JPH07322879A - New dna fragment - Google Patents

Abstract

PURPOSE:To obtain a DNA fragment useful for repairing a chromosome derived from a coryneform bacterium or a plasmid DNA, elucidating the mechanism of recombination or replication, suppression, etc., of integration of a plasmid into the chromosome by preparing a strain deficient in recombination ability. CONSTITUTION:This DNA fragment contains a gene capable of coding an RecA protein derived from a coryneform bacterium and has an amino acid sequence expressed by the formula, e.g. a DNA fragment containing the gene capable of coding the RecA protein derived from Brevibacterium flavum MJ-233 (FERM BP-1498). The DNA fragment is obtained by separation thereof from a chromosomal DNA of a natural coryneform bacterium or synthesis thereof in a DNA synthesizer.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to D derived from coryneform bacteria.
DN containing genes involved in NA repair, recombination and replication
Regarding the A fragment, more specifically, DNA repair / recombination /
A gene encoding RecA protein, which is one of the major proteins involved in replication (hereinafter referred to as "recA gene").
Sometimes referred to as)).

The recA gene is one of the important genes involved in the process of repair, recombination and replication of chromosome or plasmid DNA [Annual Review of
Genetics (Annual Review of Genetics), 16 ,
425-436 (1982)], the mechanism of repair / recombination / replication of chromosome or plasmid DNA can be elucidated by analyzing the gene function. Further, by destroying the recA gene, the integration of the plasmid into the chromosome can be suppressed, and the stability and the like of the plasmid can be improved.

[0003]

BACKGROUND OF THE INVENTION Genes involved in repair, recombination and replication of chromosomal DNA are Escherichia c
oli) [Microbiological Review, 48 ,
60-93 (1984); Molecular Microbiology, 5 , 1295-.
1299 (1991), etc.], various genes are known. Among them, the recA gene is known as a particularly important gene involved in the processes of DNA repair / recombination / replication.

The recA gene is Escherichia coli (Es
gene derived from cherichia coli [Proceeding of National Academy of Science (Proc
eeding of National Academy of Science), 77 , 2
611-2615 (1980)], a gene derived from Bacillus subtilis [Nucleic Acids Research],
18 , 4249 (1990)] and the like have been isolated. However, regarding the recA gene derived from coryneform bacterium, which is an industrially important bacterium, as far as the present inventors know, there is no conventional report.

[0005]

As one of the applications of genes involved in DNA repair / recombination / replication, for example,
It may be possible to suppress the integration of the plasmid into the chromosome by analyzing the recombination mechanism or producing a strain lacking recombination ability. However, there is no knowledge of genes involved in repair, recombination, and replication of DNA derived from coryneform bacteria, and there was no application example to industrial recombinant bacteria.

The present invention provides the above DNA derived from coryneform bacteria.
The purpose was to provide genes involved in modification, recombination, and replication.

[0007]

As a result of intensive studies to solve the above problems, the present inventors have found that rec, which is one of the genes involved in repair, recombination and replication of this DNA
The inventors have found that a DNA fragment containing the A gene can be isolated from a certain coryneform bacterium, and completed the present invention.

Thus, according to the present invention, there is provided a DNA fragment containing a gene encoding the RecA protein derived from coryneform bacteria (recA gene). Hereinafter, the present invention will be described in more detail. The "gene encoding RecA protein (recA gene)" of the present invention is DNA
Is one of the genes involved in repair, recombination, and replication of
It means a gene that encodes a protein that has a protease activity that initiates SOS induction of a gene repair pathway and that carries out a DNA strand exchange reaction necessary for recombination repair of a chromosome or a plasmid.

A DNA fragment containing the recA gene (hereinafter,
This may be abbreviated as "A fragment"), but it can be synthesized after its nucleotide sequence is determined, but it is usually cloned from a microorganism and used as a source microorganism for coryneform. -Type bacteria, such as Brevibacterium flavum MJ
-233 (FERM BP-1498), Brevibacterium ammoniage
nes), ATCC 6871, ATCC 13745, ATCC 13746, Brevibacterium devaricatum ATCC1402
0, Brevibacterium lactofermentum (Brev
ibacterium lactofermentum) ATCC 13869, Corynebacterium glutamicum
utamicum) ATCC31831 etc. can be used.

