JPH05305000A - Method of cloning pol i type dna polymerase gene - Google Patents

Abstract

PURPOSE:To provide a method of cloning the POL type DNA polymerase gene (hereinafter abbreviated to 'P gene') and a novel P gene. CONSTITUTION:The method of cloning the P gene comprises (a) the process for amplifying the P gene using the primer represented by sequence No. 1 and No. 2 in the sequence map and (b) the process for cloning the P gene to be hybridized to the amplified DNA as a probe. The P gene which is isolated from plasmid pUI1o1 or 205 or can be hybridized to the gene. The biotechnological production of DNA polymerase becomes possible using the P gene which is useful as a reagent for research in the biotechnology.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for cloning a gene encoding a pol I type DNA polymerase useful as a reagent for genetic engineering research, and a novel cloned pol I type DNA polymerase gene.

[0002]

2. Description of the Related Art DNA polymerases can be broadly classified based on their amino acid sequences into a pol I type DNA polymerase represented by Escherichia coli DNA polymerase and an α type DNA polymerase represented by human DNA polymerase α. Escherichia coli DNA has been used as a DNA polymerase that has been generally used as a reagent for genetic engineering research.
Polymerase I, its modified Klenow fragment, T4
Phage-derived DNA polymerase, T7 phage-derived D
NA Polymerase, Thermus aquaticus (Thermus
aquaticus-derived thermostable DNA polymerase [Taq polymerase] and the like, but most of them are Pol I
Type DNA polymerase. These enzymes are used for labeling specific DNAs, DNA nucleotide sequencing methods, etc. depending on the properties of the enzymes.

[0003]

Generally, a DNA polymerase has an enzyme specificity due to its origin, and there is a method of utilizing the characteristics. For example, Bacillus caldotenax is a thermophilic bacterium with an optimum growth temperature of about 70 ° C.
Type DNA polymerase is expected to be stable at high temperatures, and is expected to be useful as a reagent for genetic engineering research. However, the details of the DNA polymerase have not been clarified so far, and the production method thereof has not been established. In addition, the gene structure and amino acid sequence of this enzyme are unknown, and the isolation of the gene and the method of binding the gene to a vector to express it by genetic engineering have not been clarified. An object of the present invention is to provide a method for identifying a novel pol I type DNA polymerase gene, conveniently and efficiently cloning the gene, and a novel pol I type DNA polymerase gene isolated by the method. ..

[0004]

Means for Solving the Problems To outline the present invention, the first invention of the present invention relates to a method for cloning a pol I type DNA polymerase gene. Step: (a) Amplifying the Pol I-type DNA polymerase gene using the primers represented by SEQ ID NO: 1 or 2 of the Sequence Listing, (b) Using the amplified DNA as a probe, and hybridizing to the probe And a step of cloning the Pol type I DNA polymerase gene. The second invention of the present invention is the plasmid pUI101 or the plasmid pUI2.
No. 05 pol. Type I DNA polymerase gene. Furthermore, the third invention of the present invention relates to a pol I DNA polymerase gene capable of hybridizing to the pol I DNA polymerase gene of the second invention of the present invention.

The present inventors have proposed a method for efficiently cloning a Pol I type DNA polymerase gene in a short time without analyzing the gene structure or amino acid sequence of the Pol I type DNA polymerase of interest. Type DN
Succeeded in creating an oligonucleotide primer capable of efficiently amplifying the A polymerase gene, and by using the primer, a Pol I type DNA polymerase gene of unknown gene structure can be amplified, and using the amplified gene as a probe The present invention has been completed by discovering that by using it, the target Pol I type DNA polymerase gene can be efficiently and conveniently cloned, and that the Pol I type DNA polymerase can be obtained by genetic engineering using the cloned gene. It was

The present invention will be specifically described below. As a method for selecting a desired DNA fragment, first of all, known Pol I
The amino acid sequences of the type DNA polymerases are compared, and oligodeoxyribonucleotides are synthesized based on the region showing the common amino acid sequence. For the amino acid sequence of Pol I type DNA polymerase, see, for example, Journal of Biological Chemistry.
Chemistry), Vol. 257, pp. 1958-1964 (1982), Journal of Molecular Biology, 16th.
Volume 6, pp. 477-535 (1983), Journal of Biological Chemistry, Volume 264, Volume 6.
427-6437 (1989), and Journal of Biological Chemistry, Vol. 264, Vol.
The amino acid sequences of Escherichia coli DNA polymerase I, T7 phage-derived DNA polymerase, Thermus aquaticus-derived thermostable DNA polymerase, and Streptococcus pneumoniae-derived DNA polymerase described in pages 255 to 4263 (1989) are referred to. be able to.

Sequence Nos. 1 and 2 in the sequence listing show the sequences of the mixed primers prepared by the present inventors based on the common sequence of Pol I type DNA polymerase. The mixed primer shown in SEQ ID NO: 1 is a Pol I type D found by the present inventors.
A common primer of NA polymerase, that is, a mixed primer produced based on the amino acid sequences represented by SEQ ID NOS: 3 to 6 of the sequence listing, and the mixed primer represented by SEQ ID NO: 2 is a consensus sequence of other sites of Pol I type DNA polymerase, That is, it is prepared based on the amino acid sequences represented by SEQ ID NOs: 7 to 10 in the sequence listing. In addition, Pol I which can be used in the present invention
The primer for amplification of the type DNA polymerase gene may be any one as long as it can hybridize to the common sequence of the gene and efficiently amplify the gene, and may be a modified version of the above mix primer. It can be a built one.

[0008] Further, using this primer, Pol I type DN
As a method for amplifying the A polymerase gene, any method capable of efficiently amplifying the gene may be used, and for example, a polymerase chain reaction (PCR) method or an improved method thereof may be used. As the primer pair used in the PCR method, the above-mentioned mixed primer may be used in combination, or the primer may be used in combination with another gene structure, for example, a primer derived from a vector.

[0009] For example, the above-mentioned primer pairs of SEQ ID NOS: 1 and 2 efficiently amplify each of the above-mentioned Pol I type DNA polymerase genes derived from Escherichia coli, T7 phage, Thermus aquaticus, and Streptococcus pneumoniae by PCR. In addition, Bacillus caldolyticus (Bacillus subtilis), Bacillus caldolyticus
), Lactobacillus b
ulgaricus), Lactobacillus homohichii (Lactob)
acillus homohiochii), Lactobacillus heterohiochii), Thermus
Pol I such as Thermus thermophilus
Of microbial origin for which presence of type DNA polymerase and analysis of the DNA polymerase gene structure are still insufficient
A is also amplified, for example, the primer pair is Pol I DN
It can be used as a primer pair for PCR which is useful for cloning the A polymerase gene.

Cloning of pol I type DNA polymerase gene and transformant containing the gene, pol I type D
Acquisition of NA polymerase can be carried out by the steps exemplified below. (1) Chromosomal DNA is extracted from cells having the ability to produce Pol type I DNA polymerase. (2) Using the oligonucleotide primers for DNA polymerase gene amplification shown in SEQ ID NO: 1 and SEQ ID NO: 2 of the sequence listing prepared based on the information of the region common to the Pol I type DNA polymerase, the DNA obtained in (1) is P as a mold
Perform CR. (3) Cleave the DNA obtained in (1) with an appropriate restriction enzyme,
On the other hand, screening is performed using the DNA fragment obtained in (2) as a probe to recover the target DNA fragment. (4) The vector is cleaved with a restriction enzyme, and the DNA fragment obtained in (3) is ligated to this cleavage site. (5) A vector to which a DNA fragment is ligated is introduced into a host, and a transformant containing the target DNA fragment is selected. (6) The plasmid was taken out from the transformant obtained in (5), the DNA fragment of interest was excised, and rearranged into one continuous fragment containing the entire gene of interest as necessary based on the restriction enzyme map, This is ligated to an expression vector in the same manner as in (4). (7) The expression vector containing the target DNA fragment obtained in (6) is introduced into a host in the same manner as in (5) to obtain a transformant. (8) The transformant obtained in (7) is cultured, and DNA polymerase is produced from the cultured cells. (9) If necessary, DN is prepared from the expression type plasmid obtained in (6) by a method for preparing a defective strain using exonuclease III.
A mutant DNA polymerase expression vector in which the 5 '→ 3' exonuclease information portion in the A polymerase production information is deleted is prepared. (10) The expression vector obtained in (9) is introduced into a host to obtain a transformant. (11) The transformant obtained in (10) is cultured, and a mutant DNA polymerase is produced from the cultured cells.

