JPH09191885A - Nsp7524iii restriction-and modification-based enzymes and their genes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain new Nsp7524III restriction- and modification-based enzymes, which are both a restriction enzyme, derived from a bacterium of the genus Nostoc and having a specific amino acid sequence and activities for cleaving a DNA and a modification enzyme capable of protecting the DNA from the cleavage with the enzyme, and further their genes useful as a reagent, etc., for genetic engineering. SOLUTION: The new Nsp7524III restriction/modification-based enzymes are an Nsp7524III restriction enzyme and an Nsp7524III modification enzyme. The Nsp7524 restriction enzyme has an amino acid sequence represented by formula I and is capable of recognizing a sequence (5'-CPyCGPuG-3') comprising 6 bases of a twofold rotational symmetrical structure in a DNA base sequence and cleaving the bond between C and Py on the 5' side so as to provide a 5' sticky end. The Nsp7524III modification enzyme has an amino acid sequence represented by formula II and is capable of protecting a DNA from the cleavage with the Nsp7524III restriction enzyme. Both the enzymes and their genes are useful as a reagent, etc., for genetic engineering. The enzymes are obtained by culturing a Nostoc species PCC7524 in a culture medium or expressing expression vectors containing the respective genes capable of coding the enzymes integrated thereinto in host cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to restriction enzymes and modification enzymes useful as genetic engineering reagents, genes thereof, and methods for producing the same. More specifically, Nsp7524II
The present invention relates to an I-restriction / modification enzyme, its gene, and a method for genetically engineering Nsp7524III restriction enzyme and modification enzyme using them.

[0002]

2. Description of the Related Art Nsp7524III restriction / modification enzyme is Nostoc species PCC752
4 (hereinafter abbreviated as Nsp bacterium)
A Nsp7524III restriction enzyme having an activity of cleaving A, and a DNA obtained by the cleavage with the Nsp7524III restriction enzyme.
Nsp7524III modifying enzyme (also referred to as methylase or methyltransferase) that protects The Nsp7524III restriction enzyme is a typical type II restriction enzyme, which recognizes a sequence consisting of 6 bases having a two-fold symmetry structure (5'-CPyCGPuG-3 ') in the DNA base sequence, and C on the 5'side of the recognition sequence. It is an enzyme that cleaves between Py and Py to form a 5'overhang [Reaston (Reaston
) Et al., Gene Vol. 20, pp. 103-110 (1982)].

On the other hand, Nsp7524III modifying enzyme is DN
A specific base in the above sequence present on A is methylated and DNA is digested with Nsp7524III restriction enzyme.
It is an enzyme that has the function of protecting However, detailed properties such as a base methylated by the enzyme are not known. The method for producing the Nsp7524III restriction enzyme from Nsp bacteria is described in the above-mentioned Gene by Reston et al., But a product having such a purity that a partial amino acid sequence can be revealed has not been obtained. N
There is no report that the production method has been established for the sp7524III modifying enzyme.

Nsp7524III obtained from Nsp bacteria
The yields of restriction enzyme and Nsp7524III modification enzyme were low, and it was difficult to obtain both enzymes in large amounts. Also,
In addition to Nsp7524III restriction enzyme and modification enzyme, Nsp bacteria further contains four kinds of restriction / modification system enzymes (Nsp752
4I, Nsp7524II, Nsp7524IV, and N
sp7524V) and Nsp752
4III The procedure for producing only the restriction enzyme or the modification enzyme was complicated and difficult. Therefore Nsp752
It is expected that the genes encoding the 4III restriction enzyme and the modifying enzyme will be isolated, and a transformant microorganism containing them will be produced and used for the production of both enzymes.
4III Information for isolating genes of restriction / modification enzymes, such as partial amino acid sequences of both enzymes, has not been clarified yet.

[0005]

The first object of the present invention is to provide an Nsp7524III restriction / modification enzyme. The second object of the present invention is to provide genes for these enzymes. A third object of the present invention is Ns
A transformant introduced with the Nsp7524III restriction / modification enzyme gene suitable for industrial production of p7524III restriction enzyme and modification enzyme, particularly Escherichia coli (Escherichia coli)
(ia coli) transformant and a method for producing Nsp7524III restriction enzyme and modifying enzyme using the transformant.

[0006]

The present inventors have succeeded in cloning a DNA fragment containing an Nsp7524III restriction / modification enzyme gene from Nsp.
By culturing a microorganism into which all or part of these DNA fragments are incorporated, particularly Escherichia coli, a significant amount of Nsp7524III restriction enzyme and modifying enzyme accumulate in the cells, and a large amount of Nsp7524 is obtained from these cultures.
The inventors have found that III restriction enzymes and modified enzymes can be obtained, and completed the present invention.

That is, the gist of the present invention is (1) Nsp75 consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing.
24III restriction enzyme, (2) a polypeptide containing all or part of the amino acid sequence set forth in SEQ ID NO: 1 of the sequence listing, which has Nsp7524III restriction enzyme activity, (3) SEQ ID NO: of the sequence listing: 1 or more amino acids are added to the amino acid sequence of 1,
Nsp7524III that has been deleted or substituted
A polypeptide containing an amino acid sequence which confers a restriction enzyme activity, (4) an isolated Nsp7524III restriction enzyme gene consisting of a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, (5) in the sequence listing (4) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 2, (6) a polypeptide containing all or part of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, wherein the Nsp7524III restriction enzyme (7) Polynucleotide encoding a polypeptide having activity, (7) Polynucleotide encoding a polypeptide having Nsp7524III restriction enzyme activity, which is hybridizable with the polynucleotide described in (4) above, (8) Sequence of Sequence Listing One or more amino acids are added, deleted, or deleted in the amino acid sequence of SEQ ID NO: 1. It has been replaced,
And a polynucleotide encoding a polypeptide containing an amino acid sequence that gives Nsp7524III restriction enzyme activity, (9) an Nsp7524III modifying enzyme having the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, (10)
A polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, which is Nsp7524II.
A polypeptide having I-modifying enzyme activity, (11) An amino acid sequence having one or more amino acids added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, and giving Nsp7524III-modifying enzyme activity (12) an isolated Nsp7524III-modifying enzyme gene consisting of a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 3 of the sequence listing, (13) set forth in SEQ ID NO: 4 of the sequence listing The gene according to (12) above, which comprises a nucleotide sequence, (14)
A polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, which is Nsp7524
A polynucleotide encoding a polypeptide having III-modifying enzyme activity, (15) Nsp7524, which is hybridizable with the polynucleotide according to (12) above.
III. A polynucleotide encoding a polypeptide having a modifying enzyme activity, (16) One or more amino acids are added, deleted or substituted in the amino acid sequence of SEQ ID NO: 3 in the Sequence Listing, and Nsp752
4III A polynucleotide encoding a polypeptide containing an amino acid sequence that confers a modification enzyme activity, (17)
A transformant transformed with a vector incorporating the polynucleotide according to any one of (4) to (8) and the polynucleotide according to any one of (12) to (16), (18) A vector containing the polynucleotide according to any one of (4) to (8), and the above (12) to
(16) A transformant transformed with both the vector containing the polynucleotide according to any one of (16) and (19) transformed with the vector containing the polynucleotide according to any one of (12) to (16) above Transformant, (2
0) A method for producing Nsp7524III restriction enzyme, which comprises culturing the transformant according to (17) or (18) above, and collecting a polypeptide having Nsp7524III restriction enzyme activity from the culture. Two
1) A method for producing an Nsp7524III-modifying enzyme, which comprises culturing the transformant according to any one of (17) to (19), and collecting a polypeptide having Nsp7524III-modifying enzyme activity from the culture. It is about.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. [1] Nsp7524III Restriction / Modification Enzyme of the Present Invention In the present specification, Ns is a general term for compounds (mainly proteins) having Nsp7524III restriction enzyme activity.
The term Nsp7524III modifying enzyme is used as a general term for compounds (mainly proteins) having p7524III restriction enzyme and Nsp7524III modifying enzyme activity. The Nsp7524III restriction / modification enzyme of the present invention is produced by, for example, Nostoc species PCC7524, and has the Nsp7524III restriction enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, and SEQ ID NO: 3 in the sequence listing. Nsp75 consisting of the amino acid sequence described in 1.
24III modifying enzyme.