An example of the basic procedure for preparing the A fragment from these source microorganisms is as follows.
The A fragment is the above coryneform bacterium, for example, Brevibacterium flavum MJ-23.
3 (FERM BP-1497) strain is present on the chromosome and can be isolated and obtained from this chromosome by the method described below.

First, Brevibacterium flavum MJ
-Chromosomal DNA from a culture of the -233 strain, which is a method commonly used in itself known by Saito and Miura [Biochemica et Biophysica Acta
) 72 , 619-629 (1963)] and the like. PCR using this chromosome as a template and a common region sequence of the recA gene from Escherichia coli and Bacillus subtilis as a primer [Polymerase Chain Reaction; Mi]
chael A. Innis, PCR Protocols (PCR Protoc
ols) (1990) Academic Press Inc.].

[0012] PCR is a method for amplifying a specific DNA fragment utilizing the fact that DNA polymerase requires a primer. The principle is to use two primers (oligonucleotides) and four deoxynucleotides triphosphates [dNTPS (dATP, dCTP, dG) set in the base sequence of the common region.
TP, dTTP] using one strand of double-stranded DNA as a template,
DNA polymerase synthesizes a double-stranded region sandwiched between two primers. Practical methods include single stranded DNA by heat denaturation (denaturation), annealing of a primer to a template DNA, and synthesis of a complementary strand DNA by a DNA polymerase (extension) under conditions of optimum temperature and time. One cycle is repeated as many times as necessary to amplify a specific DNA fragment.

In the present invention, in practice, a nucleotide sequence of 18 to 27 mer, preferably 20 to 23 mer is selected from the common region sequences of recA genes derived from Escherichia coli and Bacillus subtilis to synthesize a primer. The primer is
For example, Applied Bios
ystems) 394 DNA / RNA Synthesizer (Synthes
izer) can be used for synthesis.

The amount of primers in PCR is 0.05 to 0.5 μM, preferably 0.10 to 0.
Use 30 μM. The PCR conditions are 94 ° C. for 30 to 60 seconds, preferably 60 seconds for the denaturation step, 55 ° C. for 60 to 180 seconds, preferably 55 ° C. for 90 to 150 seconds for the annealing step, and 72 for the extension step.
60 to 180 seconds at ℃, preferably 90 to 120 at 72 ℃
Seconds, and these steps as one cycle, 20 to 4
Perform 0 cycles, preferably 30 cycles.

Thus, rec of about 0.3 kb in size
A gene partial DNA fragment can be amplified. The amplified DNA fragment can be detected using agarose gel electrophoresis. The size of each DNA fragment such as the amplified DNA fragment and the restriction enzyme-digested DNA fragment can be calculated based on the standard line drawn by the migration distance on the same agarose gel with the molecular weight marker DNA fragment. In the determination of the size of each DNA fragment in the present invention, the size of a fragment of 1 kb or more is determined by λ-Hind.
Using the results obtained by 1% agarose gel electrophoresis using III digest (Takara Shuzo) as a marker, the size of fragments of about 0.1 kb to less than 1 kb was measured using pHY Marker (Takara Shuzo).
The results obtained by 4% agarose gel electrophoresis were adopted.

On the other hand, with respect to the recA gene partial DNA fragment having a size of about 0.3 kb obtained by amplifying the chromosomal DNA of Brevibacterium flavum MJ-233 by PCR, the base sequence thereof is the plasmid pGEM-. Dideoxynucleotide enzyme method using T vector (PROMEGA)
[Sanger F. et al., Proceeding of National Academy of Science
f National Academy of Science), 74 , 5463
(1977)].

The above-mentioned approximately 0.3 kb determined in this way
Regarding the nucleotide sequence of the DNA fragment of E. coli, the amino acid sequence deduced from the homology with the amino acid sequences of other microorganisms such as Escherichia coli and Bacillus subtilis is shown in the sequence listing below. 10 having the sequence shown in SEQ ID NO: 1
It is a partial DNA fragment of the recA gene composed of 318 base pairs encoding 6 amino acids.