The strain used in the present invention is Pol I type D.
Any strain that produces NA polymerase,
An example is Bacillus cardotenax YT-G strain [preserved strain of Deutsche Sammlung von Mikroorganismen: DSM406].

Hereinafter, the strain will be described as an example. Above D
Bacillus cardo tenax YT-G which is NA sample
The strain (DSM406) -derived DNA is extracted from the cultured cells that have been shake-cultured at 70 ° C. Known methods can be used for extraction, purification, digestion with restriction enzymes, and the details of the method are described in 1982, Cold Spring Harbor Laboratory, T.S. T. Maniastis
Others, Molecular Cloning, Laboratory Manual (Molecular Cloning, A Laboratory Ma
nual), pp. 75-178.

The present inventors used the Bacillus cardotenax DNA as a template and the two kinds of oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing based on a common amino acid sequence as a template to carry out a specific PCR method. Other DNA whose amino acid sequence deduced from amplification of various DNA fragments and deduced from the nucleotide sequence of the obtained fragment is known
It was found to be similar to polymerase. Therefore, the target DNA can be selected by carrying out hybridization using the DNA fragment as a probe. As a method for selection by hybridization, a known method, for example, the method described in the above-mentioned Molecular Cloning, Laboratory Manual, page 309 can be used.

By the Southern hybridization method,
The restriction enzyme fragment of the Bacillus cardotenax DNA on which the DNA polymerase gene of interest is present is analyzed, and then the selected restriction enzyme such as EcoRI or Bam is analyzed.
HI, Hinc II, HindIII, XhoI, PstI, Pv
The Bacillus cardotenax DNA digested with uII or the like is incorporated into a plasmid vector. Known plasmid vectors can be used, for example pUC1.
8, pUC19, pTV118N and the like, but are not limited thereto. A known method can be used as a means for incorporating, 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 the 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.

Based on the results of detailed analysis of the Hinc II fragment, Hind III fragment and Xho I fragment possessed by each of the three selected clones by restriction enzyme digestion, the above 3 fragments were rearranged in vitro and Continuous DNA
And the DNA fragment as an expression vector pTV118N
To obtain the desired clone. PU of the plasmid
It was named I101. Escherichia coli containing the plasmid pUI101 was cultured to obtain a crude extract of bacterial cells. The extract is 6
A sufficient amount of DNA polymerase activity was exhibited even after treatment at 0 ° C. for 20 minutes, and the E. coli crude extract containing only the expression vector did not have such activity. Therefore, there is information on the thermostable DNA polymerase production on pUI101. In addition, it was concluded that the gene carrying the information was expressed in E. coli.

The construction process of the plasmid pUI101 is shown in FIG.
Shown in. DNA polymerase gene is plasmid pUI
The NcoI fragment of 101 is encoded by an NcoI fragment of about 3.5 kb. A restriction enzyme map of the NcoI fragment is shown in FIG. 2 and its nucleotide sequence is shown in SEQ ID NO: 11 of the sequence listing.

The host HB101, which had the highest growth ability in E. coli transformed with pUI101, was selected.
It is displayed as scherichia coli HB101 / pUI101, and the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology is in charge of microscopic research
Deposited as No. 721 (FERM BP-3721).

E. coli harboring pUI101 was cultured,
The thermostable DNA polymerase can be collected from the cultured bacterial cells expressing a large amount of the thermostable DNA polymerase. From the cultured cells, for example, ultrasonic treatment, heat treatment, diethylaminoethyl (DEAE) cellulose column chromatography, and phosphocellulose column chromatography are performed, and the DNA polymerase is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It can be purified to show a single band.

The DNA polymerase obtained was SDS-P.
It is a polypeptide showing a molecular weight of about 100,000 daltons by AGE, and has 5 ′ → 3 ′ exonuclease activity in addition to DNA synthesis activity, 3 ′ → 5 ′ exonuclease activity. Amino acid analysis of the purified protein can be performed to determine its N-terminal amino acid sequence. The sequence is shown in SEQ ID NO: 12 in the sequence listing. This amino acid sequence,
From the molecular weight and the like, SEQ ID NO: 1 of the sequence listing in the NcoI fragment
The translatable region shown in 3 was determined as the structural gene of the DNA polymerase of the present invention, and the entire amino acid sequence of the DNA polymerase was clarified. The amino acid sequence is shown in SEQ ID NO: 14 of the sequence listing.

From the example of Escherichia coli DNA polymerase I, it is presumed that the 5 '→ 3' exonuclease activity exists in one domain on the amino-terminal side of the polypeptide chain. Therefore, Bacillus cardotenax on pUI101 is suspected. A plasmid in which a certain portion was partially deleted from the DNA fragment derived from the above was prepared, and the plasmid was reintroduced into Escherichia coli to select a clone showing a DNA synthesis activity and having no 5 '→ 3' exonuclease activity. .. A method for constructing a deletion plasmid is known, and Gene, Second
The method of Henicoff described in Vol. 8, pp. 351-359 (1982) can be used.

The selected plasmid was named pUI205, and the transformed E. coli was Escherichia coli H
It is displayed as B101 / pUI205, and is sent to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.
P-3720).

E. coli harboring pUI205 was cultured,
The thermostable DNA polymerase can be collected from the cultured bacterial cells expressing a large amount of the thermostable DNA polymerase. The DNA polymerase can be purified from the cultured cells by, for example, the method described above until a single band is shown by SDS-PAGE.

The DNA polymerase obtained was SDS-P.
It is a polypeptide showing a molecular weight of about 67,000 daltons by AGE and is a mutant DNA polymerase having a DNA synthesis activity, a 3 '→ 5' exonuclease activity, but not a 5 '→ 3' exonuclease activity. .. Amino acid analysis of the purified protein can be performed to determine its N-terminal amino acid sequence. The sequence is shown in SEQ ID NO: 15 of the sequence listing. Of the amino acid sequence shown in SEQ ID NO: 14 of the Sequence Listing, it was confirmed that 284 amino acids from Met of amino acid number 1 to Lys of amino acid number 284 were deleted, and the gene sequence of the mutant DNA polymerase and the entire sequence were confirmed. The amino acid sequence was clarified. The nucleotide sequence of the mutant DNA polymerase is shown in SEQ ID NO: 16 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 17 in the sequence listing.

Recombinant Escherichia coli E obtained as described above
scherichia coli HB101 / pUI101 and Esche
127 units of DNA polymerase and 212 units of DNA polymerase were obtained from 1 ml of the culture solution of richia coli HB101 / pUI205, which enabled industrial production.

As described above in detail, according to the present invention, confirmation of the pol I type DNA polymerase activity of the test substance, which was necessary for the conventional cloning of the pol I type DNA polymerase gene, and production of the polymerase were carried out. , Purification, isolation,
Steps such as determination of partial amino acid sequence and preparation of a probe are unnecessary, and the desired Pol I type DNA polymerase gene can be efficiently and conveniently isolated from the test gene.

[0027]

EXAMPLES Examples of the present invention will be given below, but the present invention is not limited to these examples.

Example 1 Two kinds of oligodeoxyribonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing were prepared by a DNA synthesizer,
Purified. Next, 100 pmol of each of these oligodeoxyribonucleotides was prepared from Escherichia coli, T7 phage, Thermus aquaticus, Thermus thermophilus, Streptococcus pneumoniae, Bacillus subtilis, Bacillus caldriticus, Lactobacillus bulgaricus, Lactobacillus homohiotii, and Lactobacillus heterohiotii. DNA1
Using 100 ng, 95 ℃ 30 seconds, 55 ℃ 1
PCR was performed for 30 minutes at 72 ° C. for 2 minutes. 5 μl
As a result of agarose gel electrophoresis analysis, a DNA fragment of about 600 base pairs was specifically amplified in each case. This DNA fragment was inserted into the SmaI opened M13 phage mp18 vector, and the respective nucleotide sequences were confirmed by the dideoxy method.