The Nsp7524III restriction enzyme of the present invention is not limited to the above-mentioned ones, and is a polypeptide containing all or part of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, which is Nsp7524III. It also includes a polypeptide having a restriction enzyme activity, and further includes 1 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
Alternatively, a polypeptide having an amino acid sequence in which a plurality of amino acids are added, deleted or substituted and which confers Nsp7524III restriction enzyme activity is also within the scope of the present invention. Further, the Nsp7524III-modifying enzyme of the present invention is not limited to the above-mentioned one, and is a polypeptide containing all or part of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, and has Nsp7524III-modifying enzyme activity. Which further comprises a polypeptide having the following formula, wherein one or more amino acids are added, deleted or substituted in the amino acid sequence of SEQ ID NO: 3 in the sequence listing, and the amino acid sequence imparts Nsp7524III modifying enzyme activity. Also included within the scope of the invention are polypeptides containing.

In general, a naturally occurring protein has polymorphisms and mutations in the gene encoding it, as well as deletions and insertions of amino acids in its amino acid sequence due to modification reactions of the produced protein in vivo and during purification. It is known that there are mutations such as additions, substitutions and the like, but nevertheless, some proteins exhibit substantially the same physiological activity and biological activity as the protein having no mutation. Those having such a difference in structure but no significant difference in their functions and activities are within the scope of the present invention. The same applies to the case where the above-described mutation is artificially introduced into the amino acid sequence of a protein. In this case, it is possible to produce a further wide variety of mutants. For example, the methionine residue present at the N-terminal of a protein expressed in E. coli is often said to be removed by the action of methionine aminopeptidase, but depending on the type of protein, the removal is not completely carried out, Both those with and without methionine residues are produced. However, the presence or absence of this methionine residue often does not affect the activity of the protein. In addition, human interleukin 2 (IL
It is known that a polypeptide obtained by substituting a certain cysteine residue in the amino acid sequence of -2) with serine retains interleukin 2 activity [Science, 224, 1431 (1984)].

Furthermore, when a protein is produced by genetic engineering, it is often expressed as a fusion protein. For example, in order to increase the expression level of the target protein, an N-terminal peptide chain derived from another protein is added to the N-terminus, or an appropriate peptide chain is added to the N-terminus or C-terminus of the target protein to be expressed. It has been attempted to facilitate purification of the target protein by using a carrier having an affinity for the peptide chain. Therefore, even a polypeptide having an amino acid sequence that is partially different from the Nsp7524III restriction / modification enzyme of the present invention, as long as it exhibits essentially the same activity as the Nsp7524III restriction / modification enzyme of the present invention, It is within the scope of the present invention.

[2] Restriction of Nsp7524III of the present invention
Modification Enzyme Gene As used herein, the gene for the Nsp7524III restriction / modification enzyme is Nsp7 having an activity of cleaving DNA.
A gene encoding a 524III restriction enzyme and Nsp75
24III Ns that protects DNA from cleavage by restriction enzymes
It contains the gene encoding the p7524III modifying enzyme. Here, the restriction / modification enzyme gene is a term indicating a gene having both a restriction enzyme gene and a modification enzyme gene, or each of these two genes. In other words, it means a restriction and / or modification enzyme gene.

The Nsp7524III restriction enzyme gene of the present invention comprises a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, for example, the base sequence set forth in SEQ ID NO: 2 in the sequence listing. Examples include, but are not limited to, genes. That is, a polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, which comprises Nsp
7524III Polynucleotide encoding a polypeptide having restriction enzyme activity, Nsp7524II of the present invention
Nsp75 capable of hybridizing to I restriction enzyme gene
24III A polynucleotide encoding a polypeptide having a restriction enzyme activity, in which one or more amino acids are added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 1 in Sequence Listing, and Nsp7524
III. Polynucleotides encoding a polypeptide containing an amino acid sequence that confers a restriction enzyme activity are also within the scope of the present invention.

The Nsp7524III-modifying enzyme gene of the present invention comprises a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing, for example, the base sequence set forth in SEQ ID NO: 4 in the sequence listing. Examples include, but are not limited to, genes. That is, a polypeptide containing all or part of the amino acid sequence set forth in SEQ ID NO: 3 of the Sequence Listing, wherein Nsp
7524III Polynucleotide encoding a polypeptide having a modifying enzyme activity, Nsp7524II of the present invention
Nsp75 capable of hybridizing to I-modifying enzyme gene
24III A polynucleotide encoding a polypeptide having a modifying enzyme activity, in which one or more amino acids are added, deleted or substituted in the amino acid sequence of SEQ ID NO: 3 in the sequence listing, and Nsp7524.
Also within the scope of the present invention are polynucleotides encoding polypeptides containing an amino acid sequence that confers III-modifying enzyme activity.

The reason why various polynucleotides are included in the scope of the present invention is as follows. That is, it is known that there are 1 to 6 types of codons (combinations of three bases) that specify amino acids on a gene for each type of amino acid. Therefore, a large number of genes encoding a certain amino acid sequence can exist. Genes are not stable in nature, and mutations in their nucleotide sequences are not uncommon. In some cases, the mutation that occurs in the gene does not change the encoded amino acid sequence (called a silent mutation), and in this case, it can be said that different genes encoding the same amino acid sequence have occurred. Therefore, even if a gene encoding a specific amino acid sequence is isolated, it is undeniable that many kinds of genes encoding the same amino acid sequence will be produced as the organism containing it is passaged.

Further, it is not difficult to artificially prepare many kinds of genes encoding the same amino acid sequence by using various genetic engineering techniques. For example, in the case of genetically engineered protein production, when the codon used in the original gene encoding the target protein is rarely used in the host, the expression level of the protein may be low. is there. In such a case, it is possible to achieve high expression of the target protein by artificially converting codons into those commonly used in the host without changing the encoded amino acid sequence. There is. As described above, it goes without saying that many kinds of genes encoding specific amino acid sequences can be artificially created, and can be generated in the natural world.
Therefore, even if the gene is not the same as the nucleotide sequence disclosed in the present invention, the present invention is not limited as long as it is a polynucleotide that coats a polypeptide showing essentially the same activity as the Nsp7524III restriction / modification enzyme of the present invention. It is included.