Next, using this partial DNA fragment of the recA gene as a probe, all the recA genes can be screened from the DNA bank derived from the Brevibacterium flavum MJ-233 chromosome by the hybridization method. First, chromosomal DNA is extracted from a culture of Brevibacterium flavum MJ-233. Use this chromosomal DNA with an appropriate restriction enzyme such as Sa
Partially decompose with u3AI. The obtained DNA fragments of various sizes (9 to 23 kb) are inserted into a lambda phage vector such as λFixII (manufactured by Toyobo Co., Ltd.) to prepare a library. Using this phage vector, Escherichia coli, for example, Escherichia coli P2
392 [Ausubel et al., Nucleic Acids Research, 7 , 1513-15]
[23] and infected with plaque, and then the above recA
Plaque hybridization is performed using the gene partial DNA fragment as a probe. Phage DNA hybridized with the recA gene partial DNA fragment was extracted by the above hybridization, and the inserted DNA fragment was excised with a restriction enzyme SalI to obtain a size of 3.0 kb.
Can be obtained.

One of the A fragments thus obtained is
A DNA fragment having a size of about 3.0 kb obtained by cleaving the chromosomal DNA of Brevibacterium flavum MJ-233 by complete digestion with the restriction enzyme SalI can be mentioned. The number of recognition sites and the size of the cleaved fragment when the DNA fragment containing the about 3.0 kb recA gene was digested with various restriction enzymes are shown in Table 1 below.

[0020]

Table 1 Number of restriction enzyme recognition sites Size of cleaved fragment (kb) BamHI 3 1.2, 1.1, 0.5, 0.2 EcoRI 2 1.3, 1.1, 0.6 HincIII 3 1.8, 0.8, 0.3, 0.1 HindIII 1 1.9, 1.1 KpnI 2 1.4, 0.9, 0.7

The base sequence of the above A fragment is, for example, the dioxynucleotide enzyme method (dideoxychain termination method).
[Sanger, F. et al., Proceeding of National Academy of Science
f National Academy of Science), 74 , 5463
(1977)] can be used to determine r from the open reading frame present in the nucleotide sequence.
The ecA gene can be confirmed. About 3.0k above
The nucleotide sequence of the recA gene whose presence was confirmed in the DNA fragment of b is shown in SEQ ID NO: 2 in the sequence listing below.

The rec of the present invention including the above-mentioned nucleotide sequence
The DNA fragment containing the A gene is not limited to that isolated from a natural coryneform bacterium chromosomal DNA, and a commonly used DNA synthesizer, for example, 394 DNA / RNA synthesizer manufactured by Applied Biosystems is used. It may be synthesized.

The DNA fragment containing the recA gene of the present invention obtained from the chromosomal DNA of Brevibacterium flavum MJ-233 as described above substantially impairs the function of the RecA protein expressed by the gene. As long as there is no, a part of the base sequence may be replaced with other bases or deleted, or a new base may be inserted, and further a part of the base sequence may be rearranged. Any of these derivatives may be a DNA containing the recA gene of the present invention.
It is included in the fragment.

The functional expression of the DNA fragment containing the recA gene of the present invention can be confirmed by examining the DNA repair function, homologous recombination ability and the like of the RecA protein expressed by the gene. The DNA repair function of the RecA protein can be confirmed, for example, as follows using the resistance to UV irradiation and the resistance to methylmethanesulfonic acid as indicators. A DNA fragment containing the recA gene of the present invention, for example, a DNA fragment having a size of about 3.0 kb, is inserted into a plasmid vector, for example, pMW118 (manufactured by Nippon Gene), and the recombinant plasmid is a recA-deficient strain Escherichia coli HB101. (Manufactured by Takara Shuzo) and plasmid vector pMW118
Escherichia coli HB1 only (made by Nippon Gene)
01 (made by Takara Shuzo) and UV
Irradiation is performed or growth is performed on a medium containing methylmethanesulfonic acid. In the strain containing the recA gene-incorporated plasmid, a larger number of viable cells were observed than in the recA gene-deficient strain, so that the DNA repair function of the RecA protein can be confirmed.

Confirmation of the homologous recombination ability of RecA protein is as follows.
For example, kanamycin resistance can be used as an index. A wild strain of Brevibacterium flavum MJ-233 and a recA gene-deficient strain of the strain, a part of a certain gene on the chromosome of the strain, for example, an aspA gene encoding aspartase, a kanamycin resistance vector, for example, The plasmid incorporated in pHSG298 (Takara Shuzo) is transformed. Since the recA gene is present in the wild-type strain, recombination occurs between the recA gene fragment on the plasmid and the homologous site on the chromosome, so that the plasmid is integrated on the chromosome and has kanamycin resistance. On the other hand, the recA-deficient strain has almost no kanamycin resistance because the integration of the plasmid into the chromosome is suppressed. When these two strains were applied to A medium containing kanamycin (50 μg / ml), colonies were observed in the transformant derived from the wild strain, but almost all growth was observed in the transformant derived from the recA-deficient strain. Absent. This gives RecA
The homologous recombination ability of the protein can be confirmed.