Example 2 (1) Preparation of Bacillus cardotenax chromosome DNA Bacillus cardotenax YT-G strain was added to 125 ml of L.
Medium (Bactotryptone 10 g / l, Yeast Extract 5 g /
l, 5 g / l of NaCl, pH 7.2) and shake-cultured at 65 ° C. overnight, and the collected cells were 4 ml of 25% sucrose, 0.1%.
The suspension was suspended in 05M Tris-HCl (pH 8.0), 800 μl of lysozyme (5 mg / ml) was added, and the mixture was allowed to stand at 20 ° C. for 1 hour. SET solution [20 mM Tris-HCl (pH
8.0), 1 mM EDTA, 150 mM NaCl] 2
After adding 4 ml, 4 ml of 5% SDS solution and 400 μl of proteinase K (10 mg / ml) were added and left at 37 ° C. for 1 hour. After phenol extraction and chloroform extraction, ethanol was added to insolubilize the long chain DNA, and the long chain DNA was wound with a sterilized toothpick and collected. By the above operation, 3.1 mg of DNA was obtained.

(2) Amplification of specific DNA fragment using PCR 100 pmol of each of the two kinds of oligodeoxyribonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing and 1 ng of Bacillus cardotenax DNA were used to make a total amount of 1
P for 00 μl 95 ° C 30 seconds, 55 ° C 1 minute, 72 ° C 2 minutes
CR was performed for 30 cycles. As a result of agarose gel electrophoresis of 5 μl, a DNA fragment of 600 base pairs was specifically amplified. This DNA fragment was incorporated into an M13 phage mp18 vector opened with SmaI, and the nucleotide sequence was analyzed by the dideoxy method.

(3) Search for target gene by genomic Southern method Using 5 μg of Bacillus cardotenax DNA per enzyme, EcoRI, BamHI, HindIII, Hin
It was digested with cII, XhoI, PstI and PvuII and subjected to agarose gel electrophoresis. The DNA in the gel was transferred to a nylon membrane and the DNA of 600 base pairs obtained by the above PCR
Hybridization was performed using the fragment as a probe. The probe was radioactively labeled by the random priming method. Hybridization is 6 x SSC, 1% S
DS, 5 x Den Hearts solution, 100 μg / ml calf thymus D
It was carried out at 65 ° C. for 5 hours in NA. 1 x SSC, 0.1% S
After washing in the DS solution for 1 hour, it was exposed to an X-ray film and an autoradiogram was taken.

(4) D containing a DNA polymerase gene
Cloning of NA fragment HindIII (2.4) which was positive in genomic Southern analysis
0 kb), Hinc II (1.45 kb) and XhoI (2.1
kb) To clone the DNA fragment into a plasmid vector, 100 μg of each Bacillus cardotenax DNA was digested with HindIII, HincII, and XhoI, and the DNA of the desired size was recovered from an agarose gel. The glass beads adsorption method was used for recovery. pTV118
N plasmids were each opened with the same enzyme, and a vector from which the terminal phosphate group had been removed by alkaline phosphatase was added and ligated with DNA ligase.
09 introduced. A target clone was selected by colony hybridization from the obtained recombinant population. Recombinant colony 5 grown on nylon membrane
0 to 200 0.5N sodium hydroxide, 1.5
After denaturing with M sodium chloride solution, it was neutralized with 1 M Tris-HCl, 1.5 M sodium chloride (pH 7.0) solution, and phage DNA was immobilized on the membrane by UV irradiation. The conditions for probe preparation and hybridization were in accordance with genomic Southern analysis.

(5) Restriction enzyme analysis on cloned fragments and rearrangement of DNA polymerase gene A restriction enzyme map was prepared for the three kinds of obtained DNA fragments. As a result, the above three fragments were Hinc II-HindIII-Xho.
It was found that they overlap each other in the order of I and continuously exist on the chromosomal DNA. Restriction enzyme cleavage sites were selected so that unnecessary ligation did not occur, and three types of DNA fragments and the vector pTV118 were selected as shown in FIG.
N was ligated at the same time to construct a plasmid incorporating a DNA fragment of about 3.5 kb containing a DNA polymerase gene, which was named pUI101. Note that FIG. 1 shows pUI10.
FIG. 3 is a process drawing showing the construction of No. 1; In addition, FIG.
FIG. 2 is a view showing a restriction enzyme map of an NcoI DNA fragment containing the DNA polymerase gene cloned in 1. Next, Escherichia coli HB101 strain was transformed with the plasmid to obtain transformant Escherichia coli HB101 / pU.
I101 (FERM BP-3721) was obtained.

(6) Cultivation of transformants and preparation of crude extract E. coli HB containing the above recombinant plasmid pUI101
101 (FERM BP-3721) was inoculated into 5 ml of L medium containing ampicillin at a concentration of 100 μg / ml, and 3
Cultured at 7 ° C. When the turbidity of the culture solution was 0.6 (A ₆₀₀ ), isopropyl-β-D-thiogalactoside (IPTG), which was an inducer, was added, and the culture was further performed for 15 hours. The cells were collected from 1 ml of the culture medium and 50 mM Tris-HCl.
(PH 8.0), washed with 25% sucrose solution. After re-dissolving in the same solution, the same amount of lysis solution [50 mM Tris-HCl (pH 7.5), 25% sucrose, 60 mM spermidine monohydrochloride, 20 mM sodium chloride, 12 mM
DTT] was added and the mixture was allowed to stand at 4 ° C. for 45 minutes. 5% more
(W / v) 20 Triton X100
μl was added, and the mixture was allowed to stand at 37 ° C. for 5 minutes, then, the supernatant was recovered by centrifugation, and allowed to stand at 60 ° C. for 20 minutes, and then centrifuged again, and the supernatant was recovered as a crude extract.

(7) Measurement of DNA polymerase activity As a reaction solution, 67 mM potassium phosphate (pH 7.
4), 6.7 mM magnesium chloride, 1 mM 2-mercaptoethanol, 20 μM activated DNA, 33 μM
dATP, dCTP, dGTP, TTP, 60 nM
[ ³ H] TTP was prepared, 150 μl of this solution was added with an appropriate amount of the crude extract, and the mixture was reacted at 60 ° C. for 5 minutes.
The reaction was stopped by adding 1 ml of 50 mM pyrophosphoric acid and 10% trichloroacetic acid. After allowing to stand in ice for 5 minutes, the whole amount was transferred onto a glass filter and suction-filtered. 10% TC
After washing several times with A, 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 from 1 ml of culture
27 units of DNA polymerase were obtained.

(8) Production of thermostable DNA polymerase by Escherichia coli into which the plasmid pUI101 was introduced. Purification was started from 2.2 g of Escherichia coli HB101 / pUI101, and the method was carried out by the method shown in Example 2- (6).
ml crude extract was obtained. After incubating these at 60 ° C. for 30 minutes, the heat-denatured proteins were centrifuged (12000 rpm, 10 minutes). Ammonium sulfate was added to this supernatant, and the fraction that precipitated at 30-80% saturation was taken and collected in DE buffer [50 mM Tris-HCl pH 7.
0, 2 mM 2-mercaptoethanol, 10% glycerol, 4 μM phenylmethanesulfonyl fluoride (PMSF)], and then added to 15 ml of DE52 (Whatman) column equilibrated with the same buffer to give 0 mM-
Fractionation was performed by elution with a linear gradient of 300 mM NaCl.
The DNA polymerase activity was examined according to Example 2- (7). Next, the active fractions were collected and dialyzed against DE buffer,
P-11 (Whatman) column 1 equilibrated with buffer
Added to 5 ml. Then, elution was performed with a linear gradient of 0 mM to 300 mM NaCl, and the active fractions were collected. When the enzyme preparation of the P11 fraction was analyzed by SDS-PAGE, the molecular weight was about 10
Giving a single band of 10,000 Daltons.

(9) Determination of N-terminal amino acid sequence by amino acid sequencer The enzyme preparation obtained in Example 2- (8) was applied to an amino acid sequencer to determine the N-terminal amino acid sequence shown in SEQ ID NO: 12 in the sequence listing.