[3] Production Method of the Present Invention To produce the Nsp7524III restriction enzyme of the present invention,
First, a transformant transformed with a vector incorporating the polynucleotide encoding the Nsp7524III restriction enzyme and the polynucleotide encoding the Nsp7524III modifying enzyme is prepared and cultured,
By collecting a polypeptide having Nsp7524III restriction enzyme activity from the culture, Nsp7524III
Restriction enzymes can be produced. In this case, the transformant may be a transformant transformed with both the vector containing the polynucleotide encoding the Nsp7524III restriction enzyme and the vector containing the polynucleotide encoding the Nsp7524III modifying enzyme.

In order to produce the Nsp7524III modifying enzyme of the present invention, a polynucleotide encoding the Nsp7524III restriction enzyme as described above and Nsp752 are used.
Ns was obtained by preparing a transformant transformed with a vector incorporating a polynucleotide encoding a 4III-modifying enzyme, culturing the transformant, and collecting a polypeptide having Nsp7524III-modifying enzyme activity from the culture.
A p7524III modifying enzyme can be produced. In this case, the transformant contains a vector containing a polynucleotide encoding a Nsp7524III restriction enzyme, and Nsp75.
Transformants transformed with both of the vectors containing the polynucleotide encoding the 24III-modifying enzyme may be used. Alternatively, a transformant transformed only with the vector containing the polynucleotide encoding the Nsp7524III modifying enzyme may be used.

In the following, as a specific embodiment of the present invention, Ns
A procedure for cloning a p7524III restriction / modification enzyme gene and expressing the gene will be exemplified. (1) Nsp bacterium (Nostoc sp. PCC) that is a DNA donor
7524) is a strain available from American Type Culture Collection (ATCC). Chromosomal DNA is extracted from cells obtained by culturing the strain, and a DNA fragment obtained by partial digestion with Sau3AI restriction enzyme is ligated to an appropriate vector. (2) For example, the Escherichia coli AP1-200 strain is transformed with the DNA library prepared in (1), and a transformant expressing the Nsp7524III modifying enzyme gene is selected. (3) Regarding the transformant obtained in (2), Nsp
Confirm 7524III restriction enzyme activity by in vitro method. (4) The transformant whose restriction enzyme activity was detected in (3) is cultured, and a large amount of Nsp7524III restriction enzyme and modifying enzyme is collected from the culture.

The Escherichia coli AP1-200 strain is a strain in which β-galactosidase is expressed when the induction of SOS repair system gene accompanying the methylation of DNA occurs in the cell [Piekarowicz].
Nucleic Acids Research
Research) Vol. 19, pp. 1831-1835 (19
91)]. It is known that the above-mentioned induction of the SOS repair system gene is caused by methylation of a base in a specific base sequence on DNA, for example, the methylation of DNA catalyzed by PstI modification enzyme, MspI modification enzyme and the like. [Heitman et al., Journal of Bacteriolog
y), Vol. 169, pp. 3243-3250, (198
7)]. Therefore, when the modified enzyme gene introduced into the strain is expressed and thereby the SOS repair system gene is induced, such a transformant is treated with 5-bromo-4-chloro, which is a substrate for β-galactosidase. It can be selected as a blue colony on a plate containing -3-indolyl-β-D-galactoside.

However, the Nsp bacterium belongs to the genus Cyanobacteria, and the gene is transcribed in Escherichia coli, which is distant in classification,
The translated and active Nsp7524III modifying enzyme is not always expressed. Further, even when expressed, the SOS repair system gene may not be induced depending on the base sequence of the DNA methylated by the modifying enzyme or the type of the base. In this system, transformation containing the modifying enzyme gene may be performed. It was unclear whether the body could be detected. As will be shown in Examples described later, the present inventors used Escherichia coli AP1-200 strain as a host for transformation in (2) to
It was found that a transformant expressing the p7524III modifying enzyme gene can be selected.

The resulting transformant Nsp7524III
The modifying enzyme activity is Nsp7524 of transformant DNA.
Confirmed by the fact that the III recognition sequence is not cleaved by the Nsp7524III restriction enzyme. However Nsp
The 7524III restriction enzyme is not commercially available, and Nsp
It is not easy to purify Nsp7524III restriction enzyme of sufficient purity from bacteria. Therefore, in the present invention, N
A with the same recognition and cleavage site as the sp7524III restriction enzyme
A vaI restriction enzyme can be used in place of the enzyme. It was unclear whether the DNA methylated by the Nsp7524III modification enzyme was cleaved by the AvaI restriction enzyme or not, but the present inventors selected the trait selected as the blue colony as shown in Examples described later. It was found that the DNA prepared from the transformant was not cleaved by AvaI restriction enzyme.

The transformant obtained here is Nsp75.
It holds a plasmid containing a DNA fragment of about 2.9 kb containing the 24III modifying enzyme gene. The plasmid was used as a plasmid pBRN3 and Escherichia coli strain AP1-200 transformed with the plasmid was Escherichia c.
oli AP1-200 / pBRN3, respectively.

In (3), Nsp7524III restriction enzyme activity of the obtained transformant is examined. Nsp75
The 24III restriction enzyme activity can be examined by the following in vitro method. That is, the transformant to be tested is cultivated, the bacterial cells are collected from the culture and disrupted by ultrasonic treatment, and then the ultracentrifugation is performed to collect the supernatant,
This is used as a sample for activity measurement. After incubating this appropriate amount with λ phage DNA (λ-DNA) as a substrate at 37 ° C., degradation of the substrate DNA can be confirmed by agarose gel electrophoresis. When the Nsp7524III restriction enzyme activity of the extract of Escherichia coli AP1-200 / pBRN3 is examined using this method, the activity of cleaving λ-DNA into a fragment of the same size as expected for Nsp7524III can be detected. This confirms that the Nsp7524III restriction / modification enzyme gene could be isolated.

In (4), Escherichia coli AP
Approximately 4,000 from 1 g of cultured cells of 1-200 / pBRN3
The unit's Nsp7524III restriction enzyme activity is detected. One unit of Nsp7524III restriction enzyme activity means the buffer [10 mM Tris-HCl (pH 7.
5) 10 mM MgCl ₂ , 1 mM DTT, 50 mM N
aCl], the amount of enzyme that completely decomposes 1 μg of λ-DNA at 37 ° C. for 1 hour. In addition, the plasmid pBRN
When the Nsp7524III restriction enzyme activity of the transformant obtained by introducing 3 into the Escherichia coli MC1061 strain was examined, a restriction enzyme activity of about 37,000 units was detected from 1 g of the cultured cells, which was about 30 times the production amount of Nsp bacteria. is there. The transformant obtained by introducing pBRN3 into the Escherichia coli MC1061 strain was Escherichia coli MC106.
It is named and displayed as 1 / pBRN3, and from September 28, 1995, the FERM P
Deposited under accession number -15209.

DNA inserted in the plasmid pBRN3
The base sequence of the fragment can be determined by a conventional method such as the dideoxy method. Furthermore, by analyzing the obtained base sequence, a region (open reading frame, O
The presence of RF) can be inferred. Plasmid pB
Approximately 2.9 kb of Sau3AI-Sa inserted in RN3
The entire nucleotide sequence of the u3AI DNA fragment is shown in SEQ ID NO: in the Sequence Listing.
It is shown in FIG. There are two consecutive ORFs in the base sequence, and they are named ORF1 and ORF2 in order from the 5'side. The nucleotide sequence of ORF1 and the amino acid sequence deduced from the sequence are shown in SEQ ID NO: 4 and SEQ ID NO: 3 of the sequence listing, respectively. The nucleotide sequence of ORF2 and the amino acid sequence deduced from the sequence are shown in SEQ ID NO: 2 and SEQ ID NO: 1 of the sequence listing, respectively. Transformants carrying plasmids lacking ORF2 are Nsp7524III
ORF1 is Nsp752 because it has no restriction enzyme activity
The 4III modifying enzyme can be presumed to have ORF2 encoding the Nsp7524III restriction enzyme.