The recA gene-deficient strain can be obtained as follows. First, a part of the recA gene of the present invention (shown in SEQ ID NO: 2 in the late sequence list) is amplified by PCR or cut out at an appropriate length to obtain, for example, a vector pHS having clamphenicol resistance.
Built in G398 (Takara Shuzo), Brevibacterium
Transform into a wild-type strain of flavum MJ-233. This is spread on A medium containing clamphenicol (3 μg / ml). As a result, a plasmid was integrated into the recA gene part, and clamphenicol became resistant.
A cA gene-deficient strain can be selected.

[0027]

The present invention has been described above.
This will be described more concretely by way of example.Example 1 Brevibacterium flavum MJ-233
Cloning of the A fragment from (A) All of Brevibacterium flavum MJ-233
Extraction of DNA Brevibacterium flavum MJ-233 (FERM
 BP-1498) bacterial cells with a platinum loop,
Adult medium A medium [Composition: Urea 2 g, (NH_Four) ₂SO_Four
7g, K₂HPO_Four0.5g, KH₂PO_Four0.5g,
MgSO_Four0.5g, FeSO_Four・ 7H₂O 6mg,
MnSO_Four・ 4-6H₂O 6mg, yeast extract 2.
5 g, casamino acid 5 g, biotin 200 μg, hydrochloric acid
Distill thiamin 200μg and glucose 20g
Dissolved in 1 liter of water] and incubate at 30-33 ° C for 12-
Shaking culture (preculture) was performed for 16 hours. Next, A medium 100
Inoculate 2% of the pre-cultured solution into ml and incubate at 31 ℃ for 10 to 16 hours.
The cells were shaken and cultured until the late logarithmic growth phase. This culture
Centrifugation (5,000 xg, 10 minutes, 15-20 ° C)
The microbial cells were collected.

The obtained cells were adjusted to a concentration of 10 mg / ml with 10 μg / ml lysozyme and NaCl-20.
mM Tris buffer (pH 8.0) and 1 mM EDTA
Suspended in an aqueous solution containing 2Na (the concentration of each component is the final concentration). Next, proteinase K was added to a final concentration of 10
The mixture was added at 0 μg / ml and kept at 37 ° C. for 1 hour. Furthermore, the final concentration of sodium dodecyl sulfate is 0.5
%, And the mixture was kept at 50 ° C. for 6 hours for lysis. An equal amount of phenol / chloroform solution was added to this lysate, and the mixture was gently shaken at room temperature for 10 minutes, and then the whole was centrifuged (3,800 xg, 15 minutes, 15 to 20 minutes).
C.) and the supernatant fraction was collected. Sodium acetate was added to this supernatant so that the concentration was 0.3 M, and then twice the amount of ethanol was slowly added. The DNA existing between the aqueous layer and the ethanol layer was scattered with a glass rod, washed with 70% ethanol, and then air-dried. 10 mM Tris buffer (pH 7.5), 1 mM EDTA · 2Na was added to the obtained DNA.
5 ml of an aqueous solution containing
Used as NA in PCR.

(B) Selection of Primer The recA gene derived from Escherichia coli and Bacillus subtilis has been well studied, and its nucleotide sequence has been determined.
Areas conserved between these two microorganisms, especially DN
In particular, the conserved region in the A base sequence was examined, the following pair of primers were selected, and Applied Biosystems' 394 D was used.
It was synthesized using an NA / RNA synthesizer. (A-1) 5'-TTY ATI GAI GCI GAR CAY GCI YT-3 '(b-1) 5'-CCI CCI GTI GTI GTI TCI GG-3' (R is A or G, Y is C Or T, where A is adenine, G is guanine, C is cytosine, T is thymine, and I is deoxyinosine.)