Example 3 (1) Preparation of partially deleted plasmid In order to eliminate the 5 ′ → 3 ′ exonuclease activity presumed to exist in the amino terminal domain of the DNA polymerase protein, the 5 ′ side of the gene was deleted. A partially deleted plasmid was created. Exonuclease
In order to use the method using III, an NcoI fragment of about 3.5 kb inserted into pUI101 was first excised, blunt-ended, and then incorporated into the HincII site of pTV118N to prepare a plasmid. Next, double-cut with 3'-protruding enzyme KpnI and 5'-protruding enzyme XbaI in the vector, and let exonuclease III act to cause a decomposition reaction only on the 3'-protruding side. Mung bean (Mung Bean) After blunting with nuclease, it was recircularized with DNA ligase. Deletion mutants of various sizes were obtained by controlling the reaction time of exonuclease III. In addition, by ligating an NcoI linker before recircularization, the initiation codon was surely put in an appropriate position, and it was constructed so that the translation frame fits with a probability of 1/3 depending on the deleted position. The plasmid manipulated as described above was introduced into Escherichia coli, 20 clones were arbitrarily selected from the obtained transformants, a crude extract was prepared, and the thermostable DNA polymerase activity was examined. The nucleotide sequence was analyzed for those having DNA synthesis activity at 60 ° C. One of the 5 active clones was selected, and its recombinant plasmid was named pUI205. The pUI205 contained the DNA fragment of about 2.2 kb shown in FIG. Next, Escherichia coli HB101 was transformed with the plasmid to obtain transformant E.
scherichia coli HB101 / pUI205 (FERM
BP-3720) was obtained. Note that FIG.
FIG. 6 is a diagram showing a restriction enzyme map of the DNA polymerase gene cloned in 5.

(2) Culture of transformant and preparation of crude extract According to the method of Example 2- (6), the above transformant (FER) was used.
MBP-3720) was cultured to prepare a crude extract.

(3) Measurement of DNA polymerase activity The DNA polymerase activity of the crude extract was measured according to the method of Example 2- (7). 212 units of DNA polymerase were obtained from 1 ml of culture.

(4) Measurement of 5 ′ → 3 ′ exonuclease activity A 322 base pair fragment obtained by cleaving plasmid pBR322 with PvuII as a substrate was phosphorylated with [γ- ³² P] ATP and polynucleotide kinase. Was prepared. 67 mM potassium phosphate (pH 7.4), 6.7 mM
The enzyme preparation of Example 3- (2) and the above substrate were mixed in a solution containing magnesium chloride and 1 mM 2-mercaptoethanol, reacted at 60 ° C. for 5 minutes, and ethanol was added to precipitate the substrate DNA. .. The radioactivity present in the supernatant was measured with a liquid scintillation counter to determine the amount of exonuclease-degraded products. No 5 '→ 3' exonuclease activity was observed in the above enzyme preparation.

(5) Production of thermostable DNA polymerase by Escherichia coli introduced with plasmid pUI205 According to the method of Example 2- (8), Escherichia coli HB101 / pU.
An enzyme preparation was obtained from the cells of I205. SD of this enzyme preparation
Analysis by S-PAGE gave a single band with a molecular weight of about 67,000 daltons.

(6) Determination of N-terminal amino acid sequence by amino acid sequencer According to the method of Example 2- (9), SEQ ID NO: 15 in the sequence listing
The N-terminal amino acid sequence shown in was determined.

Example 4 (1) Determination of Nucleotide Sequence of Bacillus cardotenax Chromosome DNA Fragment Containing Structural Gene of DNA Polymerase According to the method shown in Example 3- (1), a set of deletion mutants of various sizes was set. Was prepared, and the nucleotide sequence was decoded by the dideoxy method using them as templates. The obtained data were combined and finally the base sequence of the full length NcoI-NcoI fragment cut out from pUI101 shown in SEQ ID NO: 11 of the sequence listing was determined. In addition, pUI205 has
In the NcoI fragment of pUI101 shown in SEQ ID NO: 11 of the sequence listing, the part of base numbers 1032 to 3252 was inserted. From the N-terminal amino acid sequence determined in Example 2- (9) and Example 3- (6) and the nucleotide sequence determined in this Example, the structural gene and amino acid sequence of the DNA polymerase of the present invention can be clarified. did it.

