. in 10 μl of M MgCl ₂ solution for 5 minutes, 10 minutes,
After reacting for 15 minutes, 25 μl of ethanol and 2 μl of 20 μg / μl glycogen were added to perform ethanol precipitation, and the radioactivity of the supernatant and the precipitate was measured by a liquid scintillation counter by the Cherenkov method. When PocDNA Polymerase II was added, there was an increase in the radioactivity of the supernatant with the release of the nucleotides at the 5'end with the lapse of time.
→ 3 'exonuclease activity was observed. On the other hand, JM
When 109 Escherichia coli lysate was added, there was no increase in radioactivity and no 5 '→ 3' exonuclease activity was observed.

(10) Measurement of 3 ′ → 5 ′ exonuclease activity pUC119 was cleaved with Sau3AI and then electrophoresed on an agarose gel to recover a 341 bp fragment using Suplex-01 (Takara Shuzo). Add the 3'end of this fragment to [α
- ³² P] dCTP, radioactively labeled with dATP, dGTP, dTTP, and Klenow enzyme, except gel filtration to liberate ^[alpha-32 P] dCTP was used as a substrate in NICK column. 4 ng of this substrate was added to 20 mM Tris-hydrochloric acid (pH
6.3, or pH 7.7), 2.5 mM MgCl ₂ ,
Solution 10 of 1.25 mg / ml λ-HaeIII degradation product
0.2 U (pH of PocDNA Polymerase II in μl
6.3) or 0.05 U (pH 7.7)) and 75 ° C 5
After 10 minutes, 10 minutes and 15 minutes of reaction, ethanol precipitation was carried out in the same manner as in Example 1- (9), and the radioactivity of the supernatant and the precipitate was measured.
The reaction was performed by adding 1.25 mg / ml λ-Hae III degradation product under the condition of large excess of substrate with respect to Km. As a result, when PocDNA polymerase was added, the radioactivity of the supernatant increased with the release of the nucleotides at the 3'end over time, and 3 '→ 5' exonuclease activity was observed. On the other hand, when JM109 Escherichia coli lysate was added, there was no increase in radioactivity and no 3 ′ → 5 ′ exonuclease activity was observed. In addition, as a result of examining the ratio of 3 ′ → 5 ′ exonuclease activity to DNA polymerase activity, it was found that the pH was higher than that at pH 6.3.
The value was higher in the case of 7.7, which was about 40 times.

Example 2 (1) Cloning of DNA fragment containing DNA polymerase gene Using probe I, Southern hybridization was carried out in the same manner as in Example 1- (3). As a result, about 5.
5 kb EcoRI fragment, approximately 4.9 kb HindIII
The fragment and the PstI fragment of about 1.7 kb were positive. Within the sequence of probe I, primers I-S1 (SEQ ID NO: 13), I specific in the same direction as GC type primers 1 and 2,
-S2 (SEQ ID NO: 14) was synthesized, and PCR was performed in the same manner as in Example 1- (4) to create a restriction enzyme map. The restriction enzyme map is shown in FIG. About 4.2 kb EcoRI-H from the position of the restriction map and the primers used for PCR
It was presumed that the entire region encoding the DNA polymerase was contained on the indIII fragment. Then pTV118N
(Takara Shuzo) was cleaved with EcoRI and HindIII, and the desired clone was selected in the same manner as in Example 1- (5). A plasmid was recovered from the obtained recombinant, it was confirmed that the desired EcoRI-HindIII fragment had been inserted, and the plasmid was named pPO100-I. PPO100-I was introduced into E. coli JM109 again to prepare Escherichia coli JM109 / pPO10.
0-I (FERM P-13660) was obtained. Furthermore, p
EcoRI- cloned in PO100-I
The nucleotide sequence of the SmaI-HindIII region of the HindIII fragment was determined. The base sequence is shown in SEQ ID NO: 7 in the sequence listing. As a result, base numbers 435 to 3176,5
ORFs consisting of 914 and 879 amino acids were observed in 40 to 3176, respectively. OR consisting of 914 amino acids
The amino acid sequence of F is shown in SEQ ID NO: 1 in the sequence listing. 87
The ORF consisting of 9 amino acids corresponds to amino acids 36 to 914 in SEQ ID NO: 1.

(2) Culture of transformant and preparation of crude extract E. coli JM109 / pPO100-I was inoculated into 5 ml of L medium containing ampicillin at a concentration of 100 μg / ml, incubated at 37 ° C. for 20 hours, and 200 μl of 2
5% sucrose, 50 mM Tris-hydrochloric acid (pH 7.6),
Suspended in 10 mM NaCl, 1 mM 2-mercaptoethanol, 2 μM PMSF and added 0.5 m of lysozyme.
Add at a concentration of g / ml and let stand at 0 ℃ for 30 minutes, then 37 ℃
And allowed to stand for another 30 minutes. After freeze-thawing once, a supernatant was obtained by centrifugation (14000 rpm, 10 minutes). The obtained supernatant was heat-treated at 70 ° C. for 20 minutes, centrifuged again (14000 rpm, 10 minutes), and the supernatant was recovered to give a crude enzyme solution. When the DNA polymerase activity was measured in the same manner as in Example 1- (7), 0.22 units of activity was obtained from 1 ml of the culture solution.

(3) Modification of expression system In order to increase the expression level, a plasmid was constructed in which a DNA polymerase was directly expressed from ATG at the NcoI site of vector pET15b (Novagen). First known α
Oligonucleotides 1 and 2 for introducing NcoI sites into the two ATG regions (base numbers 435 to 437 and 540 to 542 of SEQ ID NO: 7) that are presumed to be start codons from the homology with the type DNA polymerase ( SEQ ID NOs: 15, 16) were synthesized. Site direct mutagenesis was performed using this oligonucleotide 1 or 2 and pPO100-I and Mutan-K (Takara Shuzo Co., Ltd.) to obtain NPOI in pPO100-I.
Site-introduced plasmid pPO100-IM1, p
PO100-IM2 was constructed. Next, NcoI-Afl II (1551 bp and 14
46 bp) fragment and Afl II-EcoRV (1457b
p) The fragment was purified (the Afl II site is located at base numbers 1983 to 1988 of SEQ ID NO: 7), pET-15b was cleaved with BamHI to make it blunt-ended, and then pET-15b was obtained by cutting with NcoI. It was mixed with the blunted fragment and ligated with DNA ligase. Escherichia coli HB101 was transformed with each of the mixed solutions, a plasmid was prepared from the obtained transformant, and NcoI-AflII fragment and AflII- were analyzed by restriction enzyme analysis.
The plasmid to which the EcoRV fragment was ligated was selected and pPO2 was selected.
They were named 00-I and pPO300-I. That is, p
PO200-I contains two ORFs of 914 amino acids (SEQ ID NO: 1, except that the second amino acid has been replaced by Lys to Glu), and 879 amino acids (amino acids 36 to 914 of SEQ ID NO: 1). Cage,
pPO300-I contains one ORF consisting of 879 amino acids (amino acid numbers 36 to 914 of SEQ ID NO: 1).

(4) Plasmid pPO200-I, pP
Production of thermostable DNA polymerase by Escherichia coli introduced with O300-I. Transformants obtained by introducing plasmid pPO200-I or pPO300-I into Escherichia coli HMS174 (DE3) (Novagen) were respectively HMS174 (DE3) / pP.
O200-I, HMS174 (DE3) / pPO300
-I. HMS174 (DE3) / pPO20
0-I and HMS174 (DE3) / pPO300-I each in a 2 liter baffled flask with 500 ml.
The L medium was inoculated and 0.2 ml of 1M IPTG was added when the concentration was 0.7, and the mixture was cultured for 20 hours. Example 1 from 4.4 g and 4.3 g of the bacterial cells obtained from 3 liters of the culture solution
The DNA polymerase was purified in the same manner as (8). H
From MS174 (DE3) / pPO200-I, 11250 U was obtained from the heparin active fraction, and SDS-PAGE gave two bands of approximately equal molecular weights of about 95,000 daltons and 100,000 daltons. HMS174 (DE3) / pPO
From 300-I, 13350 U was obtained from the heparin active fraction, and SDS-PAGE gave a single band having a molecular weight of about 95,000 daltons.