(C) PCR The actual PCR is a DN manufactured by Perkin Elmer Cetus.
A thermal cycler was used under the following conditions using Recombinant Taq DNA Polymerase TaKaRa Taq (Takara Shuzo) as a reaction reagent. Reaction solution: (10 ×) PCR buffer 10 μl 1.25 mM dNTP mixed solution 16 μl Template DNA MgCl ₂ 10 μl (DNA content 1 μM or less) Primers prepared in the above (B) 1 μl each (final concentration 0.25 μM) Recombinant tack DNA / Polymerase 0.5 μl Sterile distilled water 62.5 μl The above was mixed, and 100 μl of this reaction solution was subjected to PCR. PCR cycle: Denaturation process: 94 ° C. for 60 seconds Annealing process: 37 ° C. for 120 seconds Extension process: 72 ° C. for 180 seconds 30 cycles were carried out with the above as one cycle.

(D) Detection of reaction product 10 μl of the reaction solution produced in the above (C) was electrophoresed on a 2% agarose gel to detect a DNA fragment of about 0.3 kb.

(E) Cloning of amplified fragment T4 DNA ligase 10 was mixed with 4 μl of the reaction solution obtained in the above (C) and 1 μl of PCR product cloning vector pGEM-T vector (commercially available from PROMEGA).
X) Add 1 μl of buffer solution and each component of T4 DNA ligase 1 unit, and make to 10 μl with sterile distilled water,
And reacted for 3 hours to bind. Calcium chloride method [Journal of Molecular Bi
ology, 53 , 159 (1970)], and transformed into Escherichia coli JM109 (Takara Shuzo Co., Ltd.) and dissolved in a medium containing 50 mg of ampicillin [trypton 10 g, yeast extract 5 g, NaCl 5 g and agar 16 g dissolved in distilled water 11]. Smeared

The strain grown on this medium was subjected to liquid culture by a conventional method, plasmid DNA was extracted from the culture, and the plasmid was cleaved with a restriction enzyme to confirm the inserted fragment. As a result, the plasmid pGEM-T had a DN of 3.0 kb in length.
In addition to the A fragment, an insert DNA fragment having a length of about 0.3 kb was recognized.

Example 2 Determination of Nucleotide Sequence of Amplified Fragment Regarding the amplified fragment having a size of about 0.3 kb obtained in the item (E) of Example 1, its nucleotide sequence was determined by the dideoxynucleotide termination method. [Sanger,
F. et al., Proceeding of National A
cademy of Science), 74 , 5463 (1977)]
Determined by As a result of the nucleotide sequence determination, the inserted fragment showed high homology with a part of the recA gene derived from Escherichia coli and Bacillus subtilis (the region sandwiched by the primers used for PCR), and Brevibacterium flavum MJ-23.
It was confirmed that a part of the recA gene derived from 3 was cloned.

Example 3 Brevibacterium flavum
Of a DNA fragment containing the recA gene derived from MJ-233
Cloning 90 μl of a total DNA solution of Brevibacterium flavum MJ-233 obtained in the above Example 1 (A) was used at a temperature of 37 ° C. for 10 times at 37 ° C. using a restriction enzyme Sau3AI 5 units.
It was reacted for a minute and partially decomposed. This degraded DNA and λFix
II / XhoI Partial fill-in vector
Kit (Partial Fill-In Vector Kit) and Klenow
A library was prepared by using a fragment (Klenow fragment) (manufactured by Toyobo) according to the protocol. Next, a λDNA library phage solution (2-5 × 10
⁴ pfu; SM buffer dilution) and Escherichia coli P2
Equivalently mix the culture solution of 392 (manufactured by Toyobo Co., Ltd.),
Incubated for 5 minutes. Keep it warm at 50 ℃ for 3 ~
4 ml of lambda medium (10 g tryptone, 5 g yeast extract, 5 g NaCl, MgSO ₄ .7H ₂
2 g of O and 5 g of agar were dissolved in 1 liter of distilled water) and added to a λ plate (10 g of tryptone, 5 g of NaCl,
Dissolve ₂ g of MgSO ₄ .7H ₂ O and 10 g of agar in 1 liter of distilled water) and uniformly coat the mixture at 37 ° C. for 12-16
Incubated for hours.

A plaque formed on this medium was adsorbed on a nitrocellulose filter, and successively 5 minutes each, a) and b) a) a solution of 20 g of NaOH and 87.5 g of NaCl dissolved in distilled water to make 1 l. , B) tris (hydroxymethyl) aminomethane 121.
2 g and 30 g of NaCl were dissolved in distilled water and
A filter was placed on a filter paper dipped in a solution of pH 1 adjusted to 7.0 with Cl and treated, and treated. After air drying,
DNA was fixed by performing a dry heat treatment at 80 ° C. for 2 hours.