[0045]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention provides a simple and efficient method for cloning a novel DNA polymerase gene, and the gene. D that is useful as a reagent for genetic engineering research using isolated genes
Also provided is a method for genetically engineering NA polymerase.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: NO Sequence features: 1-23 S primer Sequence: GAYCCHAACY TSCARAAYAT HCC 23 SEQ ID NO: 2 Sequence length: 21 Sequence type: Nucleic acid Strand number: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Anti Sense: YES Sequence characteristics: 1-21 S primer Sequence: KASNAKYTCR TCRTGNACYT G 21 SEQ ID NO: 3 Sequence length: 8 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism name: Escherichia coli SEQ ID NO: 4 Sequence length: 8 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism name: T7 phage SEQ ID NO: 5 Sequence length: 8 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism: Thermus aquaticu
s) SEQ ID NO: 6 Sequence length: 8 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism name: Streptococcus pneumoniae (Streptoco)
ccus pneumoniae) SEQ ID NO: 7 Sequence Length: 7 Sequence Type: Amino Acid Number of Chains: Single Strand Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate Fragment Origin: Organism Name: Escherichia coli SEQ ID NO: 8 Sequence length: 7 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism name: T7 phage SEQ ID NO: 9 Sequence length: 7 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism: Thermus aquaticu
s) SEQ ID NO: 10 Sequence length: 7 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin: Organism name: Streptococcus pneumoniae (Streptoco
ccus pneumoniae) SEQ ID NO: 11 sequence length: 3252 sequence type: nucleic acid number of strands: double-stranded topology: linear sequence type: genomic DNA origin: organism name: Bacillus cardotenax
caldotenax) strain name: YT-G (DSM406) sequence: CCATGGATAT ATTACCGTAG CGAGCAAAGT GGGGCGCGGC ACCGTGTTCA CGATCCATTT 60 TCCAAAGCCG GGGCGGTAGC CGGCTTCTTT TTATCATCTC CAACTGAGAA GCCTGCCATT 120 TTTCAGCGTG ACGTGAGCAC GGGATGAATC CGCGCCTCCC ATCATGTTGG GAGAGCGTTC 180 AAGGCAAGCC GCAGGCATGG TACAATAGGA CAAGGAAGCA TCCGAGGAGG GATGAGA 237 TTG AAA AAA AAG CTT GTT TTA ATC GAC GGC AGC AGC GTG GCG TAC 282 CGC GCC TTT TTC GCC TTG CCG CTT TTG CAT AAC GAC AAA GGC ATC 327 CAT ACG AAC GCC GTC TAC GGG TTT ACG ATG ATG TTG AAT AAA ATT 372 TTG GCG GAA GAA GAG CCA ACT CAT ATG CTT GT GCG TTT GAC GCC 417 GGG AAA ACG ACG TTC CGG CAT GAA GCG TTT CAA GAG TAT AAA GGT 462 GGG CGC CAG CAG ACG CCA CCG GAG CTG TCG GAG CAG TTT CCG CTG 507 TTG CGC GAG CTG CTG AGG GCG TAT CGC ATC CCC GCC TCC GAA CTC 552 GAG AAC TAC GAA GCG GAC GAT ATT ATC GGA ACG CTT GCC GCC CGC 597 GCT GAG CAG GAA GGG TTT GAG GTG AAA GTC ATT TCC GGC GAC CGC 642 GAT CTG ACC CAG CTC GCC TCC CCC CAT GTG ACG GTG GACATT 687 AAA AAA GGG ATT ACC GAT ATC GAA CCG TAC ACG CCG GAG GCG GTC 732 CGC GAA AAA TAC GGC TTA ACT CCG GAA CAA ATC GTT GAT TTG AAA 777 GGA TTG ATG GGC GAC AAA TCG GAC AAC ATT CCC GGA GTG CCG GGC 822 ATC GGG GAA AAG ACG GCG GTC AAG CTG CTC AGG CAA TTC GGC ACG 867 GTC GAA AAC GTG CTT GCC TCC ATT GAC GAG ATC AAA GGC GAA AAG 912 TTG AAA GAA ACG CTG CGC CAA CAC CGG GAG ATG GCG CTG TTA AGC 957 AAA AAG CTC GCC GCC ATT CGC CGC GAC GCC CCG GTC GAG CTC TCG 1002 CTT GAT GAC ATC GCC TAT CAA GGG GAA GAC CGG GAG AAA GTG GTC 1047 GCT TTA TTT AAA GAG CTT GGG TTT CAA TCG TTT TTA GAG AAA ATG 1092 GAA TCG CCG TCA TCA GAA GAG GAA AAA CCG CTT GCC AAG ATG GCA 1137 TTT ACG CTT GCT GAC CGC GTG ACG GAG GAG ATG CTT GCC GAC AAG 1182 GCG GCG CTT GTC GTT GAA GTG GTC GAG GAA AAT TAT CAT GAT GCG 1227 CCG ATC GTC GGC ATC GCT GTG GTC AAC GAA CAT GGA CGG TTT CTC 1272 CTG CCG GAG ACG GCG CTT GCC GAT CCG CAG TTT GTC GCC TGG 1317 CTT GGT GAT GAA ACG AAG AAA AAA AGC ATG TTT GAC TCA AAG CGC 1362 GCG GCA GTC GCC TTG AAA TGG AAA GGA ATT GAG CTA TGC GGC GTT 1407 TCC TT T GAT TTA TTG CTG GCC GCC TAT TTG CTT GAT CCG GCG CAA 1452 GGT GTT GAT GAT GTG GCT GCC GCA GCA AAA ATG AAG CAA TAC GAA 1497 GCG GTG CGC CCG GAT GAA GCG GTG TAT GGC AAA GGG GCG AAG CGG 1542 GCC GTG CCG GAT GAG CCA GTG CTC GCC GAG CAT TTG GTC CGC AAG 1587 GCG GCG GCG ATT TGG GCG CTC GAA CGG CCG TTT TTG GAT GAG CTG 1632 CGC CGC AAC GAA CAA GAT CGG TTG CTC GTC GAG CTC GAG CAG CCG 1677 TTG TCT TCG GCG GAA ATG GAA TTT GCC GGA GTG AAA GTG 1722 GAT ACG AAG CGG CTC GAA CAG ATG GGC GAA GAG CTC GCC GAG CAG 1767 CTG CGC ACG GTC GAG CAG CGC ATT TAT GAG CTC GCC GGC CAA GAA 1812 TTC AAC ATC AAT TCA CCG AAA CAG CTC GGC GTC ATT TTA TTT GAA 1857 AAA CTG CAG CTG CCC GTC TTG AAA AAA AGC AAA ACC GGC TAC TCC 1902 ACT TCG GCG GAT GTG CTT GAA AAA CTT GCG CCT TAT CAC GAG ATC 1947 GTG GAA AAC ATT TTG CAA CAT TAC CGC CAG CTT GGC AAG TTG CAG 1992 TCG ACG TAT ATT GAA GGA TTG CTG AAA GTC GTG CGA CCC GAT ACA 2037 AAG AAG GTG CAT ACG ATT TTC AAT CAG GCG TTG ACG CAA ACC GGA 2082 CGG CTC AGC TCG ACG GAG CCG AAC TTG CAA AAC ATT CCG ATT CGG 2127 CTT GAG GAA GGA CGG AAA ATC CGC CAA GCG TTC GTG CCG TCG GAG 2172 TCT GAT TGG CTC ATT TTC GCT GCC GAC TAC TCG CAA ATT GAG TTG 2217 CGC GTC CTC GCC CAT ATT GCG GAA GAT AAT TTA ATG GAA GCG 2262 TTC CGC CGC GAT TTG GAT ATC CAT ACG AAA ACA GCG ATG GAC ATT 2307 TTC CAA GTG AGC GAG GAC GAA GTG ACG CCC AAC ATG CGC CGT CAG 2352 GCG AAG GCG GTC AAC TTT GGG ATC GTT TAC GGG AGT GAT TAC 2397 GGC TTG GCG CAA AAC TTA AAT ATT TCA CGC AAA GAG GCC GCT GAA 2442 TTC ATC GAG CGC TAC TTC GAA AGC TTC CCT GGC GTG AAG CGG TAT 2487 ATG GAA AAC ATT GTG CAA GAA GCA AAA CAG AAA GGG TAT GTG ACG 2532 ACG CTG CTG CAT CGG CGC CGC TAT TTG CCG GAT ATT ACG AGC CGC 2577 AAC TTC AAC GTC CGC AGC TTT GCT GAA CGG ATG GCG ATG AAC ACG 2622 CCG ATT CAA GGG AGC GCC GCT GAC ATT