In order to obtain a protein having Nsp7524III restriction / modification enzyme activity by genetic engineering, first, the obtained restriction / modification enzyme gene is used in a suitable host cell such as Escherichia coli or Bacillus subtilis.
us subtilis), Saccharomyces cerevisiae (Saccha
romyces cerevisiae), actinomycetes, animal cells, insect cells,
It is ligated to an expression vector that can be expressed in plant cells or the like according to a conventional method to prepare a transformant in which it is introduced into a host cell. By culturing this transformant, N
A protein having sp7524III restriction / modification enzyme activity can be expressed. Note that Nsp7524
It is desirable that the transformant for expressing the III restriction enzyme should carry the genes for both the restriction enzyme and the modified enzyme, but both genes may be located on the same vector or may be integrated on separate vectors. It may be transformed with both in the above state. On the other hand, for the expression of the Nsp7524III modifying enzyme, a transformant having only the modifying enzyme gene can be used in addition to the above transformant.

Expression of the enzyme protein by these transformants depends on Nsp7524III restriction enzyme activity or Ns
This can be confirmed by measuring the p7524III modification enzyme activity. The Nsp7524III restriction enzyme activity can be measured by the in vitro method described above. It can be confirmed by not being cleaved by a certain AvaI restriction enzyme.

From cultures of transformants Nsp7524II
When collecting restriction enzymes and modifying enzymes, for example, after collecting the cells from the culture, the enzymes are extracted by ultrasonic disruption, ultracentrifugation, etc., and then the nucleic acid removal method, salting out method, affinity chromatography method. It may be purified by a combination of a gel filtration method, an ion exchange chromatography method and the like. By this method, a large amount of Nsp7524III restriction enzyme and modification enzyme can be obtained. Depending on the expression system used, the enzyme protein expressed in the transformant may accumulate as an insoluble matter [inclusion body]. In this case, the insoluble matter is recovered, solubilized under mild denaturing conditions, for example, in the presence of a denaturing agent such as urea, and then the denaturing agent is removed to obtain an active protein. Furthermore, the target enzyme protein can be purified by performing the above-mentioned chromatography operation.

If hybridization is carried out under strict conditions using the gene obtained above as a probe,
Obtained gene (SEQ ID NO: 2, SEQ ID NO: in Sequence Listing:
Although the sequence is slightly different from 4), it is possible to obtain a similar gene encoding a polypeptide having a similar enzymatic activity,
This is also within the scope of the invention. Under such strict conditions, a nylon membrane on which DNA is immobilized is 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% SDS, 100 μg / ml salmon sperm DNA,
65 in a solution containing 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% ficoll
It means that the hybridization is carried out by keeping the temperature of the probe together with the probe for 20 hours.

As a method for obtaining a similar gene encoding the Nsp7524III restriction / modification enzyme by hybridization, for example, the following method can be mentioned.
First, DNA obtained from an appropriate gene source is connected to a plasmid or a phage vector according to a conventional method to prepare a DNA library. The transformant obtained by introducing this library into an appropriate host is cultured on a plate, the grown colonies or plaques are transferred to a nitrocellulose or nylon membrane, and after denaturing treatment, the DNA is fixed to the membrane. A probe in which this membrane is labeled with ³² P or the like in advance (the probe to be used may be a polynucleotide encoding all or part of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, For example, a polynucleotide consisting of or including all or part of the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing can be used) in a solution having the above composition and under the above conditions. Incubate and incubate for hybridization. After the hybridization is completed, the non-specifically adsorbed probe is washed away, and a clone hybridized with the probe is identified by autoradiography or the like. This operation is repeated until the hybridizing clone can be isolated. In the clone thus obtained, the gene encoding the polypeptide having the desired enzyme activity is retained.

The base sequence of the obtained gene is determined, for example, as follows, and it is confirmed whether or not it is a gene encoding the target enzyme protein. For the determination of the nucleotide sequence, in the case of a transformant prepared using a plasmid vector, if the host is Escherichia coli, it is cultured in a test tube or the like, and a plasmid is prepared according to a conventional method. The obtained plasmid is used as a template as it is, or the inserted fragment is taken out and subcloned into an M13 phage vector or the like, and then the nucleotide sequence is determined by the dideoxy method. In the case of a transformant prepared with a phage vector, the nucleotide sequence can be determined basically by the same procedure. For basic experimental methods from culture to sequencing, see, for example, T. Maniatis (T. Ma.
Niatis) et al. Molecular Cloning: A Laborato
ry Manual, 1982, Cold Spring Harbor Laboratory).

The resulting gene is the desired Nsp7524II
Whether or not it is a similar gene encoding an I restriction / modification enzyme can be determined by comparing the determined nucleotide sequence with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing, or The amino acid sequence deduced from the obtained nucleotide sequence can be compared with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing.

When the obtained gene does not contain all the regions encoding the target restriction / modification enzymes, a primer is synthesized based on the nucleotide sequence of the obtained gene, and PCR is performed using this primer. Amplify the missing area by
Alternatively, using the obtained gene fragment as a probe, DNA
By repeating the screening of the library,
The entire coding region can be obtained.

A transformant containing a similar gene encoding the Nsp7524III restriction / modification enzyme thus obtained is prepared, the enzyme protein encoded by the gene is expressed, and the expressed enzyme protein is purified. be able to. Preparation of the transformant, expression of the enzyme protein, and purification can be performed in the same manner as above. The enzyme protein thus obtained is Nsp75.
It is expected to retain 24III restriction enzyme activity or Nsp7524III modification enzyme activity.

[0036]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Nsp Bacterial Chromosomal DNA According to the method of Sutherland et al. Described in Journal of General Microbiology, Vol. 115, pp. 273-287 (1979). Were cultured to obtain wet bacterial cells. 2 g of wet Nsp bacteria was added to 10 ml of solution A [25 mM Tris-HCl (pH 8.0), 50 mM
Glucose, 10 mM EDTA] was suspended, and 1 ml of lysozyme dissolved in the same solution at 2 mg / ml was added and stirred, and the mixture was allowed to stand at 37 ° C. for 15 minutes. Next, 28 ml of solution B [100 mM NaCl, 100 mM Tris-HCl (pH 8.0)] was added to the solution, stirred, and further 4
10% SDS solution (ml) was added, and the mixture was stirred and allowed to stand at 37 ° C. for 1 hour. 1 ml of Solution C [10% SD
S, 8% Sarcosyl] and stirred,
It was left still at 60 ° C for 15 minutes.

After allowing the solution to stand, an equal volume of phenol:
Chloroform (1: 1) mixture was added and gently stirred for 10 minutes, followed by centrifugation (5,000 xg, 10 minutes).
The aqueous layer and the chloroform layer were separated. The aqueous layer is separated, an equal amount of isopropyl alcohol is added to this, and the mixture is stirred and stirred at 0 ° C.
After leaving still for 10 minutes, centrifuge (13,500 xg, 10
The precipitate was collected by This was washed with 70% ethanol and 10 ml of TE solution [10 mM Tris-HCl was used.
The chromosomal DNA was prepared by dissolving it in (pH 8.0), 1 mM EDTA], and stored at 4 ° C.