HMS174 (DE3) / pPO300-
Example 1- (9), 1- (10) using the enzyme preparation (PocDNA polymerase-I) obtained by purification from I.
The presence or absence of the accompanying nuclease activity was examined in the same manner as in. PocD for 5 '→ 3' exonuclease
Similar activity to NA polymerase II was observed. A DNA polymerase (PfuDN) derived from Pyrococcus furiosus, which is an enzyme derived from a known α-type archaeal strain.
This activity was not observed with A polymerase, Stratagene). Regarding 3 '→ 5' exonuclease, the same activity as that of PocDNA polymerase II was observed, and the activity was about 7 times higher in the reaction solution at pH 7.7 than at pH 6.3. The ratio of 3 ′ → 5 ′ exonuclease activity to DNA polymerase activity is pH 6.3.
10 times more than PocDNA Polymerase II in the reaction solution of
About 3.5 times that of fuDNA polymerase, about 14 times that of PocDNA polymerase II in the reaction solution of pH 7.7, Pfu
It was about 1.5 times that of DNA polymerase.

The 3 '→ 5' exonuclease activity is DN
It is considered to be a proofreading function of A polymerase at the time of DNA synthesis, and is an important activity in the process of separating a wrong base into a template DNA and synthesizing it when the wrong base is taken in again. P in the present invention
The ratio of the exonuclease activity to the polymerase activity of ocDNA polymerase I is the PocDN of the present invention.
The fact that the ratios of the respective activities of A polymerase II and the known Pfu DNA polymerase are significantly higher suggests that PocDNA polymerase I performs DNA synthesis with extremely high accuracy.

[0037]

INDUSTRIAL APPLICABILITY The present invention provides a gene encoding a thermostable DNA polymerase useful as a reagent for genetic engineering research, and a method for genetically engineering a thermostable DNA polymerase using the gene.

[0038]

[Sequence list]

SEQ ID NO: 1 Sequence length: 914 Sequence type: Amino acid Number of chains: Single chain Topology: linear Sequence type: Peptide Array: Met Lys Ala Gln Pro Gln Leu Ala Thr His Gln Gly Leu Thr Thr   1 5 10 15 Glu Lys Ala Val Val Asn Val Asp Ala Glu Thr Trp Ala Glu Gln                  20 25 30 His Ala Trp Ser Thr Met Val Pro Gln Ser Ser Thr Pro Pro Ala                  35 40 45 Gly Tyr Gly Asp Asp Leu Ala Gly Lys Leu Gly Ser Leu Leu Gly                  50 55 60 Gly Ser Arg Gly Ala Leu Glu Arg Leu Ser Ala Leu Pro Leu Thr                  65 70 75 Arg Lys Pro Leu Glu Ala Arg Asp Gly Val Glu Gly Phe Leu Leu                  80 85 90 Gln Thr Met Tyr Asp Gly Glu Arg Gly Val Ala Ala Ala Lys Ile                  95 100 105 Tyr Asp Asp Arg Asn Gly Ile Val Tyr Val Tyr Phe Asp Arg Thr                 110 115 120 Gly Tyr Met Pro Tyr Phe Leu Thr Asp Ile Pro Pro Asp Lys Leu                 125 130 135 Gln Glu Leu His Glu Val Val Arg His Lys Gly Phe Asp His Val                 140 145 150 Glu Val Val Glu Lys Phe Asp Leu Leu Arg Trp Gln Arg Arg Lys                 155 160 165 Val Thr Lys Ile Val Val Lys Thr Pro Asp Val Val Arg Val Leu                 170 175 180 Arg Asp Lys Val Pro Arg Ala Trp Glu Ala Asn Ile Lys Phe His                 185 190 195 His Asn Tyr Ile Tyr Asp Tyr Gly Leu Val Pro Gly Met Lys Tyr                 200 205 210 Arg Val Gly Lys Gly Arg Leu Ile Leu Leu Gly Gly Glu Ala Ser                 215 220 225 Gly Asp Asp Glu Arg His Ile Arg Glu Ile Phe Ser Gly Glu Asp                 230 235 240 Glu Ser Thr Ile Glu Met Ala Val Lys Trp Leu Ser Leu Phe Glu                 245 250 255 Gln Pro Pro Pro Lys Pro Arg Arg Leu Ala Val Asp Ile Glu Val                 260 265 270 Phe Thr Pro Phe Lys Gly Arg Ile Pro Asp Pro Ser Thr Ala Ser                 275 280 285 Tyr Pro Val Ile Ser Val Ala Met Ser Ser Asp Glu Gly Trp Arg                 290 295 300 Ala Val Tyr Val Leu Ala Arg Pro Gly Val Pro Met Asn Pro Pro                 305 310 315 Arg Gly Pro Leu Pro Glu Asn Leu His Val Glu Ile Phe Asp Asp                 320 325 330 Glu Arg Ala Leu Ile Leu Glu Ala Phe Arg Leu Ile Ser Asn Tyr                 335 340 345 Pro Val Leu Leu Thr Phe Asn Gly Asp Asn Phe Asp Leu Pro Tyr                 350 355 360 Leu Tyr Asn Arg Ala Val Lys Leu Gly Ile Pro Arg Glu Tyr Ile                 365 370 375 Pro Phe Arg Ala Arg Ser Asp Tyr Val Thr Leu Glu Tyr Gly Phe                 380 385 390 His Ile Asp Leu Tyr Lys Phe Phe Ser Thr Lys Ala Val Gln Ala                 395 400 405 Tyr Ala Phe Gly Asn Ala Tyr Gln Glu Phe Thr Leu Asp Ala Ile                 410 415 420 Ala Ser Ala Leu Leu Gly Glu His Lys Val Glu Val Glu Ser Thr                 425 430 435 Val Ser Asp Leu Pro Phe Phe Glu Leu Val Arg Tyr Asn Val Arg                 440 445 450 Asp Ala Asp Leu Thr Leu Arg Leu Thr Thr Phe Asn Asn Asp Leu                 455 460 465 Val Trp Ser Leu Ile Ile Leu Leu Met Arg Ile Ser Lys Leu Pro                 470 475 480 Leu Glu Asp Val Thr Arg Ser Gln Val Ser Ala Trp Val Lys Ser                 485 490 495 Leu Phe Tyr Trp Glu His Arg Arg Arg Gly Tyr Leu Ile Pro Ser                 500 505 510 Arg Glu Glu Ile Ile Arg Leu Lys Gly Thr Thr Arg Ser Glu Ala                 515 520 525 Leu Ile Lys Gly Lys Lys Tyr Gln Gly Ala Leu Val Leu Asp Pro                 530 535 540 Pro Ser Gly Ile Tyr Phe Asn Ile Val Val Leu Asp Phe Ala Ser                 545 550 555 Leu Tyr Pro Ser Ile Ile Lys Arg Trp Asn Leu Ser Tyr Glu Thr                 560 565 570 Val Asn Pro Val Tyr Cys Pro Glu Ser Lys Leu Val Glu Val Pro                 575 580 585 Asp Val Gly His Lys Val Cys Met Ser Ile Pro Gly Leu Thr Ser                 590 595 600 Gln Ile Val Gly Leu Leu Arg Asp Tyr Arg Val Lys Ile Tyr Lys                 605 610 615 Lys Lys Ala Lys Asp Lys Ser Leu Pro Asp Asp Val Arg Ala Trp                 620 625 630 Tyr Asn Thr Val Gln Ala Ala Met Lys Val Tyr Ile Asn Ala Ser                 635 640 645 Tyr Gly Val Phe Gly Ala Glu Ser Phe Pro Phe Tyr Ala Pro Pro                 650 655 660 Val Ala Glu Ser Val Thr Ala Ile Gly Arg Tyr Thr Ile Lys Gln                 665 670 675 Thr Leu Gln Lys Ala Gly Glu Leu Gly Leu Arg Val Leu Tyr Gly                 680 685 690 Asp Thr Asp Ser Leu Phe Ile Trp Asn Pro Asp Glu Asp Lys Leu                 695 700 705 Arg Glu Leu Gln Glu Tyr Val Glu Lys Asn Phe Gly Leu Asp Leu                 710 715 720 Glu Val Asp Lys Val Tyr Lys Phe Val Thr Phe Ser Gly Leu Lys                 725 730 735 Lys Asn Tyr Ile Gly Ala Tyr Glu Asp Gly Ser Ile Asp Val Lys                 740 745 750 Gly Met Val Ala Lys Lys Arg Asn Thr Pro Glu Phe Leu Lys Lys                 755 760 765 Glu Phe Ser Glu Met Leu Ala Val Ile Gly Ser Val Lys Ser Pro                 770 775 780 Glu Asp Phe Ile Lys Val Arg Arg Val Ile Arg Glu Arg Leu Arg                 785 790 795 Lys Val Tyr His Gly Leu Arg Asp Leu Glu Phe Asn Leu Asp Glu                 800 805 810 Leu Ala Ile Arg Met Ala Leu Asn Lys Pro Val Glu Ala Tyr Thr                 815 820 825 Lys Asn Thr Pro Gln His Val Lys Ala Ala Arg Gln Leu Ile Arg                 830 835 840 Ala Gly Val Gln Val Leu Pro Gly Asp Val Ile Ser Phe Val Lys                 845 850 855 Val Lys Gly Lys Glu Gly Val Lys Pro Val Gln Leu Ala Arg Leu                 860 865 870 Pro Glu Val Asp Val Glu Lys Tyr Val Glu Ser Met Arg Asn Val                 875 880 885 Phe Glu Gln Leu Leu Leu Ala Ile Ser Met Ser Trp Asp Glu Ile                 890 895 900 Ile Gly Ser Ser Arg Leu Glu Ala Phe Phe Ser Arg Arg Gly                 905 910