ATT AAA AAG GCG ATG ATC ATC GAT CTG AAC GCC AGA CTG AAG GAA GAG CGG CTG CAA GCG CGC CTT 2712 TTG CTG CAG GTG CAT GAC GAG CTC ATT TTG GAG GCG CCG AAA GAA 2757 GAG ATG GAG CGG CTG TGC CGG CTC GTT CCG GAA GTG ATG GAG CAA 2802 GCG GTC ACA CTT CGC GTG CCG CTC AAA GTC GAT TAC CAT TAC GGC 2847 TCG ACA TGG TAT GAC GCG AAA TAAAAAGGAG TCTTGGTGTG TGGATCGCCG 2898 GCACCCCTAA AAGGCCGGTG ATTTAAGGGG AAATACTGCT CTCCAACAGT GTTTCTCAAA 2958 TTGAAAAACC TTGCAACACC ATCACTTCAT TCCTTGTGAT TTCTCATAAA TCAAGCGAAT 3018 CCATTGTTTT TCATCAGCCT TCTAAGAAGG CCTGTGATGG AATGAAAAAG CAGTTTCACA 3078 ACGACTCTTC TCCAGTTGAG AAGCCTTGGG ACATCGAGTC GTCCTTCTCA ACCAACATGA 3138 CCGATTTTGC GAAAATCAGC GTTTCTCACC GGCCTTCTAG GCAGAATCTT TCGGTGCGAC 3198 GATTCTCGGC TGCAACTCGG: Single-chain sequence: Single-chain sequence: Long amino acid sequence: One-chain sequence of the linear chain: Amino acid sequence of the linear chain: The number of the linear chain of the linear chain: The number of the linear chain of the linear chain: The number of the linear chain of the linear chain of the linear chain: The number of the linear chain of the linear chain of the linear chain: The number of the linear chain of the linear chain of the linear chain: The number of the linear chain of the linear chain of the linear chain: The number of the linear chain of the linear chain of the linear chain: the number of the linear chain of the linear chain of the ATGAATTGGA: Type: Peptide Fragment type: N-terminal fragment Sequence: Met Lys Lys Lys Leu Val Leu Ile Asp Gly Ser Ser Val Ala Tyr 1 5 10 15 Arg Ala Phe Phe Ala Leu Pro 20 SEQ ID NO: 13 Sequence length: 2631 Sequence Type: Nucleic acid Number of strands: Double strand Topology: Linear sequence type genomic DNA origin: Biological Name: Bacillus stearothermophilus (Bacillus
caldotenax) Share name: YT-G (DSM406) Sequence: TTG AAA AAA AAG CTT GTT TTA ATC GAC GGC AGC AGC GTG GCG TAC 45 CGC GCC TTT TTC GCC TTG CCG CTT TTG CAT AAC GAC AAA GGC ATC 90 CAT ACG AAC GCC GTC TAC GGG TTT ACG ATG ATG TTG AAT AAA ATT 135 TTG GCG GAA GAA GAG CCA ACT CAT ATG CTT GTC GCG TTT GAC GCC 180 GGG AAA ACG ACG TTC CGG CAT GAA GCG TTT CAA GAG TAT AAA GGT 225 GGG CGC CAG CAG ACG CCA CCG GAG CTG TCG GAG CAG TTT CCG CTG 270 TTG CGC GAG CTG CTG AGG GCG TAT CGC ATC CCC GCC TAT GAA CTC 315 GAG AAC TAC GAA GCG GAC GAT ATT ATC GGA ACG CTT GCC GCC CGC 360 GCT GAG CAG GAA GGG TTT GAG GTG AAA GTC ATT TCC GGC GAC CGC 405 GAT CTG ACC CAG CTC GCC TCC CCC CAT GTG ACG GTG GAC ATT ACG 450 AAA AAA GGG ATT ACC GAT ATC GAA CCG TAC ACG CCG GAG GCG GTC 495 CGC GAA AAA TAC GGC TTA ACT CCG GAA CAA ATC GTT GAT TTG AAA 540 GGA TTG ATG GGC GAC AAA TCG GAC AAC ATT CCC GGA GTG CCG GGC 585 ATC GGG GAA AAG ACG GCG GTC AAG CTG CTC AGG CAA TTC GGC ACG 630 GTC GAA AAC GTG CTT GCC TCC ATT GAC GAG ATC AAA GGC GAA AAG 675 TTG AAA GAA ACG CTG CGC CAA CAC CGG GAG AT G GCG CTG TTA AGC 720 AAA AAG CTC GCC GCC ATT CGC CGC GAC GCC CCG GTC GAG CTC TCG 765 CTT GAT GAC ATC GCC TAT CAA GGG GAA GAC CGG GAG AAA GTG GTC 810 GCT TTA TTT AAA GAG CTT GGG TTT CAA TCG TTT TTA TTA GAG AAA ATG 855 GAA TCG CCG TCA TCA GAA GAG GAA AAA CCG CTT GCC AAG ATG GCA 900 TTT ACG CTT GCT GAC CGC GTG ACG GAG GAG ATG CTT GCC GAC AAG 945 GCG GCG CTT GTC GTT GAA GTG GTC GAG GAA AAT TAT CAT GAT GCG 990 CCG ATC GTC GGC ATC GCT GTG GTC AAC GAA CAT GGA CGG TTT TTC 1035 CTG CGC CCG GAG ACG GCG CTT GCC GAT CCG CAG TTT GTC GCC TGG 1080 CTT GGT GAT GAA ACG AAG AAA AAA AGC ATG TTT GAC TCA AAG CGC 1125 GCG GCA GTC GCC TTG AAA TGG AAA GGA ATT GAG CTA TGC GGC GTT 1170 TCC TTT GAT TTA TTG CTG GCC GCC TAT TTG CTT GAT CCG GCG CAA 1215 GGT GTT GAT GAT GTG GCT GCC GCA GCA AAA ATG AAG CAA TAC GAA 1260 GCG GTG CGC CCG GAT GAA GCG GTG TAT GGC AAA GGG GCG AAG CGG 1305 GCC GTG CCG GAT GAG CCA GTG CTC GCC GAG CAT TTG GTC CGC AAG 1350 GCG GCG GCG ATT TGG GCG CTC GAA CGG CCG TTT TTG GAT GAG CTG 1395 CGC CGC AGC AC GAA CAA GAT CGG TTG CTC GTC GAG CTC GAG CAG CCG 1440 TTG TCT TCG ATT TTG GCG GAA ATG GAA TTT GCC GGA GTG AAA GTG 1485 GAT ACG AAG CGG CTC GAA CAG ATG GGC GAA GAG CTC GCC GAG CAG 1530 CTG CGC ACG GT GAG CAG CGC ATT TAT GAG CTC GCC GGC CAA GAA 1575 TTC AAC ATC AAT TCA CCG AAA CAG CTC GGC GTC ATT TTA TTT GAA 1620 AAA CTG CAG CTG CCC GTC TTG AAA AAA AGC AAA ACC GGC TAC TCC 1665 ACT TCG GCG GAT GTG CTT GAA AAA CTT GCG CCT TAT CAC GAG ATC 1710 GTG GAA AAC ATT TTG CAA CAT TAC CGC CAG CTT GGC AAG TTG CAG 1755 TCG ACG TAT ATT GAA GGA TTG CTG AAA GTC GTG CGA CCC GAT ACA 1800 AAG AAG GTG CAT ACG ATT TTC AAT CAG GCG TTG ACG CAA ACC GGA 1845 CGG CTC AGC TCG ACG GAG CCG AAC TTG CAA AAC ATT CCG ATT CGG 1890 CTT GAG GAA GGA CGG AAA ATC CGC CAA GCG TTC GTG CCG TCG GAG 1935 TCT GAT TGG CTC ATT TTC GTAC GCC GCC GCC GCC TCG CAA ATT GAG TTG 1980 CGC GTC CTC GCC CAT ATT GCG GAA GAT GAC AAT TTA ATG GAA GCG 2025 TTC CGC CGC GAT TTG GAT ATC CAT ACG AAA ACA GCG ATG GAC ATT 2070 TTC CAA GTG AGC GAG GAC GAA GTG AC G CCC AAC ATG CGC CGT CAG 2115 GCG AAG GCG GTC AAC TTT GGG ATC GTT TAC GGG ATC AGT GAT TAC 2160 GGC TTG GCG CAA AAC TTA AAT ATT TCA CGC AAA GAG GCC GCT GAA 2205 TTC ATC GAG CGC TAC TTC GAA AGC TTC CCT GGC GTG AAG CGG TAT 2250 ATG GAA AAC ATT GTG CAA GAA GCA AAA CAG AAA GGG TAT GTG ACG 2295 ACG CTG CTG CAT CGG CGC CGC TAT TTG CCG GAT ATT ACG AGC CGC 2340 AAC TTC AAC GTC CGC AGC TTT GCT GAA CGG ATG GCG ATG AAC ACG 2385 CCG ATT CAA GGG AGC GCC GCT GAC ATT ATT AAA AAG GCG ATG ATC 2430 GAT CTG AAC GCC AGA CTG AAG GAA GAG CGG CTG CAA GCG CGC CTT 2475 TTG CTG CAG GTG CAT GAC GAG CTC ATT TTG GAG GCG CCG GAA 2520 GAG ATG GAG CGG CTG TGC CGG CTC GTT CCG GAA GTG ATG GAG CAA 2565 GCG GTC ACA CTT CGC GTG CCG CTC AAA GTC GAT TAC CAT TAC GGC 2610 TCG ACA TGG TAT GAC GCG AAA 2631 SEQ ID NO: 14 Array length: 877 Array