Example 2 Preparation of DNA library 25 μg of chromosomal DNA of Nsp bacterium obtained in Example 1
1 unit of Sau3AI restriction enzyme (Takara Shuzo) was added to the buffer [50 mM Tris-HCl (pH 7.5), 10
mM MgCl ₂ , 1 mM DTT, 100 mM NaCl]
After reacting at 37 ℃ for 1-10 minutes to partially digest,
Agarose gel electrophoresis was performed, and 2-7 kb of D
The NA fragment was recovered. This DNA fragment was previously Ba
D ligated to the plasmid vector pBR322 (Takara Shuzo) that had been cleaved with mHI restriction enzyme (Takara Shuzo)
An NA library was made.

Example 3 Isolation of Modification Enzyme Gene The DNA library prepared in Example 2 was used to transform Escherichia coli AP1-200. The procedure from the transformation to the selection of the desired transformant was carried out by the method described in the above-mentioned article of Nucleic Acids Research by Piekarowitz et al. However, when culturing the cells after the transformation operation on the plate, soft agar was not used, and the cell suspension was directly smeared on the plate and cultured. Among the obtained transformants, the one in which the modifying enzyme gene was expressed was 35 μg / ml of 5-bromo-4.
-Chloro-3-indolyl-β-D-galactoside and 1
They were selected as blue-colored colonies on LB plates containing 00 μg / ml ampicillin.

This transformant was cultured in 2 ml of LB medium containing 100 μg / ml of ampicillin at 42 ° C. for 16 hours, and then the cells were collected and the plasmid was extracted by the alkali-SDS method. The obtained plasmid was resistant to the treatment with AvaI restriction enzyme (Takara Shuzo), and it was confirmed that this transformant expressed the Nsp7524III modifying enzyme gene. That is, 1 μg of the obtained plasmid was 8
30 μL of buffer with 10 units of AvaI restriction enzyme [10 mM Tris-HCl (pH 7.5), 10 mM M
gCl ₂ , 1 mM DTT, 50 mM NaCl], 37
After incubating at 0 ° C., agarose gel electrophoresis was performed, and Nsp7524II was obtained on the plasmid vector pBR322 used for preparing the DNA library.
No cleavage of the plasmid was observed despite the presence of the I restriction enzyme recognition sequence. N thus obtained
The plasmid containing the sp7524III modifying enzyme gene was designated as plasmid pBRN3. In addition, Escherichia coli AP1-200 strain transformed with the plasmid
It was named Escherichia coli AP1-200 / pBRN3.

Next, the cells collected by culturing the transformant were disrupted by ultrasonic treatment and then subjected to ultracentrifugation to collect the supernatant to obtain a crude extract. About this crude extract
When the restriction enzyme activity was examined using DNA as a substrate, Ns
It was confirmed that p7524III restriction enzyme activity was present. That is, an appropriate amount of the crude extract was added to 1 μg of λ-DNA.
(Manufactured by Takara Shuzo Co., Ltd.) and 30 μL of buffer solution [10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1
mM DTT, 50 mM NaCl] at 37 ° C. followed by agarose gel electrophoresis and analysis of the reaction products reveals that λ-DNA was of the same size as expected when cleaved by Nsp7524III restriction enzyme. A DNA fragment was generated.

Example 4 Structural Analysis of Nsp7524III Restriction / Modification Enzyme Gene The plasmid pBRN3 obtained in Example 3 was about 2.9 kb.
DNA fragment derived from Nsp. Deletion containing inserted DNA fragments of various sizes, in which a part of the DNA fragments inserted in the plasmid was deleted using exonuclease (Takara Shuzo) and mung bean nuclease (Takara Shuzo) After preparing the mutant, an appropriate one was selected and the nucleotide sequence of the inserted DNA fragment portion was determined by the dideoxy method. Based on the obtained results, the nucleotide sequence of the entire DNA fragment inserted into the plasmid pBRN3 was determined.

The obtained nucleotide sequence is represented by SEQ ID NO: 5 in Sequence Listing.
Shown in Furthermore, as a result of searching an open reading frame (ORF) capable of encoding a protein, two ORFs were present in the fragment, and the base numbers 148 to 15
78 was designated as ORF1 and 1591 to 2556 were designated as ORF2. The nucleotide sequence of ORF1 is shown in SEQ ID NO: 4 in the sequence listing, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 3
Are shown below. The nucleotide sequence of ORF2 is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 1. Among the above-mentioned deletion mutants, since the Nsp7524III restriction enzyme activity is lost in the transformant containing the ORF2-deficient plasmid, ORF1 changed the Nsp7524III modification enzyme to the ORF.
2 was found to encode the Nsp7524III restriction enzyme. Nsp7 cloned in Figure 1
524III shows the structure of restriction / modification enzyme gene. In the figure, M indicates a modification enzyme gene, R indicates a restriction enzyme gene, and an arrow indicates the direction of the ORF. From the above, Nsp7524II
The structure of the I-restriction / modification enzyme gene was clarified.

Example 5 Transformants of Nsp7524III restriction enzyme and modification enzyme
Production by Escherichia coli AP1-200 obtained in Example 3
/ PBRN3 containing 100 µg / ml ampicillin 10
0 ml of LB medium was inoculated and cultured at 42 ° C. for 16 hours.
1 g of cells obtained by collecting the cells was added to 5 ml of 20 mM Tris-HCl (pH: 10 mM 2-mercaptoethanol).
The cells were suspended in 7.5), sonicated to disrupt the cells, and the supernatant was recovered by ultracentrifugation (100,000 × g, 30 minutes).
When the activity of the supernatant was measured, Nsp7524III restriction enzyme was produced in an amount of about 4,000 units per gram of wet cells. For the activity measurement, an appropriate amount of the obtained supernatant was added to 1 μg of λ-D.
30 μL buffer with NA [10 mM Tris-HC
1 (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT,
Incubation at 37 ° C. in 50 mM NaCl] followed by agarose gel electrophoresis to analyze the reaction products. One unit of restriction enzyme was the amount of enzyme that completely decomposed 1 μg of λ-DNA per hour under the above reaction conditions. Further, Escherichia coli MC1061 / pBRN3, which is a transformant obtained by introducing the plasmid pBRN3 into Escherichia coli MC1061.
When the activity of (FERM P-15209) was examined by the same method, Nsp7524III restriction enzyme was produced in an amount of about 37,000 units per gram of wet cells.
The production amount was about 30 times that of the sp. As described above, a mass production system for Nsp7524III restriction enzyme and modification enzyme was completed.

[0046]

INDUSTRIAL APPLICABILITY The present invention limits Nsp7524III
A modified enzyme and its gene are provided. With a transformant transformed with a plasmid containing the gene,
It becomes possible to efficiently produce a large amount of Nsp7524III restriction enzyme and modification enzyme useful in genetic engineering.

[0047]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 322 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Sequence: Met Ser Ser His Arg His His Leu Gln Ser Ser Asp Asp Leu Val 5 10 15 Thr Thr Tyr Glu Ala Thr Arg Ala Gly Phe Ile Ala Leu Ala Leu 20 25 30 Glu Lys Asn Arg Arg Ala Thr Pro Tyr Val Ala Glu Ala Arg Ile 35 40 45 Leu Gln Glu Ala Ala Ser Gln Ala Glu Lys Pro Ala Asp Leu Leu 50 55 60 Asn Val Lys Gly Ile Glu Met Gly Leu Leu Thr Ala Ala Gly Leu 65 70 75 Ser Glu Lys Ser Leu Ala His Leu Met Ala Glu Asp Lys Ile Glu 80 85 90 Ala Ile Asn Gly Leu Ile Arg Asn Phe Leu Glu Pro Thr Gly Thr 95 100 105 Asn Phe Val Glu Glu Leu Val Phe Arg Phe Leu Leu Thr Arg Gly 110 115 120 Asp Thr Leu Gly Gly Ser Met Arg Asn Ile Gly Gly Ala Leu Ala 125 130 135 Gln Arg Lys Leu Thr Arg Ala Ile Leu Ser Thr Leu Thr Ile Ala 140 145 150 Gly Gln Lys Tyr Gln Trp Gln His Ser Lys Thr Lys Lys Trp Ile 155 160 165 Ala Met Thr Asn Asp Asp Thr Asp Ile Glu Leu Ser Leu Arg Gly 170 175 18 0 Ile Thr Trp Glu Ser Glu Phe Gly Asn Arg Arg Val Ile Tyr Asn 185 190 195 Leu Thr Val Pro Leu Val Lys Ser Asn Val Asp Leu Cys Leu Phe 200 205 210 Asn Leu Ala Pro Lys Glu Leu Val Ala Asn Gln Ser Ser Ala Ile 215 220 225 Asp Pro Ser Val Val Ala Pro Tyr Ala Ile Ala Leu Gly Glu Leu 230 235 240 Lys Gly Gly Ile Asp Pro Ala Gly Ala Asp Glu His Trp Lys Thr 245 250 265 Ala Gln Ala Ala Leu Asn Arg Ile Arg Glu Ala Phe Ser Arg Val 260 265 270 Gly Tyr Ser Pro Leu Thr Phe Phe Val Gly Ser Ala Ile Ala Lys 275 280 285 Arg Met Ala Gly Glu Ile Trp Ser Gln Leu Glu Asn Gly Thr Leu 290 295 300 Ser Asn Ala Ala Asn Leu Asn Glu Glu His Gln Val Ala Ser Ile 305 310 315 Ser Arg Trp Leu Tyr Gly Leu 320

SEQ ID NO: 2 Sequence length: 966 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Nostoc species Strain name : PCC7524 sequence: ATGAGTTCTC ACCGTCACCA TCTTCAATCT AGTGATGATC TTGTAACCAC TTATGAAGCG 60 ACTCGTGCGG GTTTTATCGC ACTCGCCCTG GAAAAGAATC GACGGGCTAC ACCTTACGTT 120 GCAGAAGCCA GAATACTTCA AGAGGCTGCT AGTCAAGCCG AAAAACCTGC TGATTTGTTA 180 AACGTGAAAG GGATTGAGAT GGGATTATTA ACTGCGGCGG GACTGTCTGA AAAATCTCTG 240 GCTCATTTGA TGGCGGAGGA TAAAATTGAA GCTATCAATG GTCTCATCAG AAATTTTCTT 300 GAACCAACAG GCACCAATTT TGTGGAAGAA TTAGTCTTTA GATTTTTGCT GACTCGTGGT 360 GATACCCTGG GTGGCTCAAT GCGTAACATT GGAGGAGCTT TAGCACAAAG AAAACTAACT 420 CGTGCTATCC TTTCGACTTT GACAATTGCT GGTCAGAAAT ATCAATGGCA ACATTCAAAA 480 ACGAAAAAAT GGATAGCGAT GACAAATGAT GACACAGATA TTGAGTTGTCCTTACGTTGATATATATTAGTATCTATATTACTATACTATCTACTTACTATCTACTTACTATCTACTTACTTACCTACTTACCTACTTACCTACTTACATCAGAGTA 540 ATCACTAGTA TC CGAAGGAGTT GGTAGCCAAT 660 CAATCCAGCG CAATTGATCC ATCTGTAGTT GCACCATATG CGATCGCACT AGGTGAACTC 720 AAAGGCGGAA TCGATCCCGC AGGCGCAGAT GAGCATTGGA AAACCGCACA GGCTGCACTC 780 AATCGCATAC GTGAAGCATT TTCCAGAGTT GGGTATTCGC CTTTGACTTT CTTCGTAGGA 840 TCAGCGATCG CAAAAAGAAT GGCTGGTGAA ATCTGGAGTC AACTAGAGAA TGGGACTCTT 900 AGTAATGCTG CCAACTTGAA CGAGGAGCAT CAAGTTGCGT CTATTTCTCG TTGGCTATAC 960 GGTCTA 966

SEQ ID NO: 3 Sequence length: 477 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence: Met Val Ser Leu Ser Ser Ile Ser Asn Ile Thr Ser Glu Val Glu 5 10 15 Ser Asn Ala Leu Arg Glu Ile Asp His Leu Asp Lys Gln Leu Gln 20 25 30 Ser His Phe Gln Thr Lys Phe Val Ile Gln Pro Ser Leu Thr Arg 35 40 45 Leu Leu Val Ser Phe Gln Ala Asn Lys Thr Arg Pro Ile Tyr Arg 50 55 60 Trp Tyr Lys Tyr Lys Glu Ala Phe Ser Ala Ser Leu Val Glu Leu 65 70 75 Leu Cys Gln Lys Tyr Gly Ile Thr Gln Gly Lys Val Leu Asp Pro 80 85 90 Phe Ala Gly Ser Gly Thr Ala Leu Phe Ala Thr Ile Asp Ile Gly 95 100 105 Ile Asp Ala Asp Gly Ile Glu Leu Leu Pro Ile Gly Gln Gln Ile 110 115 120 Ile Asp Thr Lys Lys Ile Leu Asp Val Glu Phe Thr Pro Asp Asp 125 130 135 Phe Thr Arg Leu Lys Thr Trp Leu Asp Leu Gln Val Trp Lys Gln 140 145 150 Phe Glu Thr Glu Val Thr Leu Pro Glu Leu Arg Ile Thr Lys Gly 155 160 165 Ala Tyr Ser Gln Ala Thr Lys Thr Ala Ile Glu Gln Tyr Leu Glu 170 175 180 Ala Cys Asn His Glu Asn His Arg Val Lys Ser Val Leu His Phe 185 190 195 Ala Leu Leu Cys Val Leu Glu Ser Val Ser Tyr Thr Arg Lys Asp 200 205 210 Gly Gln Tyr Leu Arg Trp Asp Tyr Arg Ser Gly Arg Gly Gln Gly 215 220 225 Lys Lys Pro Phe Asn Lys Gly Thr Ile Leu Glu Phe Asp His Ala 230 235 240 Ile Thr Asn Lys Ile Ser Glu Ile Ile Asn Asp Leu Glu Pro Ser 245 250 255 Thr Gln Gln Leu Glu Leu Phe Ser Phe Lys Lys Ser Gln Val Gln 260 265 270 Ile Asn Leu Arg Lys Gly Ser Cys Leu Asn Ile Met Pro Cys Leu 275 280 285 Pro Asn Ala Val Tyr Asp Ala Ile Ile Thr Ser Pro Pro Tyr Cys 290 295 300 Asn Arg Tyr Asp Tyr Thr Arg Thr Tyr Ala Leu Glu Leu Ala Leu 305 310 315 Leu Gly Ile Ser Glu Gln Glu Leu Ile Asn Leu Arg Gln Glu Met 320 325 330 Leu Ser Cys Thr Val Glu Asn Arg Pro Lys Asp Leu Leu Ala Ile 335 340 345 Asn Gln Asp Trp Glu Leu Ala Leu Arg Val Ala Asp Ser Gln Thr 350 355 360 Leu Leu Gln Ala Ile Leu Lys Tyr Leu Asp Asn Gln Asn Val Gln 365 370 375 Gly Leu Leu Asn Asn Lys Gly Ile Pro Arg Met Val Arg Gly Tyr 380 385 390 Phe Tyr Glu Met Ala Cys Val Ile Gln Glu Cys Phe Arg Val Leu 395 400 405 Lys Pro Gly Ala Phe Leu Leu Met Val Asn Asp Asn Val Arg Tyr 410 415 420 Ala Gly Ala Ser Ile Ser Val Asp Met Ile Leu Ser Asp Phe Ala 425 430 435 Glu Lys Leu Gly Phe Val Val Glu Ser Ile Leu Val Leu Pro Ser 440 445 450 Asp Lys Gly Asn Ser Ser Gln Gln Met Gly Asn His Gly Arg Glu 455 460 465 Pro Leu Arg Lys Cys Val Tyr Val Trp Lys Lys Gln 470 475