SEQ ID NO: 2 Array length: 803 Sequence type: Amino acid Number of chains: Single chain Topology: linear Sequence type: Peptide Array: Met Thr Glu Thr Ile Glu Phe Val Leu Leu Asp Ser Ser Tyr Glu   1 5 10 15 Ile Leu Gly Lys Glu Pro Val Val Ile Leu Trp Gly Ile Thr Leu                  20 25 30 Asp Gly Lys Arg Val Val Leu Leu Asp His Arg Phe Arg Pro Tyr                  35 40 45 Phe Tyr Ala Leu Ile Ala Arg Gly Tyr Glu Asp Met Val Glu Glu                  50 55 60 Ile Ala Ala Ser Ile Arg Arg Leu Ser Val Val Lys Ser Pro Ile                  65 70 75 Ile Asp Ala Lys Pro Leu Asp Lys Arg Tyr Phe Gly Arg Pro Arg                  80 85 90 Lys Ala Val Lys Ile Thr Thr Met Ile Pro Glu Ser Val Arg His                  95 100 105 Tyr Arg Glu Ala Val Lys Lys Ile Glu Gly Val Glu Asp Ser Leu                 110 115 120 Glu Ala Asp Ile Arg Phe Ala Met Arg Tyr Leu Ile Asp Lys Arg                 125 130 135 Leu Tyr Pro Phe Thr Val Tyr Arg Ile Pro Val Glu Asp Ala Gly                 140 145 150 Arg Asn Pro Gly Phe Arg Val Asp Arg Val Tyr Lys Val Ala Gly                 155 160 165 Asp Pro Glu Pro Leu Ala Asp Ile Thr Arg Ile Asp Leu Pro Pro                 170 175 180 Met Arg Leu Val Ala Phe Asp Ile Glu Val Tyr Ser Arg Arg Gly                 185 190 195 Ser Pro Asn Pro Ala Arg Asp Pro Val Ile Ile Val Ser Leu Arg                 200 205 210 Asp Ser Glu Gly Lys Glu Arg Leu Ile Glu Ala Glu Gly His Asp                 215 220 225 Asp Arg Arg Val Leu Arg Glu Phe Val Glu Tyr Val Arg Ala Phe                 230 235 240 Asp Pro Asp Ile Ile Val Gly Tyr Asn Ser Asn His Phe Asp Trp                 245 250 255 Pro Tyr Leu Met Glu Arg Ala Arg Arg Leu Gly Ile Lys Leu Asp                 260 265 270 Val Thr Arg Arg Val Gly Ala Glu Pro Thr Thr Ser Val Tyr Gly                 275 280 285 His Val Ser Val Gln Gly Arg Leu Asn Val Asp Leu Tyr Asp Tyr                 290 295 300 Ala Glu Glu Met Pro Glu Ile Lys Met Lys Thr Leu Glu Glu Val                 305 310 315 Ala Glu Tyr Leu Gly Val Met Lys Lys Ser Glu Arg Val Ile Ile                 320 325 330 Glu Trp Trp Arg Ile Pro Glu Tyr Trp Asp Asp Glu Lys Lys Arg                 335 340 345 Gln Leu Leu Glu Arg Tyr Ala Leu Asp Asp Val Arg Ala Thr Tyr                 350 355 360 Gly Leu Ala Glu Lys Met Leu Pro Phe Ala Ile Gln Leu Ser Thr                 365 370 375 Val Thr Gly Val Pro Leu Asp Gln Val Gly Ala Met Gly Val Gly                 380 385 390 Phe Arg Leu Glu Trp Tyr Leu Met Arg Ala Ala Tyr Asp Met Asn                 395 400 405 Glu Leu Val Pro Asn Arg Val Glu Arg Arg Gly Glu Ser Tyr Lys                 410 415 420 Gly Ala Val Val Leu Lys Pro Leu Lys Gly Val His Glu Asn Val                 425 430 435 Val Val Leu Asp Phe Ser Ser Met Tyr Pro Ser Ile Met Ile Lys                 440 445 450 Tyr Asn Val Gly Pro Asp Thr Ile Val Asp Asp Pro Ser Glu Cys                 455 460 465 Pro Lys Tyr Gly Gly Cys Tyr Val Ala Pro Glu Val Gly His Arg                 470 475 480 Phe Arg Arg Ser Pro Gly Phe Phe Lys Thr Val Leu Glu Asn                 485 490 495 Leu Leu Lys Leu Arg Arg Gln Val Lys Glu Lys Met Lys Glu Phe                 500 505 510 Pro Pro Asp Ser Pro Glu Tyr Arg Leu Tyr Asp Glu Arg Gln Lys                 515 520 525 Ala Leu Lys Val Leu Ala Asn Ala Ser Tyr Gly Tyr Met Gly Trp                 530 535 540 Ser His Ala Arg Trp Tyr Cys Lys Arg Cys Ala Glu Ala Val Thr                 545 550 555 Ala Trp Gly Arg Asn Leu Ile Leu Thr Ala Ile Glu Tyr Ala Arg                 560 565 570 Lys Leu Gly Leu Lys Val Ile Tyr Gly Asp Thr Asp Ser Leu Phe                 575 580 585 Val Val Tyr Asp Lys Glu Lys Val Glu Lys Leu Ile Glu Phe Val                 590 595 600 Glu Lys Glu Leu Gly Phe Glu Ile Lys Ile Asp Lys Ile Tyr Lys                 605 610 615 Lys Val Phe Phe Thr Glu Ala Lys Lys Arg Tyr Val Gly Leu Leu                 620 625 630 Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala Val Arg Gly                 635 640 645 Asp Trp Cys Glu Leu Ala Lys Glu Val Gln Glu Lys Ala Ala Glu                 650 655 660 Ile Val Leu Asn Thr Gly Asn Val Asp Lys Ala Ile Ser Tyr Ile                 665 670 675 Arg Glu Val Ile Lys Gln Leu Arg Glu Gly Lys Val Pro Ile Thr                 680 685 690 Lys Leu Ile Ile Trp Lys Thr Leu Ser Lys Arg Ile Glu Glu Tyr                 695 700 705 Glu His Asp Ala Pro His Val Met Ala Ala Arg Arg Met Lys Glu                 710 715 720 Ala Gly Tyr Glu Val Ser Pro Gly Asp Lys Val Gly Tyr Val Ile                 725 730 735 Val Lys Gly Ser Gly Ser Val Ser Ser Arg Ala Tyr Pro Tyr Phe                 740 745 750 Met Val Asp Pro Ser Thr Ile Asp Val Asn Tyr Tyr Ile Asp His                 755 760 765 Gln Ile Val Pro Ala Ala Leu Arg Ile Leu Ser Tyr Phe Gly Val                 770 775 780 Thr Glu Lys Gln Leu Lys Ala Ala Ala Thr Val Gln Arg Ser Leu                 785 790 795 Phe Asp Phe Phe Ala Ser Lys Lys                 800