type: Number of amino acid chains: Single-stranded topology: Linear array types: Peptide sequence: Met Lys Lys Lys Leu Val Leu Ile Asp Gly Ser Ser Val Ala Tyr 1 5 10 15 Arg Ala Phe Phe Ala Leu Pro Leu Leu His Asn Asp Lys Gly Ile 20 25 30 His Thr Asn Ala Val Tyr Gly Phe Thr Met Met Leu Asn Lys Ile 35 40 45 Leu Ala Glu Glu Glu Pro Thr His Met Leu Val Ala Phe Asp Ala 50 55 60 Gly Lys Thr Thr Phe Arg His Glu Ala Phe Gln Glu Tyr Lys Gly 65 70 75 Gly Arg Gln Gln Thr Pro Pro Glu Leu Ser Glu Gln Phe Pro Leu 80 85 90 Leu Arg Glu Leu Leu Arg Ala Tyr Arg Ile Pro Ala Tyr Glu Leu 95 100 105 Glu Asn Tyr Glu Ala Asp Asp Ile Ile Gly Thr Leu Ala Ala Arg 110 115 120 Ala Glu Gln Glu Gly Phe Glu Val Lys Val Ile Ser Gly Asp Arg 125 130 135 Asp Leu Thr Gln Leu Ala Ser Pro His Val Thr Val Asp Ile Thr 140 145 150 Lys Lys Gly Ile Thr Asp Ile Glu Pro Tyr Thr Pro Glu Ala Val 155 160 165 Arg Glu Lys Tyr Gly Leu Thr Pro Glu Gln Ile Val Asp Leu Lys 170 175 180 Gly Leu Met Gly Asp Lys Ser Asp Asn Ile Pro Gly Val Pro Gly 185 190 195 Ile Gly Glu Lys Thr Ala Val Lys Leu Leu Arg Gln Phe Gly Thr 200 205 210 Val Glu As n Val Leu Ala Ser Ile Asp Glu Ile Lys Gly Glu Lys 215 220 225 Leu Lys Glu Thr Leu Arg Gln His Arg Glu Met Ala Leu Leu Ser 230 235 240 Lys Lys Leu Ala Ala Ile Arg Arg Asp Ala Pro Val Glu Leu Ser 245 250 255 Leu Asp Asp Ile Ala Tyr Gln Gly Glu Asp Arg Glu Lys Val Val 260 265 270 Ala Leu Phe Lys Glu Leu Gly Phe Gln Ser Phe Leu Glu Lys Met 275 280 285 Glu Ser Pro Ser Ser Glu Glu Glu Lys Pro Leu Ala Lys Met Ala 290 295 300 Phe Thr Leu Ala Asp Arg Val Thr Glu Glu Met Leu Ala Asp Lys 305 310 315 Ala Ala Leu Val Val Glu Val Val Glu Glu Asn Tyr His Asp Ala 320 325 330 Pro Ile Val Gly Ile Ala Val Val Asn Glu His Gly Arg Phe Phe 335 340 345 Leu Arg Pro Glu Thr Ala Leu Ala Asp Pro Gln Phe Val Ala Trp 350 355 360 Leu Gly Asp Glu Thr Lys Lys Lys Ser Met Phe Asp Ser Lys Arg 365 370 375 Ala Ala Val Ala Leu Lys Trp Lys Gly Ile Glu Leu Cys Gly Val 380 385 390 Ser Phe Asp Leu Leu Leu Ala Ala Tyr Leu Leu Asp Pro Ala Gln 395 400 405 Gly Val Asp Asp Val Ala Ala Ala Ala Lys Met Lys Gln Tyr Glu 410 415 420 Al a Val Arg Pro Asp Glu Ala Val Tyr Gly Lys Gly Ala Lys Arg 425 430 435 Ala Val Pro Asp Glu Pro Val Leu Ala Glu His Leu Val Arg Lys 440 445 450 Ala Ala Ala Ile Trp Ala Leu Glu Arg Pro Phe Leu Asp Glu Leu 455 460 465 Arg Arg Asn Glu Gln Asp Arg Leu Leu Val Glu Leu Glu Gln Pro 470 475 480 Leu Ser Ser Ile Leu Ala Glu Met Glu Phe Ala Gly Val Lys Val 485 490 495 Asp Thr Lys Arg Leu Glu Gln Met Gly Glu Glu Leu Ala Glu Gln 500 505 510 Leu Arg Thr Val Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln Glu 515 520 525 Phe Asn Ile Asn Ser Pro Lys Gln Leu Gly Val Ile Leu Phe Glu 530 535 540 Lys Leu Gln Leu Pro Val Leu Lys Lys Ser Lys Thr Gly Tyr Ser 545 550 555 Thr Ser Ala Asp Val Leu Glu Lys Leu Ala Pro Tyr His Glu Ile 560 565 570 Val Glu Asn Ile Leu Gln His Tyr Arg Gln Leu Gly Lys Leu Gln 575 580 585 Ser Thr Tyr Ile Glu Gly Leu Leu Lys Val Val Arg Pro Asp Thr 590 595 600 Lys Lys Val His Thr Ile Phe Asn Gln Ala Leu Thr Gln Thr Gly 605 610 615 Arg Leu Ser Ser Thr Glu Pro Asn Leu Gln Asn Ile Pro Ile Arg 620 62 5 630 Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe Val Pro Ser Glu 635 640 645 Ser Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln Ile Glu Leu 650 655 660 Arg Val Leu Ala His Ile Ala Glu Asp Asp Asn Leu Met Glu Ala 665 670 675 Phe Arg Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp Ile 680 685 690 Phe Gln Val Ser Glu Asp Glu Val Thr Pro Asn Met Arg Arg Gln 695 700 705 Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr 710 715 720 Gly Leu Ala Gln Asn Leu Asn Ile Ser Arg Lys Glu Ala Ala Glu 725 730 735 Phe Ile Glu Arg Tyr Phe Glu Ser Phe Pro Gly Val Lys Arg Tyr 740 745 750 Met Glu Asn Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Val Thr 755 760 765 Thr Leu Leu His Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser Arg 770 775 780 Asn Phe Asn Val Arg Ser Phe Ala Glu Arg Met Ala Met Asn Thr 785 790 795 Pro Ile Gln Gly Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Ile 800 805 810 Asp Leu Asn Ala Arg Leu Lys Glu Glu Arg Leu Gln Ala Arg Leu 815 820 825 Leu Leu Gln Val His Asp Glu Leu Ile Leu Glu Ala Pro Lys Gl u 830 835 840 Glu Met Glu Arg Leu Cys Arg Leu Val Pro Glu Val Met Glu Gln 845 850 855 Ala Val Thr Leu Arg Val Pro Leu Lys Val Asp Tyr His Tyr Gly 860 865 870 Ser Thr Trp Tyr Asp Ala Lys 875 SEQ ID NO: : 15 Array length: 10 Array type: Number of amino acid chains: Single-stranded topology: Linear array types: Peptide fragment type: N-terminal fragment SEQ ID NO: 16 sequence length: 1779 sequence type: nucleic acid number of strands: double strand topology: linear sequence type: genomic DNA origin: organism name: Bacillus cardotenax (Bacillus c)
aldotenax) Share name: YT-G (DSM406) Sequence: ATG GAA TCG CCG TCA TCA GAA GAG GAA AAA CCG CTT GCC AAG ATG 45 GCA TTT ACG CTT GCT GAC CGC GTG ACG GAG GAG ATG CTT GCC GAC 90 AAG GCG GCG CTT GTC GTT GAA GTG GTC GAG GAA AAT TAT CAT GAT 135 GCG CCG ATC GTC GGC ATC GCT GTG GTC AAC GAA CAT GGA CGG TTT 180 TTC CTG CGC CCG GAG ACG GCG CTT GCC GAT CCG CAG TTT GTC GCC 225 TGG CTT GGT GAT GAA ACG AAG AAA AAA AGC ATG TTT GAC TCA AAG 270 CGC GCG GCA GTC GCC TTG AAA TGG AAA GGA ATT GAG CTA TGC GGC 315 GTT TCC TTT GAT TTA TTG CTG GCC GCC TAT TTG CTT GAT CCG GCG 360 CAA GGT GTT GAT GAT GTG GCT GCC GCA GCA AAA ATG AAG CAA TAC 405 GAA GCG GTG CGC CCG GAT GAA GCG GTG TAT GGC AAA GGG GCG AAG 450 CGG GCC GTG CCG GAT GAG CCA GTG CTC GCC GAG CAT TTG GTC CGC 495 AAG GCG GCG GCG ATT TGG GCG CTC GAA CGG CCG TTT TTG GAT GAG 540 CTG CGC CGC AAC GAA CAA GAT GG TTG CTC GTC GAG CTC GAG CAG 585 CCG TTG TCT TCG ATT TTG GCG GAA ATG GAA TTT