Using the recA partial fragment obtained by the PCR method as a probe, plaque hybridization was carried out using the filter prepared above. At this time, the probe was produced using a random labeling kit (Takara Shuzo).
Plaque hybridization can be carried out by a conventional method [Molecular Cloning, cold
Spring Harbor Laboratory (Cold Spring Ha
rbor Laboratory) (1989)]. As a result, positive plaques could be selected, and phage DNA was extracted from the plaques to confirm an insert fragment having a size of about 3.0 kb which was excised by the restriction enzyme SalI. This was inserted into the SalI site of the plasmid pMW118 and used for confirmation of the entire recA gene region. The number of recognition sites and the size of the cleaved fragment when the DNA fragment having a size of about 3.0 kb was cleaved with various restriction enzymes are shown in Table 1 above.
It was as shown in. The restriction enzyme cleavage point map is shown in FIG.

Example 4 Confirmation of the entire region of recA gene In order to identify the site of the recA gene present on the DNA fragment of about 3.0 kb obtained in Example 3, the following sequence was used. It was determined. 14 μl of a DNA fragment solution having a size of about 3.0 kb was digested with the restriction enzyme Sa
Using u3AI5units, reaction was carried out at 37 ° C. for 1 to 5 minutes for partial decomposition. On the other hand, cloning vector pUC1
18 (commercially available from Takara Shuzo) was cleaved with a restriction enzyme BamHI and dephosphorylated. This vector was mixed with the partially digested DNA fragment described above, and 50 mM Tris buffer (p
H7.6), 10 mM dithiothreitol, 1 mM AT
P, 10 mM MgCl ₂ and T4 DNA ligase
1 unit of each component (concentration of each component is the final concentration) was added, and reacted at 16 ° C. for 10 hours to bind.
DNA fragment solution 1 of about 3.0 kb in size as above
4 μl of the restriction enzyme TaqI 50 units,
Partial decomposition was carried out by reacting for 5 to 8 minutes. The cloning vector pUC118 was cleaved with the restriction enzyme AccI, dephosphorylated and ligated to the partially degraded DNA fragment in the same manner as above.

Using the plasmid mixture obtained above, the calcium chloride method [Journal of Molecular Biology, 53 ,
159 (1970)] by Escherichia coli JM1
09 (Takara Shuzo) was transformed and ampicillin 50μ
The solution was applied to a medium containing 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 16 g of agar dissolved in 1 l of distilled water containing g / ml.

The strain grown on this medium is liquid-cultured by a conventional method, plasmid DNA is extracted from the culture solution, and its nucleotide sequence is determined by the dideoxy nucleotide method (Side method) using pUC118 (Takara Shuzo).
er F. et al. , Proceeding of National Academy of Science
onal Academy of Science), 74 , 5463 (197)
7)] was determined as follows.

Plasmid DNA extracted from the above culture solution
Catalyst 800 Molecular Biology Lab Station; manufactured by Applied Biosystems (CATA
LYST800 Moleculer Biology Labostation ； Applied Bio
system) to react according to the protocol,
The nucleotide sequence of the inserted DNA fragment of each plasmid was determined by a 373A DNA sequencer (manufactured by Applied Biosystems). The sequence analysis software INHERIT; Applied Bios
(manufactured by ystems) to determine the entire base sequence.

From the existence of the open reading frame in the base sequence, it was revealed that the recA gene DNA is composed of 1017 base pairs encoding 338 amino acids having the base sequence shown in SEQ ID NO: 2 in the late sequence listing. became. FIG. 1 shows the restriction enzyme cleavage points of the DNA fragment of the present invention having a size of about 3.0 kb, the orientation of the nucleotide sequence of the inserted DNA fragment of each of the above-mentioned plasmids and the position of RecA gene DNA. .

[0043]

INDUSTRIAL APPLICABILITY The transformant strain in which the DNA fragment containing the recA gene of the present invention has been incorporated improves the gene recombination ability of the microorganism, and thus, for example, a homologous site with a gene into which transformation or the like has been introduced from the outside is introduced. The frequency of recombination in plants will increase, and breeding efficiency will be improved. In addition, by using the DNA fragment containing the recA gene of the present invention to delete or weaken the recombination ability of the host, it is possible to suppress the integration of the artificially introduced plasmid into the chromosome. It is expected to contribute to the stabilization of the plasmid within.