SEQ ID NO: 3 Array length: 20 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: WSSYTSTACC CSWSSATCAT 20

SEQ ID NO: 4 Array length: 20 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: TCNGTRTCNC CRTARATNAC 20

SEQ ID NO: 5 Array length: 416 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Array: AGC CTG TAC CCC AGT ATC ATA AAG AGG TGG AAC CTA AGC TAC GAG 45 Ser Leu Tyr Pro Ser Ile Ile Lys Arg Trp Asn Leu Ser Tyr Glu   1 5 10 15 ACC GTA AAC CCC GTA TAC TGC CCC GAA TCG AAG CTA GTG GAG GTT 90 Thr Val Asn Pro Val Tyr Cys Pro Glu Ser Lys Leu Val Glu Val                  20 25 30 CCC GAT GTA GGG CAT AAG GTG TGC ATG AGC ATA CCC GGC CTG ACC 135 Pro Asp Val Gly His Lys Val Cys Met Ser Ile Pro Gly Leu Thr                  35 40 45 TCG CAG ATA GTT GGC CTG CTT AGG GAC TAT CGA GTC AAG ATA TAC 180 Ser Gln Ile Val Gly Leu Leu Arg Asp Tyr Arg Val Lys Ile Tyr                  50 55 60 AAG AAG AAG GCC AAG GAT AAG AGT CTG CCG GAT GAT GTT AGA GCA 225 Lys Lys Lys Ala Lys Asp Lys Ser Leu Pro Asp Asp Val Arg Ala                  65 70 75 TGG TAT AAT ACA GTC CAG GCA GCC ATG AAG GTG TAT ATA AAT GCC 270 Trp Tyr Asn Thr Val Gln Ala Ala Met Lys Val Tyr Ile Asn Ala                  80 85 90 AGC TAT GGA GTC TTC GGG GCC GAG AGC TTC CCG TTC TAC GCG CCG 315 Ser Tyr Gly Val Phe Gly Ala Glu Ser Phe Pro Phe Tyr Ala Pro                  95 100 105 CCG GTA GCG GAG AGC GTC ACA GCC ATA GGC AGG TAT ACT ATC AAG 360 Pro Val Ala Glu Ser Val Thr Ala Ile Gly Arg Tyr Thr Ile Lys                 110 115 120 CAG ACG CTG CAG AAG GCT GGC GAA CTA GGG CTC CGC GTG ATC TAT 405 Gln Thr Leu Gln Lys Ala Gly Glu Leu Gly Leu Arg Val Leu Tyr                 125 130 135 GGC GAT ACG GA 416 Gly Asp Thr

SEQ ID NO: 6 Array length: 422 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Array: TCC ATG TAC CCG AGC ATA ATG ATA AAG TAC AAC GTG GGC CCC GAC 45 Ser Met Tyr Pro Ser Ile Met Ile Lys Tyr Asn Val Gly Pro Asp   1 5 10 15 ACT ATA GTC GAC GAC CCC TCG GAG TGC CCA AAG TAC GGC GGC TGC 90 Thr Ile Val Asp Asp Pro Ser Glu Cys Pro Lys Tyr Gly Gly Cys                  20 25 30 TAT GTA GCC CCC GAG GTC GGG CAC CGG TTC CGT CGC TCC CCG CCA 135 Tyr Val Ala Pro Glu Val Gly His Arg Phe Arg Arg Ser Pro Pro                  35 40 45 GGC TTC TTC AAG ACC GTG CTC GAG AAC CTA CTG AAG CTA CGC CGA 180 Gly Phe Phe Lys Thr Val Leu Glu Asn Leu Leu Lys Leu Arg Arg                  50 55 60 CAG GTA AAG GAG AAG ATG AAG GAG TTT CCG CCT GAC AGC CCC GAG 225 Gln Val Lys Glu Lys Met Lys Glu Phe Pro Pro Asp Ser Pro Glu                  65 70 75 TAC AGG CTC TAC GAT GAG CGC CAG AAG GCG CTC AAG GTT CTT GCG 270 Tyr Arg Leu Tyr Asp Glu Arg Gln Lys Ala Leu Lys Val Leu Ala                  80 85 90 AAC GCG AGC TAT GGC TAC ATG GGG TGG AGC CAT GCC CGC TGG TAC 315 Asn Ala Ser Tyr Gly Tyr Met Gly Trp Ser His Ala Arg Trp Tyr                  95 100 105 TGC AAA CGC TGC GCC GAG GCT GTC ACA GCC TGG GGC CGT AAC CTT 360 Cys Lys Arg Cys Ala Glu Ala Val Thr Ala Trp Gly Arg Asn Leu                 110 115 120 ATA CTG ACA GCT ATC GAG TAT GCC AGG AAG CTC GGC CTA AAG GTG 405 Ile Leu Thr Ala Ile Glu Tyr Ala Arg Lys Leu Gly Leu Lys Val                 125 130 135 ATA TAT GGG TAC ACC GA 422 Ile Tyr Gly Asp Thr                 140