GCC GGA GTG AAA 630 GTG GAT ACG AAG CGG CTC GAA CAG ATG GGC GAA GAG CTC GCC GAG 675 CAG CTG CGC ACG GTC GAG CAG CGC ATT GA G CTC GCC GGC CAA 720 GAA TTC AAC ATC AAT TCA CCG AAA CAG CTC GGC GTC ATT TTA TTT 765 GAA AAA CTG CAG CTG CCC GTC TTG AAA AAA AGC AAA ACC GGC TAC 810 TCC ACT TCG GCG GAT GTG CTT GAA AAA CTT GCG CCT TAT CAC GAG 855 ATC GTG GAA AAC ATT TTG CAA CAT TAC CGC CAG CTT GGC AAG TTG 900 CAG TCG ACG TAT ATT GAA GGA TTG CTG AAA GTC GTG CGA CCC GAT 945 ACA AAG AAG GTG CAT ACG ATT TTC AAT CAG GCG TTG ACG CACG ACC 990 GGA CGG CTC AGC TCG ACG GAG CCG AAC TTG CAA AAC ATT CCG ATT 1035 CGG CTT GAG GAA GGA CGG AAA ATC CGC CAA GCG TTC GTG CCG TCG 1080 GAG TCT GAT TGG CTC ATT TTC GCT GCC GAC TAC TCG CAA ATT GAG 1 TTG CGC GTC CTC GCC CAT ATT GCG GAA GAT GAC AAT TTA ATG GAA 1170 GCG TTC CGC CGC GAT TTG GAT ATC CAT ACG AAA ACA GCG ATG GAC 1215 ATT TTC CAA GTG AGC GAG GAC GAA GTG ACG CCC AAC ATG CGC CGT 1260 CAG AAG GCG GTC AAC TTT GGG ATC GTT TAC GGG ATC AGT GAT 1305 TAC GGC TTG GCG CAA AAC TTA AAT ATT TCA CGC AAA GAG GCC GCT 1350 GAA TTC ATC GAG CGC TAC TTC GAA AGC TTC CCT GGC GTG AAG CGG 1395 TAT ATG G AA AAC ATT GTG CAA GAA GCA AAA CAG AAA GGG TAT GTG 1440 ACG ACG CTG CTG CAT CGG CGC CGC TAT TTG CCG GAT ATT ACG AGC 1485 CGC AAC TTC AAC GTC CGC AGC TTT GCT GAA CGG ATG GCG ATG AAC 1530 ACG CCG ATT CAA GGG AGC GCC GCT GAC ATT ATT AAA AAG GCG ATG 1575 ATC GAT CTG AAC GCC AGA CTG AAG GAA GAG CGG CTG CAA GCG CGC 1620 CTT TTG CTG CAG GTG CAT GAC GAG CTC ATT TTG GAG GCG CCG AAA 1665 GAA GAG ATG GAG CGG CTG AAA TGC CGG CTC GTT CCG GAA GTG ATG GAG 1710 CAA GCG GTC ACA CTT CGC GTG CCG CTC AAA GTC GAT TAC CAT TAC 1755 GGC TCG ACA TGG TAT GAC GCG AAA 1779 SEQ ID NO: 17 Array length: 593 Array type: Number of amino acid chains: Single-stranded topology: Linear array types: Peptide sequence: Met Glu Ser Pro Ser Ser Glu Glu Glu Lys Pro Leu Ala Lys Met 1 5 10 15 Ala Phe Thr Leu Ala Asp Arg Val Thr Glu Glu Met Leu Ala Asp 20 25 30 Lys Ala Ala Leu Val Val Glu Val Val Glu Glu Asn Tyr His Asp 35 40 45 Ala Pro Ile Val Gly Ile Ala Val Val Asn Glu His Gly Arg Phe 50 55 60 Phe Leu Arg Pro Glu Thr Ala Leu Ala Asp Pro Gln Phe Val Ala 65 70 75 Trp Leu Gly Asp Glu Thr Lys Lys Lys Ser Met Phe Asp Ser Lys 80 85 90 Arg Ala Ala Val Ala Leu Lys Trp Lys Gly Ile Glu Leu Cys Gly 95 100 105 Val Ser Phe Asp Leu Leu Leu Ala Ala Tyr Leu Leu Asp Pro Ala 110 115 120 Gln Gly Val Asp Asp Val Ala Ala Ala Ala Lys Met Lys Gln Tyr 125 130 135 Glu Ala Val Arg Pro Asp Glu Ala Val Tyr Gly Lys Gly Ala Lys 140 145 150 Arg Ala Val Pro Asp Glu Pro Val Leu Ala Glu His Leu Val Arg 155 160 165 Lys Ala Ala Ala Ile Trp Ala Leu Glu Arg Pro Phe Leu Asp Glu 170 175 180 Leu Arg Arg Asn Glu Gln Asp Arg Leu Leu Val Glu Leu Glu Gln 185 190 195 Pro Leu Ser Ser Ile Leu Ala Glu Met Glu Phe Ala Gly Val Lys 200 205 210 Val Asp Th r Lys Arg Leu Glu Gln Met Gly Glu Glu Leu Ala Glu 215 220 225 Gln Leu Arg Thr Val Glu Gln Arg Ile Tyr Glu Leu Ala Gly Gln 230 235 240 Glu Phe Asn Ile Asn Ser Pro Lys Gln Leu Gly Val Ile Leu Phe 245 250 255 Glu Lys Leu Gln Leu Pro Val Leu Lys Lys Ser Lys Thr Gly Tyr 260 265 270 Ser Thr Ser Ala Asp Val Leu Glu Lys Leu Ala Pro Tyr His Glu 275 280 285 Ile Val Glu Asn Ile Leu Gln His Tyr Arg Gln Leu Gly Lys Leu 290 295 300 Gln Ser Thr Tyr Ile Glu Gly Leu Leu Lys Val Val Arg Pro Asp 305 310 315 Thr Lys Lys Val His Thr Ile Phe Asn Gln Ala Leu Thr Gln Thr 320 325 330 Gly Arg Leu Ser Ser Thr Glu Pro Asn Leu Gln Asn Ile Pro Ile 335 340 345 Arg Leu Glu Glu Gly Arg Lys Ile Arg Gln Ala Phe Val Pro Ser 350 355 360 Glu Ser Asp Trp Leu Ile Phe Ala Ala Asp Tyr Ser Gln Ile Glu 365 370 375 Leu Arg Val Leu Ala His Ile Ala Glu Asp Asp Asn Leu Met Glu 380 385 390 Ala Phe Arg Arg Asp Leu Asp Ile His Thr Lys Thr Ala Met Asp 395 400 405 Ile Phe Gln Val Ser Glu Asp Glu Val Thr Pro Asn Met Arg Arg 410 415 420 Gl n Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp 425 430 435 Tyr Gly Leu Ala Gln Asn Leu Asn Ile Ser Arg Lys Glu Ala Ala 440 445 450 Glu Phe Ile Glu Arg Tyr Phe Glu Ser Phe Pro Gly Val Lys Arg 455 460 465 Tyr Met Glu Asn Ile Val Gln Glu Ala Lys Gln Lys Gly Tyr Val 470 475 480 Thr Thr Leu Leu His Arg Arg Arg Tyr Leu Pro Asp Ile Thr Ser 485 490 495 Arg Asn Phe Asn Val Arg Ser Phe Ala Glu Arg Met Ala Met Asn 500 505 510 Thr Pro Ile Gln Gly Ser Ala Ala Asp Ile Ile Lys Lys Ala Met 515 520 525 Ile Asp Leu Asn Ala Arg Leu Lys Glu Glu Arg Leu Gln Ala Arg 530 535 540 Leu Leu Leu Gln Val His Asp Glu Leu Ile Leu Glu Ala Pro Lys 545 550 555 Glu Glu Met Glu Arg Leu Cys Arg Leu Val Pro Glu Val Met Glu 560 565 570 Gln Ala Val Thr Leu Arg Val Pro Leu Lys Val Asp Tyr His Tyr 575 580 585 Gly Ser Thr Trp Tyr Asp Ala Lys 590

[Brief description of drawings]

FIG. 1 is a process diagram showing the construction of pUI101.

FIG. 2 DNA cloned in pUI101
It is a figure which shows the restriction enzyme map of a polymerase gene.

FIG. 3: DNA cloned in pUI205
It is a figure which shows the restriction enzyme map of a polymerase gene.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Ikuno Kato, Inventor Ikunoshin Kato 3-4-1 Seta, Otsu City, Shiga Prefecture

Claims (3)

[Claims] 1. A method for cloning a pol I-type DNA polymerase gene, comprising the steps of: (a) using the primer represented by SEQ ID NO: 1 or 2 of the pol I-type DNA polymerase gene, respectively. A method for cloning a type I DNA polymerase gene, comprising the steps of: (a) using amplified DNA as a probe and cloning a type I DNA polymerase gene that hybridizes to the probe. 2. A Pol I type DNA polymerase gene isolated from plasmid pUI101 or plasmid pUI205. 3. A Pol I type DNA polymerase gene hybridizable to the Pol I type DNA polymerase gene according to claim 2.