[0044]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 318 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Brevibacterium flavum Strain name: MJ-233 Sequence characteristics Characteristic symbol: CDS Location: 1-318 Method for determining characteristic: E sequence GAT CCA GAT TAT GCT CGC AAG CTT GGT GTA GAT ACT GAT GCG CTT CTG GTT TCG Asp Pro Asp Tyr Ala Arg Lys Leu Gly Val Asp Thr Asp Ala Leu Leu Val Ser 1 5 10 15 CAG CCA GAC ACT GGT GAG CAA GCA CTA GAA ATC GCC GAC ATG CTG GTT CGT TCC Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile Ala Asp Met Leu Val Arg Ser 20 25 30 35 GGC GCA ATC GAC ATC ATC GTG ATT GAC TCG GTG GCT GCG CTG ACA CCA AAG GCT Gly Ala Ile Asp Ile Ile Val Ile Asp Ser Val Ala Ala Leu Thr Pro Lys Ala 40 45 50 GAA ATT GAA GGC GAA ATG GGC GAT AGC CAC GTT GGT CTT CAG GCC CGC CTC ATG Glu Ile Glu Gly Glu Met Gly Asp Ser His Val Gly Leu Gln Ala Arg Leu Met 55 60 65 70 AGC CAG GCG CTT CGT AAG ATG A CA GGT GCG CTG TAC AAC TCG GGT ACC ACC GCG Ser Gln Ala Leu Arg Lys Met Thr Gly Ala Leu Tyr Asn Ser Gly Thr Thr Ala 75 80 85 90 ATC CTC ATT AAC CAG CTG CGT GAA AAG ATC GGT GTG ATG TTC GGT TCC Ile Leu Ile Asn Gln Leu Arg Glu Lys Ile Gly Val Met Phe Gly Ser 95 100 105