SEQ ID NO: 4 Sequence length: 1431 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Nostoc species Strain name : PCC7524 sequence: GTGGTCAGTT TGTCAAGTAT CTCAAACATC ACCTCTGAAG TGGAAAGCAA TGCACTGAGG 60 GAAATTGATC ATCTTGACAA GCAATTACAA TCTCACTTTC AAACCAAATT TGTAATTCAG 120 CCTTCACTTA CCCGGCTTTT AGTTAGTTTT CAAGCTAACA AAACTAGACC TATTTATCGG 180 TGGTACAAGT ACAAGGAAGC TTTCTCCGCC TCTCTGGTTG AGCTTTTATG CCAAAAATAT 240 GGAATTACCC AAGGCAAGGT TTTAGATCCC TTTGCAGGTA GTGGAACTGC TTTGTTTGCT 300 ACTATTGACA TTGGTATTGA TGCTGATGGT ATTGAATTGT TACCTATTGG TCAACAAATA 360 ATTGATACCA AAAAAATTTT AGATGTAGAA TTTACACCAG ATGATTTCAC ACGGCTGAAA 420 ACTTGGTTGG ATTTGCAGGT ATGGAAACAA TTTGAAACAG AAGTAACTCT ACCAGAGTTA 480 AGAATTACAA AAGGGGCTTA TTCTCAAGCA ACTAAAACGG CAATTGAGGAATAGATCGATCGATCGATCGATCGATCGAGGTCATGCTTGGCA ATGCAATG CC TGCGTTGGGA TTACCGTTCT 660 GGTCGAGGAC AAGGAAAGAA ACCTTTCAAT AAAGGTACTA TTTTAGAATT TGATCATGCC 720 ATTACTAATA AAATTAGCGA AATTATCAAT GATTTAGAAC CCTCAACACA ACAGTTAGAG 780 CTTTTTTCAT TCAAAAAAAG TCAAGTTCAA ATCAATCTCC GCAAAGGATC TTGTCTTAAT 840 ATCATGCCTT GTCTGCCCAA TGCTGTATAC GATGCGATTA TTACATCACC ACCTTACTGT 900 AATCGATATG ACTATACTCG CACCTATGCT TTAGAATTGG CTTTATTAGG CATTTCTGAA 960 CAAGAATTAA TTAATCTGCG TCAGGAAATG TTGAGTTGTA CAGTTGAAAA TCGTCCCAAA 1020 GATTTACTAG CTATTAATCA AGATTGGGAA TTAGCTTTAA GAGTTGCTGA TTCACAAACT 1080 CTATTGCAAG CCATCTTAAA ATATTTAGAC AACCAAAATG TGCAAGGTTT ATTAAACAAT 1140 AAAGGTATCC CAAGAATGGT GAGGGGATAC TTTTATGAAA TGGCTTGCGT GATTCAAGAA 1200 TGCTTTCGTG TCCTTAAACC TGGAGCATTT TTGTTGATGG TGAATGACAA TGTGCGTTAT 1260 GCAGGTGCAA GCATTTCAGT AGACATGATT CTCTCGGACT TTGCTGAAAA GTTAGGCTTT 1320 GTAGTTGAAA GTATTCTCGT TTTACCAAGT GATAAAGGCA ATAGTAGTCA ACAAATGGGA 1380 AATCATGGAC GCGAGCCTTT GAGGAAATGT GTTTATGTAT GGAAAAAACA A 1431

SEQ ID NO: 5 Sequence length: 2890 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Nostoc species Strain name : features of PCC7524 sequence: 2875 th N unknown nucleotide sequence: GATCACGTCT CTTATGATAG TGTATGAAGG CTAAATTTTT GCAAACATTT TGTAGTTGAG 60 AAACCACAAT CAACTAAAAC CAACACCAAT GTACATATTG GACTGATATA CTATTGAGGC 120 CAGTTAATTC TCCAGGAATT TGTAGCCGTG GTCAGTTTGT CAAGTATCTC AAACATCACC 180 TCTGAAGTGG AAAGCAATGC ACTGAGGGAA ATTGATCATC TTGACAAGCA ATTACAATCT 240 CACTTTCAAA CCAAATTTGT AATTCAGCCT TCACTTACCC GGCTTTTAGT TAGTTTTCAA 300 GCTAACAAAA CTAGACCTAT TTATCGGTGG TACAAGTACA AGGAAGCTTT CTCCGCCTCT 360 CTGGTTGAGC TTTTATGCCA AAAATATGGA ATTACCCAAG GCAAGGTTTT AGATCCCTTT 420 GCAGGTAGTG GAACTGCTTT GTTTGCTACT ATTGACATTG GTATTGATGC TGATGGTATT 480 GAATTGTTAC CTATTGGTCA ACAAATAATT GATACCAAAA AAATTTTAGA TGTAGAATTT 540 ACACCAGATG ATTTCACACG GCTGAAAACT TGGTTGGATT TGCAGGTATG GAAA CAATTT 600 GAAACAGAAG TAACTCTACC AGAGTTAAGA ATTACAAAAG GGGCTTATTC TCAAGCAACT 660 AAAACGGCAA TTGAGCAATA TCTTGAAGCT TGTAATCATG AAAATCATAG GGTTAAATCA 720 GTTTTGCATT TTGCTTTACT CTGTGTTTTG GAATCCGTGA GTTATACCCG CAAAGATGGA 780 CAATACCTGC GTTGGGATTA CCGTTCTGGT CGAGGACAAG GAAAGAAACC TTTCAATAAA 840 GGTACTATTT TAGAATTTGA TCATGCCATT ACTAATAAAA TTAGCGAAAT TATCAATGAT 900 TTAGAACCCT CAACACAACA GTTAGAGCTT TTTTCATTCA AAAAAAGTCA AGTTCAAATC 960 AATCTCCGCA AAGGATCTTG TCTTAATATC ATGCCTTGTC TGCCCAATGC TGTATACGAT 1020 GCGATTATTA CATCACCACC TTACTGTAAT CGATATGACT ATACTCGCAC CTATGCTTTA 1080 GAATTGGCTT TATTAGGCAT TTCTGAACAA GAATTAATTA ATCTGCGTCA GGAAATGTTG 1140 AGTTGTACAG TTGAAAATCG TCCCAAAGAT TTACTAGCTA TTAATCAAGA TTGGGAATTA 1200 GCTTTAAGAG TTGCTGATTC ACAAACTCTA TTGCAAGCCA TCTTAAAATA TTTAGACAAC 1260 CAAAATGTGC AAGGTTTATT AAACAATAAA GGTATCCCAA GAATGGTGAG GGGATACTTT 1320 TATGAAATGG CTTGCGTGAT TCAAGAATGC TTTCGTGTCC TTAAACCTGG AGCATTTTTG 1380 TTGATGGTGA ATGACAATGT GCGTTATGCA GGTGCAAGCA TTTCAGTAGA CATGATTCTC 1440 T CGGACTTTG CTGAAAAGTT AGGCTTTGTA GTTGAAAGTA TTCTCGTTTT ACCAAGTGAT 1500 AAAGGCAATA GTAGTCAACA AATGGGAAAT CATGGACGCG AGCCTTTGAG GAAATGTGTT 1560 TATGTATGGA AAAAACAATA ACTTGAATCT ATGAGTTCTC ACCGTCACCA TCTTCAATCT 1620 AGTGATGATC TTGTAACCAC TTATGAAGCG ACTCGTGCGG GTTTTATCGC ACTCGCCCTG 1680 GAAAAGAATC GACGGGCTAC ACCTTACGTT GCAGAAGCCA GAATACTTCA AGAGGCTGCT 1740 AGTCAAGCCG AAAAACCTGC TGATTTGTTA AACGTGAAAG GGATTGAGAT GGGATTATTA 1800 ACTGCGGCGG GACTGTCTGA AAAATCTCTG GCTCATTTGA TGGCGGAGGA TAAAATTGAA 1860 GCTATCAATG GTCTCATCAG AAATTTTCTT GAGCCAGCAG GCACAAATTT TGTGGAAGAA 1920 TTAGTCTTTA GATTTTTGCT GACTCGTGGT GATACCCTGG GTGGCTCAAT GCGTAACATT 1980 GGAGGAGCTT TAGCACAAAG AAAACTAACT CGTGCTATCC TTTCGACTTT GACAATTGCT 2040 GGTCAGAAAT ATCAATGGCA ACATTCAAAA ACGAAAAAAT GGATAGCGAT GACAAATGAT 2100 GACACAGATA TTGAGTTGTC CTTACGTGGA ATCACTTGGG AGAGTGAGTT TGGTAATCGT 2160 ACACTAATCT ACAACCTAAC TGTTCCTTTG GTTAAAAGTA ATGTGGATTT GTGTTTATTT 2220 AATCTTGCTC CGAAGGAGTT GGTAGCCAAT CAATCCAGCG CAATTGATCC ATCTGTAGTT 2280 GCACCAT ATG CGATCGCACT AGGTGAACTC AAAGGCGGAA TCGATCCCGC AGGCGCAGAT 2340 GAGCATTGGA AAACCGCACA GGCTGCACTC AATCGCATAC GTGAAGCATT TTCCAGAGTT 2400 GGGTATTCGC CTTTGACTTT CTTCGTAGGA TCAGCGATCG CAAAAAGAAT GGCTGGTGAA 2460 ATCTGGAGTC AACTAGAGAA TGGGACTCTT AGTAATGCTG CCAACTTGAA CGAGGAGCAT 2520 CAAGTTGCGT CTATTTCTCG TTGGCTATAC GGTCTATGAG AGCAACAGCT ACTGAGTATT 2580 GGGCTGCAAT AATAAGAAAG AAAAAACTCA GCCAGAGAAT ACATTTTCAT TACTGATAAA 2640 AGTAATGCAG TTGGAATATC TTACAACTTA CTTTTAGCTG CTTCTTTCTG CGCTGTCTCA 2700 CTATATTTTT TATATAGTTG AGTGCGGATT GTAGCTTCTT GATAGCGGCT ATGTTCTGGT 2760 GGTACTGCTG CCATTAAATC AGAAGCTCTT TGCCACATGG CAGCTAGTTC TAACCATTGA 2820 GTTGAGCTTG TAGCTGTTTT TCCAGCAGCA GAGGCTTGGT TAGCGATTCG CACGNTGCCG 2880 CAAATGGATC

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction enzyme map of Nsp7524III restriction / modification enzyme gene of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/21 C12R 1:19) (C12N 9/16 C12R 1:19) (72) Inventor Ikunoshin Kato 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Mina Shuzo Co., Ltd.

Claims (21)

[Claims] 1. An Nsp7524III restriction enzyme comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 2. A polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, wherein N is N.
A polypeptide having sp7524III restriction enzyme activity. 3. A polypeptide having an amino acid sequence which has Nsp7524III restriction enzyme activity, wherein one or more amino acids are added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing. 4. An isolated Nsp7524III restriction enzyme gene comprising a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 5. The gene according to claim 4, comprising the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing. 6. A polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, wherein N is N.
A polynucleotide encoding a polypeptide having sp7524III restriction enzyme activity. 7. A polynucleotide encoding a polypeptide having Nsp7524III restriction enzyme activity, which is hybridizable with the polynucleotide according to claim 4. 8. A polypeptide comprising an amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, in which one or more amino acids are added, deleted or substituted, and which has an amino acid sequence which confers Nsp7524III restriction enzyme activity. A polynucleotide. 9. An Nsp7524III modifying enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. 10. A polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing,
A polypeptide having Nsp7524III modifying enzyme activity. 11. A polypeptide comprising an amino acid sequence which has one or more amino acids added, deleted or substituted in the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, and which has an amino acid sequence imparting Nsp7524III modifying enzyme activity. 12. An isolated Nsp7524III-modifying enzyme gene comprising a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. 13. The gene according to claim 12, comprising the nucleotide sequence set forth in SEQ ID NO: 4 in the sequence listing. 14. A polypeptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing,
A polynucleotide encoding a polypeptide having Nsp7524III modification enzyme activity. 15. A polynucleotide encoding a polypeptide having Nsp7524III-modifying enzyme activity, which is hybridizable with the polynucleotide according to claim 12. 16. A polypeptide encoding an amino acid sequence of SEQ ID NO: 3 in the sequence listing, in which one or more amino acids are added, deleted or substituted, and which has an amino acid sequence which confers Nsp7524III modifying enzyme activity. A polynucleotide. 17. A trait transformed with a vector incorporating the polynucleotide according to any one of claims 4 to 8 and the polynucleotide according to any one of claims 12 to 16. Convertible. 18. A vector comprising the polynucleotide according to any one of claims 4 to 8, and 12 to 12.
A transformant transformed with both of the vectors containing the polynucleotide according to claim 16. 19. A transformant transformed with the vector containing the polynucleotide according to any one of claims 12 to 16. 20. The transformant according to claim 17 or 18 is cultured, and Nsp7524II is extracted from the culture.
A method for producing Nsp7524III restriction enzyme, which comprises collecting a polypeptide having I restriction enzyme activity. 21. A transformant according to any one of claims 17 to 19 is cultured, and Nsp7524 is extracted from the culture.
A method for producing an Nsp7524III-modifying enzyme, which comprises collecting a polypeptide having a III-modifying enzyme activity.