SEQ ID NO: 7 Sequence length: 3437 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear Sequence type: Genomic DNA Origin: Organism name: Pyrodictium occultum (Pyrodicti)
um occultum) strain name: DSM2709 ^T sequence: CCCGGGCCAC TCCATCCATA GGCTCAAGGC GCTCCAGGCT CCTTTTAAAC ATTACATGCA 60 ATTCTAAGGG ACTCTGCGCG CGGCTTAGGT CACCCACCTT ATACGGTGAT ACGTGGGAGC 120 TGGATAGGGG GCGGTGCGTG GTTAGGCGCT CAAAGAGGCG TGGAGGGGAG CGCGACCTAC 180 TCGAGTTCCT AGCTGGTGGC GTAACCGGCG CCCGCAGGGC TAAGGGCCGA ACCACCGAGA 240 GCGGGGATGG TACGGGCAGC GAGAGGGATG GTGCTAAGCC CCTCTGGGAG GGGAATACGG 300 CCAGGAGGGC CGGGGTGGAG AGGCTATACG ATAACAGCCT CTACGAACTG TTATCGGAAA 360 TATCCTCATC TAGGAGACGC GGGTCTAGCC ATCCAAGAGA CGATGATCGG GAGGGGGCTG 420 ATCTCACTGG CGGC ATG AAG GCT CAG CCG CAG CTT GCT ACG CAC CAA GGG 470 Met Lys Ala Gln Pro Gln Leu Ala Thr His GTG AAG 5G GLA GAG GCA GAA ACC TGG 515 Leu Thr Thr Glu Lys Ala Val Val Asn Val Asp Ala Glu Thr Trp 15 20 25 GCT GAG CAG CAT GCA TGG AGC ACT ATG GTG CCT CAG AGC TCT ACG 560 Ala Glu Gln His Ala Trp Ser Thr Met Val Pro Gln Ser Ser Thr 30 35 40 CCC CCC GCG GGG TAT GGA GAT GAT CTG GCA GGG AAG CTG GGT TCG 605 Pro Pro Ala Gly T yr Gly Asp Asp Leu Ala Gly Lys Leu Gly Ser 45 50 55 CTG CTA GGG GGC TCA CGG GGT GCC CTT GAG AGA CTT TCC GCT CTC 650 Leu Leu Gly Gly Ser Arg Gly Ala Leu Glu Arg Leu Ser Ala Leu 60 65 70 CCG CTT ACG CGC AAA CCC CTG GAA GCG CGT GAT GGG GTT GAG GGT 695 Pro Leu Thr Arg Lys Pro Leu Glu Ala Arg Asp Gly Val Glu Gly 75 80 85 TTC CTG CTT CAA ACA ATG TAT GAC GGG GAG AGG GGT GTT GCG GCG 740 Phe Leu Leu Gln Thr Met Tyr Asp Gly Glu Arg Gly Val Ala Ala 90 95 100 GCT AAG ATA TAT GAC GAC CGT AAT GGC ATT GTC TAC GTC TAC TTT 785 Ala Lys Ile Tyr Asp Asp Arg Asn Gly Ile Val Tyr Val Tyr Phe 105 110 115 GAT AGG ACT GGT TAC ATG CCA TAC TTT CTA ACC GAT ATA CCA CCG 830 Asp Arg Thr Gly Tyr Met Pro Tyr Phe Leu Thr Asp Ile Pro Pro 120 125 130 GAC AAG CTG CAG GAG CTT CAC GAG GTG GTG CGG CAT AAG GGG TTC 875 Asp Lys Leu Gln Glu Leu His Glu Val Val Arg His Lys Gly Phe 135 140 145 GAC CAT GTT GAG GTT GTG GAG AAG TTT GAT CTC CTG CGT TGG CAG 920 Asp His Val Glu Val Val Glu Lys Phe Asp Leu Leu Arg Trp Gln 150 155 160 CGT AGG AAG GTT ACT AAG ATC GTT GTA AAG ACC CCC GAT GTG GTG 965 Arg Arg Lys Val Thr Lys Ile Val Val Lys Thr Pro Asp Val Val 165 170 175 AGG GTG CTC CGT GAC AAG GTT CCA CGC GCC TGG GAG GCC AAT ATA 1010 Arg Val Leu Arg Asp Lys Val Pro Arg Ala Trp Glu Ala Asn Ile 180 185 190 AAG TTT CAC CAC AAC TAT ATA TAT GAT TAT GGG CTA GTG CCT GGA 1055 Lys Phe His His Asn Tyr Ile Tyr Asp Tyr Gly Leu Val Pro Gly 195 200 205 ATG AAG TAC CGC GTC GGG AAG GGC AGG CTA ATC CTC CTG GGG GGA 1100 Met Lys Tyr Arg Val Gly Lys Gly Arg Leu Ile Leu Leu Gly Gly 210 215 220 GAG GCT AGC GGG GAC GAT GAG CGC CAT ATA CGC GAG ATA TTC AGT 1145 Glu Ala Ser Gly Asp Asp Glu Arg His Ile Arg Glu Ile Phe Ser 225 230 235 GGT GAG GAT GAA AGC ACT ATT GAG ATG GCA GTA AAA TGG CTC TCC 1190 Gly Glu Asp Glu Ser Thr Ile Glu Met Ala Val Lys Trp Leu Ser 240 245 250 CTG TTT GAG CAG CCT CCC CCT AAG CCT CGT AGA CTT GCA GTG GAC 1235 Leu Phe Glu Gln Pro Pro Pro Lys Pro Arg Arg Leu Ala Val Asp 255 260 265 ATC GAG GTA TTC ACT CCC TTC AAG GGC CGT ATA CC A GAC CCT TCC 1280 Ile Glu Val Phe Thr Pro Phe Lys Gly Arg Ile Pro Asp Pro Ser 270 275 280 ACA GCC AGC TAC CCT GTA ATC AGT GTA GCT ATG TCC TCG GAC GAG 1325 Thr Ala Ser Tyr Pro Val Ile Ser Val Ala Met Ser Ser Asp Glu 285 290 295 GGG TGG CGC GCG GTC TAT GTG CTG GCG CGC CCG GGC GTG CCT ATG 1370 Gly Trp Arg Ala Val Tyr Val Leu Ala Arg Pro Gly Val Pro Met 300 305 310 AAT CCC CCG CGT GGC CCA TTA CCC GAG AAT CTA CAC GTA GAG ATA 1415 Asn Pro Pro Arg Gly Pro Leu Pro Glu Asn Leu His Val Glu Ile 315 320 325 TTC GAC GAT GAG CGT GCA CTC ATA TTG GAG GCG TTC CGG CTT ATA 1460 Phe Asp Asp Glu Arg Ala Leu Ile Leu Glu Ala Phe Arg Leu Ile 330 335 340 TCA AAC TAC CCG GTG CTG CTC ACC TTC AAC GGT GAT AAC TTT GAC 1505 Ser Asn Tyr Pro Val Leu Leu Thr Phe Asn Gly Asp Asn Phe Asp 345 350 355 CTC CCC TAC CTC TAC AAC CGG GCA GTA AAA CTA GGC ATA CCA CGC 1550 Leu Pro Tyr Leu Tyr Asn Arg Ala Val Lys Leu Gly Ile Pro Arg 360 365 370 GAG TAC ATA CCA TTC CGT GCT AGA AGC GAC TAT GTG ACA TTG GAG 1595 Glu Tyr Ile Pro Phe Ar g Ala Arg Ser Asp Tyr Val Thr Leu Glu 375 380 385 TAC GGC TTC CAT ATA GAC CTC TAT AAG TTC TTC AGC ACC AAG GCG 1640 Tyr Gly Phe His Ile Asp Leu Tyr Lys Phe Phe Ser Thr Lys Ala 390 395 400 GTT CAG GCA TAT GCC TTC GGC AAC GCT TAC CAG GAG TTC ACC CTT 1685 Val Gln Ala Tyr Ala Phe Gly Asn Ala Tyr Gln Glu Phe Thr Leu 405 410 415 GAT GCT ATA GCC TCT GCG TTG CTG GGG GAG CAC AAG GTG GAG GTC 1730 Asp Ala Ile Ala Ser Ala Leu Leu Gly Glu His Lys Val Glu Val 420 425 430 GAG TCT ACT GTA AGC GAC CTA CCA TTC TTT GAG CTG GTC AGG TAT 1775 Glu Ser Thr Val Ser Asp Leu Pro Phe Phe Glu Leu Val Arg Tyr 435 440 445 AAT GTG CGT GAC GCT GAT CTA ACC CTT AGG CTA ACA ACG TTC AAC 1820 Asn Val Arg Asp Ala Asp Leu Thr Leu Arg Leu Thr Thr Phe Asn 450 455 460 AAC GAC CTG GTA TGG TCC CTT ATC ATA CTG CTA ATG CGT ATC TCC 1865 Asn Asp Leu Val Trp Ser Leu Ile Ile Leu Leu Met Arg Ile Ser 465 470 475 AAG CTG CCT CTG GAG GAT GTC ACG AGA AGC CAG GTC TCA GCT TGG 1910 Lys Leu Pro Leu Glu Asp Val Thr Arg Ser Gln Val Ser Ala Trp 480 485 490 GTG AAG AGC TTA TTC TAC TGG GAG CAT AGG AGG AGG GGC TAC CTA 1955 Val Lys Ser Leu Phe Tyr Trp Glu His Arg Arg Arg Gly Tyr Leu 495 500 505 ATA CCA TCA AGG GAG GAG ATA ATA CGG CTT AAG GGC ACC ACC CGC 2000 Ile Pro Ser Arg Glu Glu Ile Ile Arg Leu Lys Gly Thr Thr Arg 510 515 520 TCT GAA GCC CTG ATA AAG GGT AAG AAG TAT CAG GGG GCG CTA GTC 2045 Ser Glu Ala Leu Ile Lys Gly Lys Lys Tyr Gln Gly Ala Leu Val 525 530 535 CTT GAC CCG CCT AGC GGC ATA TAC TTC AAC ATA GTG GTG CTT GAC 2090 Leu Asp Pro Pro Ser Gly Ile Tyr Phe Asn Ile Val Val Leu Asp 540 545 550 TTC GCC AGC CTG TAC CCC AGT ATA ATA AAG AGG TGG AAC CTA AGC 2135 Phe Ala Ser Leu Tyr Pro Ser Ile Ile Lys Arg Trp Asn Leu Ser 555 560 565 TAC GAG ACC GTA AAC CCC GTA TAC TGC CCC GAA TCG AAG CTA GTG 2180 Tyr Glu Thr Val Asn Pro Val Tyr Cys Pro Glu Ser Lys Leu Val 570 575 580 GAG GTT CCC GAT GTA GGG CAT AAG GTG TGC ATG AGC ATA CCC GGC 2225 Glu Val Pro Asp Val Gly His Lys Val Cys Met Ser Ile Pro Gly 585 590 595 CTG ACC TCG CAG ATA GTT GGC CTG CTT AGG GAC TAT CGA GTC AAG 2270 Leu Thr Ser Gln Ile Val Gly Leu Leu Arg Asp Tyr Arg Val Lys 600 605 610 ATA TAC AAG AAG AAG GCC AAG GAT AAG AGT CTG CCG GAT GAT GTT 2315 Ile Tyr Lys Lys Lys Ala Lys Asp Lys Ser Leu Pro Asp Asp Val 615 620 625 AGA GCA TGG TAT AAT ACA GTC CAG GCA GCC ATG AAG GTG TAT ATA 2360 Arg Ala Trp Tyr Asn Thr Val Gln Ala Ala Met Lys Val Tyr Ile 630 635 640 AAT GCC AGC TAT GGA GTC TTC GGG GCC GAG AGC TTC CCG TTC TAC 2405 Asn Ala Ser Tyr Gly Val Phe Gly Ala Glu Ser Phe Pro Phe Tyr 645 650 655 GCG CCG CCG GTA GCG GAG AGC GTC ACA GCC ATA GGC AGG TAT ACT 2450 Ala Pro Pro Val Ala Glu Ser Val Thr Ala Ile Gly Arg Tyr Thr 660 665 670 ATC AAG CAG ACG CTG CAG AAG GCT GGC GAA CTA GGG CTC CGC GTG 2495 Ile Lys Gln Thr Leu Gln Lys Ala Gly Glu Leu Gly Leu Arg Val 675 680 685 CTC TAT GGC GAT ACG GAC TCA CTA TTC ATA TGG AAT CCA GAT GAG 2540 Leu Tyr Gly Asp Thr Asp Ser Leu Phe Ile Trp Asn Pro Asp Glu 690 695 700 GAT AAG CTG CGG GAG CTG CAA GAG TAT GTA GAG AAG AAC TTT GGC 2585 Asp Lys Leu Arg Glu Leu Gln Glu Tyr Val Glu Lys Asn Phe Gly 705 710 715 CTA GAC CTT GAG GTT GAT AAG GTC TAT AAA TTC GTG ACA TTT AGC 2630 Leu Asp Leu Glu Val Asp Lys Val Tyr Lys Phe Val Thr Phe Ser 720 725 730 GGC CTG AAG AAG AAC TAT ATA GGC GCC TAC GAG GAT GGA AGC ATC 2675 Gly Leu Lys Lys Asn Tyr Ile Gly Ala Tyr Glu Asp Gly Ser Ile 735 740 745 GAT GTC AAG GGT ATG GTC GCT AAG AAG CGT AAT ACG CCG GAG TTC Asp Val Lys Gly Met Val Ala Lys Lys Arg Asn Thr Pro Glu Phe 750 755 760 CTC AAG AAG GAG TTT AGC GAG ATG CTA GCA GTT ATA GGC TCT GTT 2765 Leu Lys Lys Glu Phe Ser Glu Met Leu Ala Val Ile Gly Ser Val 765 770 775 AAG AGC CCT GAG GAC TTC ATA AAG GTG AGG AGA GTT ATA CGT GAA 2810 Lys Ser Pro Glu Asp Phe Ile Lys Val Arg Arg Val Ile Arg Glu 780 785 790 AGG CTG CGC AAA GTA TAC CAT GGC CTG CGC GAC CTG GAG TTC AAC 2855 Arg Leu Arg Lys Val Tyr His Gly Leu Arg Asp Leu Glu Phe Asn 795 800 805 TTA GAC GAG CTA GCC ATA AGG ATG GCT TTA AAC AAG CCC GTT GAG 2900 Leu Asp Glu Leu Ala Ile Arg Met Ala Leu Asn Lys P ro Val Glu 810 815 820 GCC TAT ACC AAG AAT ACG CCC CAG CAT GTG AAG GCT GCG CGG CAG 2945 Ala Tyr Thr Lys Asn Thr Pro Gln His Val Lys Ala Ala Arg Gln 825 830 835 CTC ATA AGG GCG GGG GTG CAG GTG CTG CCA GGT GAT GTC ATA TCC 2990 Leu Ile Arg Ala Gly Val Gln Val Leu Pro Gly Asp Val Ile Ser 840 845 850 TTC GTT AAA GTG AAG GGC AAG GAG GGT GTT AAG CCG GTC CAA CTC 3035 Phe Val Lys Val Lys Gly Lys Glu Gly Val Lys Pro Val Gln Leu 855 860 865 GCA AGA CTG CCG GAG GTT GAT GTA GAG AAG TAT GTG GAG AGC ATG 3080 Ala Arg Leu Pro Glu Val Asp Val Glu Lys Tyr Val Glu Ser Met 870 875 880 AGG AAT GTG TTT GAA CAG CTA CTG CTT GCA ATA AGT ATG TCG TGG 3125 Arg Asn Val Phe Glu Gln Leu Leu Leu Ala Ile Ser Met Ser Trp 885 890 895 GAT GAG ATA ATA GGC TCC TCG AGG CTT GAG GCC TTC TTT AGC CGC 3170 Asp Glu Ile Ile Gly Ser Ser Arg Leu Glu Ala Phe Phe Ser Arg 900 905 910 CGG GGC TAGCTTGAAG AAAGCTATCT TTTCCGGCTT CTACGCCTCT TCTTAGGCCT 3226 Arg Gly CTCCTCTAGC TCTTCCAAGC CTTCCTCGAG TCGCTTGATC TCCTCTTCGC CAAGCGATAG 3286 CTGTGCCT CC CCGCCCATCT TCTTGGGCTC TTTTGTTGCC TCTATGGCGT ACTCCTCTAG 3346 CTGTCGTTTT GCCGCCTCGT CAAGGTAGTA GGATATCACA TACATGCGTC TGTCAAGGTA 3406 TGCGTCCTCG AGCCATGCAT TATCGAAGCT T 3437

SEQ ID NO: 8 Sequence length: 3068 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear Sequence type: Genomic DNA Origin: Organism name: Pyrodictium occultum (Pyrodicti)
um occultum) strain name: DSM2709 ^T sequence features: SEQ: CTGCAGCTCT CGGGGCTACA GCTCCTTCGT GCTCGAGGTG GAGGCCGGCA ACTATCCAGC 60 GCAGAGCCTA TATGCGAGAA GCTCCTTCAA GCCCGTCATG ATAGTGCCCG ACTACTATGG 120 CGAGGGCCGG CACGCTGTGG TCATGGCGTT GTTGGGGGAG AGGCCCTGCT GCCTAGACGG 180 CTAGCCGTCC TCATGCGTTA GCGGGCAGAG GCAGGCAATG ATATACGATT ATGTAGGGGC 240 GGGTGGTGGT AGATTCTCCA GGGCAGAGCC AGCCC ATG ACA GAG ACT ATA GAG TTC 296 Met Thr Glu Thr Ile Glu Phe 1 5 GTG CTG CTA GAC TCT AGC TAC GAG ATA CTG GGG AAG GAG CCG GTA 341 Val Leu Leu Asp Ser Ser Tyr Glu Ile Leu Gly Lys Glu Pro Val 10 15 20 GTA ATC CTC TGG GGG ATA ACG CTT GAC GGT AAA CGT GTC GTG CTT 386 Val Ile Leu Trp Gly Ile Thr Leu Asp Gly Lys Arg Val Val Leu 25 30 35 CTA GAC CAC CGC TTC CGC CCC TAC TTC TAC GCC CTC ATA GCC CGG 431 Leu Asp His Arg Phe Arg Pro Tyr Phe Tyr Ala Leu Ile Ala Arg 40 45 50 GGC TAT GAG GAT ATG GTG GAG GAG ATA GCA GCT TCC ATA AGG AGG 476 Gly Tyr Glu Asp Met Val Glu Glu Ile Ala Ala Ser Ile Arg Arg 55 60 65 CTT AGT GTG GTC AAG AGT CC G ATA ATA GAT GCC AAG CCT CTT GAT 521 Leu Ser Val Val Lys Ser Pro Ile Ile Asp Ala Lys Pro Leu Asp 70 75 80 AAG AGG TAC TTC GGC AGG CCC CGT AAG GCG GTG AAG ATT ACC ACT 566 Lys Arg Tyr Phe Gly Arg Pro Arg Lys Ala Val Lys Ile Thr Thr 85 90 95 ATG ATA CCC GAG TCT GTT AGA CAC TAC CGC GAG GCG GTG AAG AAG 611 Met Ile Pro Glu Ser Val Arg His Tyr Arg Glu Ala Val Lys Lys 100 105 110 ATA GAG GGT GTG GAG GAC TCC CTC GAG GCA GAT ATA AGG TTT GCA 656 Ile Glu Gly Val Glu Asp Ser Leu Glu Ala Asp Ile Arg Phe Ala 115 120 125 ATG AGA TAT CTG ATA GAT AAG AGG CTC TAC CCG TTC ACG GTT TAC 701 Met Arg Tyr Leu Ile Asp Lys Arg Leu Tyr Pro Phe Thr Val Tyr 130 135 140 CGG ATC CCC GTA GAG GAT GCG GGC CGC AAT CCA GGC TTC CGT GTT 746 Arg Ile Pro Val Glu Asp Ala Gly Arg Asn Pro Gly Phe Arg Val 145 150 155 GAC CGT GTC TAC AAG GTT GCT GGC GAC CCG GAG CCC CTA GCG GAT 791 Asp Arg Val Tyr Lys Val Ala Gly Asp Pro Glu Pro Leu Ala Asp 160 165 170 ATA ACG CGG ATC GAC CTT CCC CCG ATG AGG CTG GTA GCT TTT GAT 836 Ile Thr Arg Ile Asp Leu Pro Pro Met Arg Leu Val Ala Phe Asp 175 180 185 ATA GAG GTG TAT AGC AGG AGG GGG AGC CCT AAC CCT GCA AGG GAT 881 Ile Glu Val Tyr Ser Arg Arg Gly Ser Pro Asn Pro Ala Arg Asp 190 195 200 CCA GTG ATA ATA GTG TCG CTG AGG GAC AGC GAG GGC AAG GAG AGG 926 Pro Val Ile Ile Val Ser Leu Arg Asp Ser Glu Gly Lys Glu Arg 205 210 215 CTC ATA GAA GCT GAA GGC CAT GAC GAC AGG AGG GTT CTG AGG GAG 971 Leu Ile Glu Ala Glu Gly His Asp Asp Arg Arg Val Leu Arg Glu 220 225 230 TTC GTA GAG TAC GTG AGA GCC TTC GAC CCC GAC ATA ATA GTG GGC 1016 Phe Val Glu Tyr Val Arg Ala Phe Asp Pro Asp Ile Ile Val Gly 235 240 245 TAT AAC AGT AAC CAC TTC GAC TGG CCC TAC CTA ATG GAG CGC GCC 1061 Tyr Asn Ser Asn His Phe Asp Trp Pro Tyr Leu Met Glu Arg Ala 250 255 260 CGT AGG CTC GGG ATT AAG CTC GAC GTT ACA CGC CGT GTG GGG GCA 1106 Arg Arg Leu Gly Ile Lys Leu Asp Val Thr Arg Arg Val Gly Ala 265 270 275 GAG CCC ACC ACC AGC GTC TAC GGC CAC GTC TCG GTG CAG GGT AGG 1151 Glu Pro Thr Thr Ser Val Tyr Gly His Val Ser Val Gln Gly Arg 280 285 290 CTG AAC GTG GAC CTC TAC GAC TAT GCC GAG GAG ATG CCG GAG ATA 1196 Leu Asn Val Asp Leu Tyr Asp Tyr Ala Glu Glu Met Pro Glu Ile 295 300 305 AAG ATG AAG ACG CTT GAG GAG GTA GCG GAG TAC CTA GGC GTT ATG 1241 Lys Met Lys Thr Leu Glu Glu Val Ala Glu Tyr Leu Gly Val Met 310 315 320 AAG AAG AGC GAG CGT GTG ATA ATA GAG TGG TGG AGG ATA CCC GAG 1286 Lys Lys Ser Glu Arg Val Ile Ile Glu Trp Trp Arg Ile Pro Glu 325 330 335 TAC TGG GAT GAC GAG AAG AAG AGG CAG CTG CTA GAG CGC TAC GCG 1331 Tyr Trp Asp Asp Glu Lys Lys Arg Gln Leu Leu Glu Arg Tyr Ala 340 345 350 CTC GAC GAT GTG AGG GCT ACC TAC GGC CTC GCG GAA AAG ATG CTA 1376 Leu Asp Asp Val Arg Ala Thr Tyr Gly Leu Ala Glu Lys Met Leu 355 360 365 CCG TTC GCC ATA CAG CTC TCC ACT GTT ACG GGT GTG CCT CTC GAC 1421 Pro Phe Ala Ile Gln Leu Ser Thr Val Thr Gly Val Pro Leu Asp 370 375 380 CAG GTA GGT GCT ATG GGC GTA GGC TTC CGC CTA GAG TGG TAT CTC 1466 Gln Val Gly Ala Met Gly Val Gly Phe Arg Leu Glu Trp Tyr Leu 385 390 395 ATG CGT GCA GCC TAC GAT ATG A AC GAG CTG GTG CCG AAC CGG GTG 1511 Met Arg Ala Ala Tyr Asp Met Asn Glu Leu Val Pro Asn Arg Val 400 405 410 GAG AGG AGG GGG GAG AGC TAC AAG GGT GCA GTA GTG TTA AAG CCT 1556 Glu Arg Arg Gly Glu Ser Tyr Lys Gly Ala Val Val Leu Lys Pro 415 420 425 CTC AAG GGA GTC CAT GAG AAT GTT GTG GTG CTC GAT TTC AGT TCC 1601 Leu Lys Gly Val His Glu Asn Val Val Val Leu Asp Phe Ser Ser 430 435 440 ATG TAC CCG AGC ATA ATG ATA AAG TAC AAC GTG GGC CCC GAC ACT 1646 Met Tyr Pro Ser Ile Met Ile Lys Tyr Asn Val Gly Pro Asp Thr 445 450 455 ATA GTC GAC GAC CCC TCG GAG TGC CCA AAG TAC GGC GGC TGC TAT 1691 Ile Val Asp Asp Pro Ser Glu Cys Pro Lys Tyr Gly Gly Cys Tyr 460 465 470 GTA GCC CCC GAG GTC GGG CAC CGG TTC CGT CGC TCC CCG CCA GGC 1736 Val Ala Pro Glu Val Gly His Arg Phe Arg Arg Ser Pro Pro Gly 475 480 485 TTC TTC AAG ACC GTG CTC GAG AAC CTA CTG AAG CTA CGC CGA CAG 1781 Phe Phe Lys Thr Val Leu Glu Asn Leu Leu Lys Leu Arg Arg Gln 490 495 500 GTA AAG GAG AAG ATG AAG GAG TTT CCG CCT GAC AGC CCC GAG TAC 1826 Val L ys Glu Lys Met Lys Glu Phe Pro Pro Asp Ser Pro Glu Tyr 505 510 515 AGG CTC TAC GAT GAG CGC CAG AAG GCG CTC AAG GTT CTT GCG AAC 1871 Arg Leu Tyr Asp Glu Arg Gln Lys Ala Leu Lys Val Leu Ala Asn 520 525 530 GCG AGC TAT GGC TAC ATG GGG TGG AGC CAT GCC CGC TGG TAC TGC 1916 Ala Ser Tyr Gly Tyr Met Gly Trp Ser His Ala Arg Trp Tyr Cys 535 540 545 AAA CGC TGC GCC GAG GCT GTC ACA GCC TGG GGC CGT AAC CTT ATA 1961 Lys Arg Cys Ala Glu Ala Val Thr Ala Trp Gly Arg Asn Leu Ile 550 555 560 CTG ACA GCT ATC GAG TAT GCC AGG AAG CTC GGC CTA AAG GTT ATA 2006 Leu Thr Ala Ile Glu Tyr Ala Arg Lys Leu Gly Leu Lys Val Ile 565 570 575 TAT GGA GAC ACC GAC TCC CTC TTC GTG GTC TAT GAC AAG GAG AAG 2051 Tyr Gly Asp Thr Asp Ser Leu Phe Val Val Tyr Asp Lys Glu Lys 580 585 590 GTT GAG AAG CTG ATA GAG TTT GTC GAG AAG GAG CTG GGC TTT GAG 2096 Val Glu Lys Leu Ile Glu Phe Val Glu Lys Glu Leu Gly Phe Glu 595 600 605 ATA AAG ATA GAC AAG ATC TAC AAG AAA GTG TTC TTC ACG GAG GCT 2141 Ile Lys Ile Asp Lys Ile Tyr Lys Lys Val Phe Ph e Thr Glu Ala 610 615 620 AAG AAG CGC TAT GTA GGT CTC CTC GAG GAC GGA CGT ATA GAC ATC 2186 Lys Lys Arg Tyr Val Gly Leu Leu Glu Asp Gly Arg Ile Asp Ile 625 630 635 GTG GGC TTT GAA GCA GTC CGC GGC GAC TGG TGC GAG CTG GCT AAG 2231 Val Gly Phe Glu Ala Val Arg Gly Asp Trp Cys Glu Leu Ala Lys 640 645 650 GAG GTG CAG GAG AAG GCG GCT GAG ATA GTG TTG AAT ACG GGG AAC 2276 Glu Val Gln Glu Lys Ala Ala Glu Ile Val Leu Asn Thr Gly Asn 655 660 665 GTG GAC AAG GCT ATA AGC TAC ATA AGG GAG GTA ATA AAG CAG CTC 2321 Val Asp Lys Ala Ile Ser Tyr Ile Arg Glu Val Ile Lys Gln Leu 670 675 680 CGC GAG GGC AAG GTG CCA ATA ACA AAG CTT ATC ATA TGG AAG ACG 2366 Arg Glu Gly Lys Val Pro Ile Thr Lys Leu Ile Ile Trp Lys Thr 685 690 695 695 CTG AGC AAG AGG ATA GAG GAG TAC GAG CAT GAC GCG CCT CAT GTG 2411 Leu Ser Lys Arg Ile Glu Glu Tyr Glu His Asp Ala Pro His Val 700 705 710 ATG GCT GCA CGG CGT ATG AAG GAG GCA GGC TAC GAG GTG TCT CCC 2456 Met Ala Ala Arg Arg Met Lys Glu Ala Gly Tyr Glu Val Ser Pro 715 720 725 GGC GAT AAG GTG GGC TAC GTC ATA GTT AAG GGT AGC GGG AGT GTG 2501 Gly Asp Lys Val Gly Tyr Val Ile Val Lys Gly Ser Gly Ser Val 730 735 740 TCC AGC AGG GCC TAC CCC TAC TTC ATG GTT GAT CCA TCG ACC ATC 2546 Ser Ser Arg Ala Tyr Pro Tyr Phe Met Val Asp Pro Ser Thr Ile 745 750 755 GAC GTC AAC TAC TAT ATT GAC CAC CAG ATA GTG CCG GCT GCT CTG 2591 Asp Val Asn Tyr Tyr Ile Asp His Gln Ile Val Pro Ala Ala Leu 760 765 770 AGG ATA CTC TCC TAC TTC GGA GTC ACC GAG AAA CAG CTC AAG GCG 2636 Arg Ile Leu Ser Tyr Phe Gly Val Thr Glu Lys Gln Leu Lys Ala 775 780 785 GCG GCT ACG GTG CAG AGA AGC CTC TTC GAC TTC TTC GCC TCA AAG 2681 Ala Ala Thr Val Gln Arg Ser Leu Phe Asp Phe Phe Ala Ser Lys 790 795 800 AAA TAGCTCCTCC ACCCGGCTAG CTTTATTAAA CGCGTAGGCA CAAGCTCTCC 2734 Lys GAGAGGCCTG GAGGGTAAGG GGTGCAATAG AGCCAGCCTC TCCGCCGAGG CCGTGCGCTC 2794 TTGGGTGGCT TGGAATGATC CTCGCATCCT GGAGATCCTT GGCGTGGATA GTAAGGCGTG 2854 TCGACGTAGT ACTCGAGGTT GTCGATGCGC GCGACCCGGT CTCGACAAGG AGCCTGCGGC 2914 TAGAGAGGAT GGTGCAGAGC CTAGGGAAGC GCCTCCTAAT AGTCA TCAAT AAGGCTGACC 2974 TGGTGCCCCG CGGGGTCGCT GAGAAGTGGA AGCGCATCCT CGAGGATCAG GGTTACCGTA 3034 CTGTCTACAT GGCTGCCCGC GATCACAAGG GTAC 3068

SEQ ID NO: 9 Array length: 23 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GTACATATTG TCGTTAGAAC GCG 23

SEQ ID NO: 10 Array length: 23 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: TAATACGACT CACTATAGGG AGA 23

SEQ ID NO: 11 Array length: 18 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GGGGTCGTCG ACTATAGT 18

SEQ ID NO: 12 Array length: 18 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: ATACTGACAG CTATCGAG 18

SEQ ID NO: 13 Array length: 18 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GTATACGGGG TTTACGGT 18

SEQ ID NO: 14 Array length: 18 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: ATCAAGCAGA CGCTGCAG 18

SEQ ID NO: 15 Array length: 30 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: ATCTCACTGG CGCCATGGAG GCTCAGCCGC 30

SEQ ID NO: 16 Array length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: TGCATGGAGC ACCATGGTGC CTCAG 25

[Brief description of drawings]

FIG. 1 Approximately 3.1k inserted into pPO500-II
It is a figure which shows the restriction enzyme map of the DNA fragment of b, and the position of the primer used for PCR.

FIG. 2 Approximately 4.2k inserted into pPO100-I
It is a figure which shows the restriction enzyme map of the DNA fragment of b, and the position of the primer used for PCR.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:01) (56) Reference literature JP-A-6-343466 (JP, A) Abstracts of the General Meeting of the American Society for. Microbiology, 1993, Vol. 93, No. 0, pp. 197 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/09 C12N 9/00-9/99 JISST file (JOIS) EUROPAT (QUESTEL) BIOSIS / MEDLINE / WPIDS (STN) ) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq

Claims (4)

(57) [Claims] 1. An amino acid sequence represented by SEQ ID NO: 1 in Sequence Listing, or a part thereof , and SEQ ID NO: in Sequence Listing
Amino acid sequence represented by amino acid numbers 36 to 914 of 1
A DNA polymerase gene encoding an amino acid sequence containing 2. A DNA polymerase gene capable of hybridizing to the DNA polymerase gene according to claim 1 under strict conditions. 3. A transformant carrying a plasmid containing the DNA polymerase gene according to claim 1 or 2 is cultured, and the DNA according to claim 1 or 2 is obtained from the culture.
A method for producing a DNA polymerase, which comprises collecting a DNA polymerase encoded by a polymerase gene. 4. Amino acid represented by SEQ ID NO: 1 in Sequence Listing
Sequence or a part of it, and the sequence number in the sequence listing
Amino acid sequence represented by amino acid numbers 36 to 914 of 1
A DNA polymerase comprising an amino acid sequence containing