SEQ ID NO: 2 Sequence length: 1017 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Brevibacterium flavum Strain name: MJ-233 Characteristic of the sequence Characteristic symbol: CDS Location: 1-1017 Method of determining the characteristic: E Sequence ATG CGT CTG GGT GAT GAG AAT CGT CCG CCA ATC CAG ACC ATC TCA 45 Met Arg Leu Gly Asp Glu Asn Arg Pro Pro Ile Gln Thr Ile Ser 1 5 10 15 TCT GGT AAC ACC GCG ATT GAT ATC GCC TTG GGT ATC GGT GGA TTC 90 Ser Gly Asn Thr Ala Ile Asp Ile Ala Leu Gly Ile Gly Gly Phe 20 25 30 CCA CGT GGT CGA ATC GTT GAG GTG TAT GGC CCA GAA TCA TCA GGT 135 Pro Arg Gly Arg Ile Val Glu Val Tyr Gly Pro Glu Ser Ser Gly 35 40 45 AAA ACC ACC GTT GCA CTG CAC GCT ATT GCG CAG GCA CAA AAG GCC 180 Lys Thr Thr Val Ala Leu His Ala Ile Ala Gln Ala Gln Lys Ala 50 55 60 GGC GGC ATC GCT GCA TTC ATT GAC GCC GAG CAC GCG TTG GAT CCA 225 Gly Gly Ile Ala Ala Phe Ile A sp Ala Glu His Ala Leu Asp Pro 65 70 75 GAT TAT GCT CGC AAG CTT GGT GTA GAT ACT GAT GCG CTT CTG GTT 270 Asp Tyr Ala Arg Lys Leu Gly Val Asp Thr Asp Ala Leu Leu Val 80 85 90 TCG CAG CCA GAC ACT GGT GAG CAA GCA CTA GAA ATC GCC GAC ATG 315 Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile Ala Asp Met 95 100 105 CTG GTT CGT TCC GGC GCA ATC GAC ATC ATC GTG ATT GAC TCG GTG 360 Leu Val Arg Ser Gly Ala Ile Asp Ile Ile Val Ile Asp Ser Val 110 115 120 GCT GCG CTG ACA CCA AAG GCT GAA ATT GAA GGC GAA ATG GGC GAT 405 Ala Ala Leu Thr Pro Lys Ala Glu Ile Glu Gly Glu Met Gly Asp 125 130 135 AGC CAC GTT GGT CTT CAG GCC CGC CTC ATG AGC CAG GCG CTT CGT 450 Ser His Val Gly Leu Gln Ala Arg Leu Met Ser Gln Ala Leu Arg 140 145 150 AAG ATG ACA GGT GCG CTG TAC AAC TCG GGT ACC ACC GCG ATC TTC 495 Lys Met Thr Gly Ala Leu Tyr Asn Ser Gly Thr Thr Ala Ile Phe 155 160 165 ATT AAC CAG CTG CGT GAA AAG ATC GGT GTG ATG TTC GGT TCC CCA 540 Ile Asn Gln Leu Arg Glu Lys Ile Gly Val Met Phe Gly Ser Pro 170 175 180 GAA ACC ACC ACC GGT GGT AAG GCC CTG AAG TTC TAC GCA TCT GTT 585 Glu Thr Thr Thr Gly Gly Lys Ala Leu Lys Phe Tyr Ala Ser Val 185 190 195 CGT TGT GAC ATT CGA CGA ATC CAG ACT CTG AAG GAC GGA CAG GAT 630 Arg Cys Asp Ile Arg Arg Ile Gln Thr Leu Lys Asp Gly Gln Asp 200 205 210 GCC ATT GGT AAC CGC ACC CGC TTG AAG GTC GTT AAG AAC AAG GTC 675 Ala Ile Gly Asn Arg Thr Arg Leu Lys Val Val Lys Asn Lys Val 215 220 225 TCC CCA CCG TTC AAG ATC GCT GAA TTC GAC ATC ATG TAC GGC GAA 720 Ser Pro Pro Phe Lys Ile Ala Glu Phe Asp Ile Met Tyr Gly Glu 230 235 240 GGC ATC TCC CGT GAA TCC TCC GTC ATT GAC TTG GCA GTG GAC AAC 765 Gly Ile Ser Arg Glu Ser Ser Val Ile Asp Leu Ala Val Asp Asn 245 250 255 GGC ATT GTG AAG AAG TCA GGT TCC TGG TTC ACC TAC GAG GGT GAA 810 Gly Ile Val Lys Lys Ser Gly Ser Trp Phe Thr Tyr Glu Gly Glu 260 265 265 270 CAG CTT GGT CAA GGT AAG GAA AAG GTG CGT CTT TCC CTC AAG GAG 855 Gln Leu Gly Gln Gly Lys Glu Lys Val Arg Leu Ser Leu Lys Glu 275 280 285 AAC CCT GAA CTC ACC GAT GAG CTG GAA GAT AAG ATC TTC AAG 900 Asn Pro Glu Leu Thr Asp Glu Leu Glu Asp Lys Ile Phe Lys Lys 290 295 300 CTG GGA GTA GGC AAG TAC GCT GCA GCC TCA GAT GAA CTC ACC GAC 945 Leu Gly Val Gly Lys Tyr Ala Ala Ala Ser Asp Glu Leu Thr Asp 305 310 315 GAA CCA GTA GAG CTC GTG CCT AAC GTT GAC TTC GAT GAT GAA GCC 990 Glu Pro Val Glu Leu Val Pro Asn Val Asp Phe Asp Asp Glu Ala 320 325 330 GAC ACC GAA GCA GAC GCT GAA GAC TAA 1017 Asp Thr Glu Ala Asp Ala Glu Asp 335

[0046]

[Brief description of drawings]

FIG. 1 is a DNA fragment of the present invention having a size of about 3.0 kb.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:13) (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Inside the Tsukuba Research Institute, Mitsubishi Petrochemical Co., Ltd.

Claims (5)

[Claims] 1. A DNA fragment containing a gene encoding a RecA protein derived from a coryneform bacterium. 2. The DNA fragment according to claim 1, wherein the coryneform bacterium is Brevibacterium flavum MJ-233. 3. The DN according to claim 1 or 2, which has a restriction enzyme SalI recognition site at both ends and is cleaved by the following restriction enzyme to the size of the following cleavage fragment with the number of the following restriction enzyme sites.
A fragment. Restriction enzyme number of recognition sites Size of cleavage fragment (kb) BamHI 3 1.2, 1.1, 0.5, 0.2 EcoRI 2 1.3, 1.1, 0.6 HincII 3 1.8, 0 .8, 0.3, 0.1 HindIII 1 1.9, 1.1 KpnI 2 1.4, 0.9, 0.7 4. A DNA containing a gene encoding RecA protein represented by the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing.
fragment. 5. A DNA fragment containing a gene encoding the RecA protein represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing.