JP3449758B2 - Insertion sequence - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to a novel coryneform bacterium.
It relates to an insertion sequence having a regular DNA base sequence. Tran
Transposable elements such as sposons and insertion sequences are
Found in nuclear cells. Transposable elements on the chromosome
It is possible to transpose and insert into various sites.
Ability of these transposable elements in gene analysis
It can be used as a means. An example of usage is insertion
Preparation of input mutants, gene mapping, promoter search
・ Insertion of genetic information ・ Destruction of specific genes
It can be used as an industrially useful factor. [0002] 2. Description of the Related Art One of transposition sequences in microorganisms is insertion.
The input sequence has several features. They are approximately
It has a size of 1-2 kb,
There is an inverted repeat (inverted repeat sequence).
When inserted into the microbial chromosome,
There is a duplication of sequences. Microbial insertion sequence
From bacteria (Mol. Gen. Genet. Vol. 122, 267-277 (19
73)), derived from Shigella [J. Bacteriol. Vol.
090-4099 (1990)), those derived from acetic acid bacteria (Mol.Microbio
l. Vol. 9, 211-218 (1993)]
Researched well in [Journal Vol.7, 577-584 (1993)]
However, there is a requirement for insertion sequences from coryneform bacteria.
There is no knowledge. [0003] A technique for isolating transposable elements derived from microorganisms has been proposed.
Using a sacB gene product derived from Bacillus subtilis
(J. Bacteriol. Vol. 164, 918-921 (198
5); J. Bacteriol. Vol. 174, 5462-5465 (1992)]. sand
That is, the sacB gene product is transferred to sucrose in the medium.
Therefore, Escherichia coli produced by induction and carrying this gene
When cultured in a medium containing sucrose, the expression of this gene
Is known to lyse Escherichia coli and cause growth inhibition.
Gen. Genet. Vol. 191, 138-144 (1983)
 ]. Therefore, plasmids containing the sacB gene are
Plates transformed with enterobacteria and containing sucrose
Extract plasmids from E. coli grown on top
And the sacB gene on this plasmid was deleted or
One that has an insertion mutation is obtained. This insertion mutation
The transposable element can be isolated from the reaction.
Similarly, in coryneform bacteria, the sacB gene is
Is known to cause growth inhibition [J. Bacterio
l. Vol. 174, 5462-5465 (1992)].
It is possible to isolate transposable elements from Neisseria bacteria. [0004] SUMMARY OF THE INVENTION The conversion from coryneform bacteria
There has been no previous report on the position factor, and coryneform bacterial chromosomes
It is difficult to analyze the above genetic information using a simple method
Met. Industrially important microorganisms, especially coryneform bacteria
Isolation of transposable elements in products
Research can be accelerated, and its usefulness is high. [0005] Means for Solving the Problems The present inventors have solved the above problems.
As a result of intensive research to solve the point, sacB genetic
By using the method of using a new element, a new transposition factor
Isolating the inserted sequence having the sequence shown in SEQ ID NO: 1
Thus, the present invention was completed. Thus book
According to the invention, Corynebacterium glutamicum (Cor
ynebacterium glutamicum)
It has the DNA base sequence shown in 1 above.
An insertion sequence is provided. Hereinafter, the present invention will be described in more detail.
This will be described in detail. [0006] The "insertion sequence" of the present invention refers to a coryneform bacterium.
Present on chromosome, on chromosome or plasmid
It means a DNA base sequence that can be transposed up
It is. Insertion sequence of the present invention (hereinafter referred to as “A fragment”
May be abbreviated) to the source microorganism, coryne
An example of the basic operation for preparing from a bacterium is as follows.
The A fragment is the coryneform bacterium described above, for example,
Corynebacterium glutamicum ATCC31831
This strain is present on the chromosome of the strain,
Plasmid having B gene and capable of replicating in coryneform bacteria
Using, for example, the plasmid pCRY30-sacB
Transform and spread on plates containing sucrose
Plumer than sucrose resistant strains
By extracting the codes, they can be separated and acquired. First, Bacillus subtilis, for example, Marbu
rg) strain [Biochem. Biophys. Res. Commun.,119, 795-8
00 (1984)]. this
Using chromosomal DNA as a template, known sacB and sa
Includes cR (sacB gene expression control gene) gene
The region is amplified and isolated by the PCR method. The amplified DNA fragment
Into a shuttle vector of Escherichia coli and coryneform bacteria,
Escherichia coli (Escherichia coli) J
M109 (commercially available from Takara Shuzo)
To get. Plasmid DNA from the resulting transformant
And coryneform bacteria using the plasmid, for example,
Corynebacterium glutamicum ATCC31831
On a plate containing sucrose
Extract plasmids from growing sucrose-resistant strains
Put out. Of these plasmids, the original plasmid
Select the one that is larger in size and add this book
Inserted into the sacB and sacR gene regions of the mid
A fragment is isolated by isolating the DNA fragment
Can be obtained. [0008] As one of the A fragments thus obtained,
The coryneform bacterium, for example, Corynebacterium
On the chromosomal DNA of Glutamicum ATCC 31831 strain
Fragments that are present and about 1.5 kb in size can be mentioned.
You. The inserted sequence of about 1.5 kb was digested with various restriction enzymes.
The number of recognition sites and the size of the fragment
It is shown in Table 1. [0009] [Table 1] [0010] The function of the fragment A of the present invention is as follows.
For example, it originally exists on the coryneform chromosome,
DNA fragments not present on the plasmid used for the transformation
Under certain conditions, fragments are transferred from chromosome to plasmid
Can be proved by observing the dislocation phenomenon
You. Conversely, the inserted sequence transposed on the plasmid is stained.
The function of fragment A is further confirmed by transposition on the body.
Can be recognized. In this specification, the restriction enzyme
The "number of recognition sites" by the element is a DNA fragment or plasmid
Are completely digested with restriction enzymes, and those digests are
1% agarose gel electrophoresis and 5
% For polyacrylamide gel electrophoresis and separable
The value determined from the number of fragments was adopted. [0011] The "size of the cut fragment"
When using agarose gel electrophoresis
Contains the DNA of E. coli lambda phage (λphage).
Of known molecular weight obtained by digestion with the restriction enzyme HindIII
Drawing of DNA fragments on the same agarose gel as migration distance
Standard polyacrylamide gel
If electrophoresis is used, use E. coli
Restriction enzyme for 74 phage (φx174phage) DNA
DNA fragment of known molecular weight obtained by cleavage with HaeIII
Of the migration distance on the same polyacrylamide gel
Each of the cleaved DNA fragments or plasmids
Calculate the size of the DNA fragment. Plasmid size
Is calculated by adding the sizes of the cut fragments. What
In determining the size of each DNA fragment, 1 kb or more
For fragment size, 1% agarose gel electrophoresis
Approximately 0.1 kb to 1 kb
4% polyacrylamide for full fragment size
The results obtained by electrophoresis were employed. On the other hand, the above-mentioned Corynebacterium gluta
Insertion sequence derived from chromosomal DNA of micam ATCC31831
The nucleotide sequence of the column is the plasmid pUC118 or pUC118.
Dideoxynucleotide yeast using 119 (Takara Shuzo)
Elementary method (dideoxy chain termination method, Sanger, F. et.
 al., Proc. Natl. Acad. Sci. USA, Vol. 74, 5463 (197
7)]. Determined in this way
Insert having the nucleotide sequence of the above-mentioned DNA fragment of about 1.5 kb
The input sequence has the sequence shown in SEQ ID NO: 1 in the sequence listing below.
Things. A fragment of the present invention comprising the above nucleotide sequence
Was isolated from the natural coryneform chromosomal DNA.
Not only, but also a commonly used DNA synthesizer, for example,
Applied Biosystems
Using 394 DNA / RNA synthesizer
May be done. Further, as described above, Corynebacterium
Obtained from the chromosomal DNA of Rutamicam ATCC31832
The inserted sequence of the present invention has the function of the inserted sequence substantially.
Some bases in the base sequence may be replaced by other bases unless otherwise impaired.
May be substituted or deleted.
Or a new base may be inserted,
A part of the base sequence may be transposed,
All of these derivatives are included in the insertion sequences of the present invention.
Is what is done. Here, the dying used for the isolation of the fragment A of the present invention is described.
Has a sacB gene from Bacillus subtilis and replicates in coryneform bacteria
Possible plasmids, such as plasmid pCRY30-s
Method for constructing acB, coli transformable with the plasmid
Necrotic bacteria, a method for culturing the transformant, and the like will be described. Destination
SacB and sacR gene regions
As a shuttle vector for enterobacteria and coryneform bacteria, for example,
For example, a plasmid described in JP-A-3-210184
pCRY30; described in JP-A-2-276575
Plasmids pCRY21, pCRY2KE, pCRY2
KX, pCRY31, pCRY3KE and pCRY3
KX: Plasmid described in JP-A-1-191686
PCRY2 and pCRY3; JP-A-58-6767.
PAM330 described in JP-A No. 9;
PHM1519 described in JP-A No. 5;
No. 900, pAJ655, pAJ611 and
And pAJ1844; see JP-A-57-134500.
PCG1 described; described in JP-A-58-35197
PCG2; described in JP-A-57-183799.
pCG4 and pCG11 can be used. The above plasmid vector pCRY30 was prepared.
Brevibacterium stationi
(Brevibacterium stationis) IFO12144 (F
ERM BP-2515) to plasmid pBY503
(For details of this plasmid, see JP-A-1-9578.
No. 5) DNA was extracted and purified with restriction enzyme XhoI.
The size of the gene containing the gene responsible for the replication and proliferation function of rasmid is about
A 4.0 kb DNA fragment (replication functional region) was cut out and excised.
Approximately 2.1 kb in size with restriction enzymes EcoRI and KpnI
DNA fragment containing the gene responsible for the stabilization function of the plasmid
Cut out a piece (stabilizing function area). Plot both of these sections
EcoRI-K of Rasmid pHSG298 (Takara Shuzo)
By incorporating at the pnI and SalI sites,
Plasmid vector pCRY30 can be prepared
You. Next, the aforementioned Escherichia coli and coryneform bacterium
Of sacB and sacR gene regions into vector
For example, there is only one entry in the plasmid vector.
Cleavage site with the restriction enzyme, the fragment and
Replace the cleaved shuttle vector with S1 nucleus
To blunt ends or a suitable adapter
DNA ligase treatment in the presence of
It can be performed by connecting. SacB into the above plasmid pCRY30
And the introduction of a DNA fragment containing the sacR gene region,
A restriction enzyme that does not cut the gene region, such as BamHI
When the site is amplified by PCR,
This restriction enzyme site is added to the end of the primer used for
In addition, the amplified DNA fragment of the gene region is
BamHI-treated plasmid and plasmid pCRY30
Was cleaved with the restriction enzyme BamHI and DNA ligated.
Can be carried out by linking the two. This plastic
Using E. coli (Escherichia
Coli) JM109 (Takara Shuzo)
A transformant is obtained, and plasmid DNA is extracted from the transformant.
And B. subtilis used for isolation of fragment A of the present invention.
Increased replication in coryneform bacteria with the sacB gene from the fungus
A transposable plasmid can be constructed. The thus constructed plasmid p
Cry30 has sacB and sa of about 2.0 kb in size.
The recombinant plasmid into which the cR gene region has been introduced is called pC
It was named RY30-sacB. Plasmid pCRY3
For details of the method for producing 0-sacB, see the Examples described later.
1 will be described. The bacterium having the sacB gene derived from Bacillus subtilis
Transformed with a plasmid capable of replicating and growing in line-type bacteria
Examples of coryneform bacteria that can be obtained include Corynebacterium
Glutamicum ATCC 31831, Brevibacterium
・ Flavum MJ-233 (FERM BP-1497)
And its derived strain, Brevibacterium ammoniage
Nes (Brevibacterium ammoniagenes) ATCC687
1, the same ATCC 13745, the same ATCC 13746;
Levibacterium divicatam
aricatum) ATCC14020; Brevibacterium
Lactofermentum (Brevibacterium lactofermentu
m) Using ATCC 13869 etc. as a host coryneform bacterium
Can be Among these coryneform bacteria, the present invention
For isolation of fragment A, Corynebacterium glutamicum
Most preferably, ATCC31831 is used as a host.
No. The above-mentioned recombinant plus of the host coryneform bacterium
Transformation with a mid can be carried out by a method known per se, for example, Ca
lvin, N.M. and Hanawalt, P.C., Journal of Bacter
iology,170, 2796 (1988); Ito, K., Nishida, T. and
Izaki. K., Agricultural and Biological Chemistry,
52, 293 (1988), etc.
Energize the main microorganism with pulse waves (Satoh, Y. et al., Journa
l of Industrial Microbiology,Five , 159 (1990))
It can be done by doing. The transformant thus obtained was prepared as described above.
On a plate containing 2-15% sucrose
Each sucrose resistant to grow and grow
Culture the strain in liquid and use the usual
Method, such as the alkali-SDS method.
And analyze the plasmid with appropriate restriction enzymes.
And a plasmid larger in size than the original
Select the sacB and sacR genes of the plasmid
By isolating the DNA fragment inserted into the region
The fragment A of the present invention can be separated and obtained. Here, the cultivation of the above transformant is carried out using a carbon source,
It can be performed in a normal nutrient medium containing sources, inorganic salts, etc.
Sucrose is used as a carbon source. Nitrogen source
As, for example, ammonia, ammonium sulfate, chloride
Ammonium, ammonium nitrate, urea, etc.
Use alone or as a mixture. Examples of inorganic salts include:
For example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, sulfuric acid
Magnesium acid or the like is used. In addition, peptone and meat
, Yeast extract, corn steep liquor, casamino
Add nutrients such as various vitamins such as acid and biotin to the medium
can do. Culturing is usually carried out by aeration stirring or shaking (liquid culture).
Under aerobic conditions of about 25 ° C to about 35 ° C
Can be done with PH during cultivation should be around 7-8
PH can be adjusted during culture by adding acid or alkali.
Can be done. The carbon source concentration at the start of culture
It is 12% by volume. The culture thus obtained is prepared as described above.
Smear on plate containing 2-15% sucrose
By culturing at 33 ° C. for 1 to 3 days, fragment A is
Introduced sucrose resistant strains can be obtained
You. [0026] The present invention has been described above.
This will be described more specifically with an example. Embodiment 1 Coryneform cell having sacB and sacR gene regions
Construction of plasmid capable of replication in bacteria (A) Extraction of total DNA of Bacillus subtilis Semi-synthetic medium A medium [Composition: 2 g of urea, (NH_Four )_Two SO
_Four 7g, K_Two HPO_Four0.5g, KH_Two PO_Four 0.5
g, MgSO_Four 0.5g, FeSO_Four ・ 7H_Two O6mg,
MnSO_Four ・ 4-6H_Two O 6mg, yeast extract 2.5
g, casamino acid 5 g, biotin 200 μg, thiamic acid hydrochloride
200 μg, glucose 20 g, distilled water 1 liter]
1 liter of the Bacillus subtilis strain Marburg
The cells were cultured until the early logarithmic phase, and the cells were collected. The obtained fungus
The body was treated with 10 mM NaCl- containing 10 mg / ml lysozyme.
20 mM Tris buffer (pH 8.0) -1 mM EDTA · 2N
a. Suspended in 15 ml of solution a. Next, proteinase K was added to the final
Add the solution to a concentration of 100 μg / ml, and add
Incubated for hours. Further add sodium dodecyl sulfate to final concentration
Add so that the degree becomes 0.5%, and keep at 50 ° C for 6 hours
And lysed. Add an equal volume of phenol / clo
Add roform solution and gently shake at room temperature for 10 minutes
After that, the whole amount was centrifuged (5,000 × g, 20 minutes,
10 to 12 ° C.), and the supernatant fraction is collected and subjected to sodium acetate
Was added to a concentration of 0.3 M, and then 2 times the amount of ethanol
Was slowly added. Between the aqueous and ethanol layers
The existing DNA is taken up with a glass rod and 70% ethanol
And then air-dried. A 10 mM bird was added to the obtained DNA.
Buffer (pH 7.5)-5 ml of 1 mM EDTA · 2Na solution
And allowed to stand at 4 ° C. overnight.
Used for (B) Selection of primer Nucleotide sequence of sacB and sacR gene regions of Bacillus subtilis
Has already been determined [Mol. Gen. Genet. Vol. 200, 22
0-228 (1985)]. Based on this sequence, sacB and s
The following can amplify a DNA fragment containing the entire acR gene region:
Select a pair of primers for Applied Bio
394 DNA / RNA manufactured by Applied Biosystems
Synthesized using a synthesizer. Note that these
A recognition site for the restriction enzyme BamHI is added to the end of the immer.
Has been added.   (A-1) 5'-CCAGGATCCG ATCCTTTTTA ACCCATCAC-3 '(SEQ ID NO: 2)   (B-1) 5'-CCCGGATCCA AAAGGTTAGG AATACGGTT-3 '(SEQ ID NO: 3) (In the sequence, A is adenine, G is guanine, C is cytosine
And T represent thymine. ) Using this primer
PCR was performed using the chromosomal DNA prepared in (A) as a template.
In the presence of the sacB and sacR gene regions.
Thus, a reaction product of about 2.0 kb is expected to be obtained. (C) PCR reaction (sacB and sacB
Amplification of R gene region) The actual PCR reaction was performed by PerkinElmer Cetus.
Performed under the following conditions using a DNA thermal cycler
Was. Reaction solution: 50 mM KCl 10 mM Tris-HCl (pH 8.4) 1.5 mM MgCl_Two 5 µl of template DNA Each of the primers prepared in the above (B) 0.25 μM dNTPs 200μM each Taq DNA polymerase (Takara Shuzo) 2.5units The above was mixed to make 100 μl. PCR cycle: Denaturation process: 94 ° C for 60 seconds Annealing process: 37 ° C for 120 seconds Extension process: 72 ° C, 180 seconds The above was regarded as one cycle, and 30 cycles were performed. (D) Detection of reactants 10 μl of the reaction solution produced in the above (C) was added to 2% agarose
Approximately 2.0 kb amplification by gel electrophoresis
DNA fragments were detected. (E) Construction of pCRY30 Plasmid vector pCRY30 is disclosed in JP-A-3-210.
It was prepared by the method described in JP-A-184. (F) Construction of pCRY30-sacB An amplified DNA fragment of about 2.0 kb in size obtained in (C) above
And the pCRY30 obtained in the above (E).
 Mix 0.5 μg and add 5 units of BamHI restriction enzyme.
The mixture was mixed and reacted at 37 ° C. for 1 hour to completely digest. 6
After treatment at 5 ° C for 15 minutes to inactivate the enzyme, 50 mM
Squirrel buffer (pH 7.6), 10 mM dithiothreitol,
1 mM ATP, 10 mM MgCl_Two And T4 DNA rigger
Add 1 unit of each component (concentration of each component is final concentration)
Yes) and allowed to react at 4 ° C. for 15 hours for binding. Obtained
Using a mixed plasmid solution, the calcium chloride method (J. Mol.
Biol., Vol. 53, 159 (1970)].
JM109 (Takara Shuzo) was transformed with kanamycin 5
Medium containing 0 mg [10 g of tryptone, yeast extract
5 g of lacto, 5 g of NaCl and 16 g of agar were distilled water
Dissolved in 1 liter]. Growing strains on this medium
After performing liquid culture by a conventional method, plasmid DNA is
Extract and cut the plasmid with the restriction enzyme BamHI
Then, the inserted fragment was confirmed. As a result, the plasmid pCR
In addition to the 8.6 kb DNA fragment of Y30,
A 2.0 kb insert was observed. This recombinant plasmid
This was named pCRY30-sacB. Plasmid p
FIG. 1 shows a map of restriction sites for CRY30-sacB restriction enzyme.
You. Embodiment 2 A simple insertion sequence from Corynebacterium glutamicum
Separation (A) Isolation of sacB resistant strain The plasmid DNA prepared in Example 1 was used for coryneform cells.
The cells were introduced and transformed. Host Coryne used
Type bacteria is Corynebacterium glutamicum ATCC
31831, Brevibacterium flavum MJ-23
3 (FERMBP-1497) plasmid pBY50
2 removed strains. The electric pulse method was used for transformation.
The coryneform bacterium was added to 100 ml of A medium [composition: urea 2
g, ammonium sulfate 7 g, monopotassium phosphate 0.5
g, dipotassium phosphate 0.5 g, MgSO_Four ・ 7H_Two O
  0.5 g, MnSO_Four ・ 4-6H_TwoO 6mg, FeS
O_Four ・ 7H_Two O 6mg, yeast extract 1g, casamino acid 1
g, biotin 200 μg and thiamine hydrochloride 100 μg
Dissolve in ionic water to make 1 liter (pH 7.4)]
After culturing until the beginning of logarithmic growth, penicillin G was added at 1 unit / m
l, and cultured with shaking for another 2 hours. Culture
The cultured cells were collected by centrifugation, and 20 ml of a solution for pulse
Composition: 272 mM sucrose, 7 mM KH_Two PO_Four, 1mM
MgCl_Two PH 7.4]. Centrifuge again
The cells were collected and suspended in 5 ml of the pulse solution,
5 ml of cells and 50 ml of the plasmid solution obtained in (A) above
μl, and allowed to stand on ice for 20 minutes. Genepa
2500 volts 2 using Luther (manufactured by Bio-Rad)
Set to 5μF, and after applying the pulse, let stand on ice for 20 minutes.
Was. After incubating the whole amount at 30 ° C for 1 hour, 10% shoe
Agar medium containing sucrose and 50 μg / ml kanamycin
Smeared. The cells were cultured at 30 ° C. for 2 days. (B) Isolation of inserted sequence Emerging sucrose and kanamycin resistant strains
From the plasmid described in JP-A-1-95785.
It was prepared by the method. Cut this plasmid with various restriction enzymes.
And the size was confirmed.
About 1.5 kb larger than the plasmid used for the transformation
The plasmid was present. In addition, analysis using restriction enzymes
And the inserted sequence having a size of about 1.5 kb
Present in offspring and inactivates the sacB gene product
Turned out to be. Embodiment 3 Salts of the inserted sequence from Corynebacterium glutamicum
Base sequencing The inserted sequence of about 1.5 kb obtained in Example 2 was identified.
To determine the nucleotide sequence, pUC118 or pUC118
Dideoxynucleotide using UC119 (Takara Shuzo)
The method was determined according to the strategy shown in FIG. this
As a result, the sequence described in SEQ ID NO: 1 was clarified. Main distribution
On the top row is an open reading of 436 amino acids.
Frame (ORF) was present. The ends of this insert sequence
Has 24bp inverted repeat, and
There is a direct repeat of 8bp which is the target sequence
did. The inserted sequence of about 1.5 kb in size is
Number of recognition sites and size of digested fragments when digested with restriction enzymes
The size was as shown in Table 1 above. Turn off the restriction enzyme
The breakpoint map is shown in FIG. Embodiment 4 (A) Confirmation of existence of inserted sequence Using the isolated insert as a probe, Corynebacterium
Mu glutamicum ATCC 31831, Brevibacterium
Umm Flabham MJ-233 (FERM BP-149)
For the chromosomal DNA of 7), Southern hybridization
Was conducted. The probe is a random labeling kit.
(Manufactured by Takara Shuzo). Southern Hive
Redidation is carried out by a conventional method [“Molecular Cloning”,
Cold Spring Harbor Laboratory Press (1989)]
went. As a result, the inserted sequence is Corynebacterium
・ Glutamicum ATCC31831-derived chromosome and high
It turned out to be bridging. That is, the insertion of the present invention
The input sequence is Corynebacterium glutamicum ATCC
31831 exists in the chromosome,
This shows that it is possible to translocate upward. Ma
Brevibacterium flavum MJ-233 (FE
RM BP-1497) -derived chromosome
Brevibacterium flavum MJ-23
3 (FERM BP-1497)
Is consistent with the failure to isolate the inserted sequence. (B) Preparation of plasmid for detecting transposition function The inserted sequence of about 1.5 kb obtained above was determined.
Based on the sequence obtained, BamHI
Synthesis of oligonucleotide having site, PCR method
Amplified by Obtained DNA fragment and plasmid pH
SG398 (Takara Shuzo) was mixed with the restriction enzyme BamHI.
  Mix 5 units and react at 37 ° C for 1 hour.
Digested. Treated at 65 ° C. for 15 minutes to inactivate the enzyme
Then, 50 mM Tris buffer (pH 7.6), 10 mM dithios
Reitol, 1 mM ATP, 10 mM MgCl_Two And T4
Add 1 unit of each component of DNA ligase (concentration of each component)
Is the final concentration).
I let you. Using the resulting plasmid mixture, calcium chloride
Method (J. Mol. Biol., Vol. 53, 159 (1970)).
Transforms Richia coli JM109 (commercially available from Takara Shuzo)
Medium containing 50 mg of chloramphenicol [trypto
10 g, yeast extract 5 g, NaCl 5
g and 16 g of agar in 1 liter of distilled water]
did. The culture grown on this medium is liquid-cultured by a standard method,
Extract plasmid DNA from nutrient solution and control the plasmid.
Cleavage was performed with the restriction enzyme BamHI, and the inserted fragment was confirmed.
As a result, the size of the plasmid pHSG398 was 2.7.
In addition to DNA fragment of kb, insert fragment of about 1.5 kb in size
Was observed. Next, the plasmid constructed in the same manner
Is a restriction enzyme NheI that cuts this plasmid at one place.
With kanamycin resistance gene cassette (Pharma
Made in Shea), insert chloramphenicol and kanamaishi
Plasmid resistant to both drugs
pHSG-Tn-Km was prepared. The NheI site has an insertion of about 1.5 kb.
It is located near the end of the sequence and has a DNA fragment of about 1.5 kb.
Open reading frame (ORF)
Do not destroy. Therefore, replication capable of growing in coryneform bacteria
Coryneform bacteria can be isolated using this plasmid without
After transforming, transformants were obtained using kanamycin resistance as an index.
Once obtained, this plasmid is inserted into the coryneform bacterial chromosome.
The resulting strain is obtained. Of these, chloramphenicol feeling
In the competent strain, only the inserted sequence is transposed on the chromosome,
Judge that HSG-Tn-Km as a whole is not included
A, the A fragment obtained by the present invention can be translocated to a chromosome.
It is shown that. (C) Confirmation of dislocation Introduction of plasmid pHSG-Tn-Km into coryneform bacteria
Then, the cells were transformed. Host coryneform bacteria used
Is a Brevibacterium flavae without this insertion sequence
Plus of MJ-233 (FERM BP-1497)
Mid pBY502 deleted strain. For transformation described above
The electric pulse method was used. 150 kanamycin resistant strains
was gotten. Of these, only 8 are chloramphenicol
Chloramphenicol-sensitive
Gender. This means that the plasmid pHSG-Tn-
The insertion sequence present on Km is a kanamycin resistance gene
Both show transposition on the chromosome. [Sequence list] SEQ ID NO: 1 Sequence length: 1469 Sequence type: nucleic acid Number of chains: double strand Topology: linear Sequence type: Genomic DNA origin Organism name: Corynebacterium glutamicum (Coryneb
acterium glutamicum) Stock Name: ATCC 31831 Array features Characteristic symbol: insertion seq Location: 1-1469 How the features were determined: E Array CCTTTTGCGG CCCTTCCGGT TTTGGGGTAC ATCACAGAAC CTGGGCTAGC GGTGTAGACC 60 CGAAAATAAA CGAGCCTTTT GTCAGGGTTA AGGTTTAGGT ATCTAAGCTA ACCAAACACC 120 AACAAAAGGC TCTACCC ATG AAG TCT ACC GGC AAC ATC ATC GCT GAC ACC 170                    Met Lys Ser Thr Gly Asn Ile Ile Ala Asp Thr                      1 5 10 ATC TGC CGC ACT GCG GAA CTA GGA CTC ACC ATC ACC GGC GCT TCC GAT 218 Ile Cys Arg Thr Ala Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp              15 20 25 GCA GGT GAT TAC ACC CTG ATC GAA GCA GAC GCA CTC GAC TAC ACC TCC 266 Ala Gly Asp Tyr Thr Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser          30 35 40 ACC TGC CCA GAA TGC TCC CAA CCT GGG GTG TTT CGT AAT CAC ACC CAC 314 Thr Cys Pro Glu Cys Ser Gln Pro Gly Val Phe Arg Asn His Thr His      45 50 55 CGG ATG CTC ATT GAT TTA CCC ATC GTC GGG TTT CCC ACC AAA CTG TTT 362 Arg Met Leu Ile Asp Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe  60 65 70 75 ATC CGT CTA CCT CGC TAC CGC TGC ACC AAC CCC ACA TGT AAG CAA AAG 410 Ile Arg Leu Pro Arg Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys                  80 85 90 TAT TTC CAA GCA GAA CTA AGC TGC GCT GAC CAC GGT AAA AAG GTC ACC 458 Tyr Phe Gln Ala Glu Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr              95 100 105 CAC CGG GTC ACC CGC TGG ATT TTA CAA CGC CTT GCT ATT GAC CGG ATG 506 His Arg Val Thr Arg Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met         110 115 120 AGT GTT CAC GCA ACC GCG AAA GCA CTT GGG CTA GGG TGG GAT TTA ACC 554 Ser Val His Ala Thr Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr     125 130 135 TGC CAA CTA GCC CTC GAT ATG TGC CGT GAG CTG GTC TAT AAC GAT CCT 602 Cys Gln Leu Ala Leu Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro 140 145 150 155 CAC CAT CTT GAT GGA GTG TAT GTC ATT GGG GTG GAT GAG CAT AAG TGG 650 His His Leu Asp Gly Val Tyr Val Ile Gly Val Asp Glu His Lys Trp                 160 165 170 TCA CAT AAT AGG GCT AAG CAT GGT GAT GGG TTT GTC ACC GTG ATT GTC 698 Ser His Asn Arg Ala Lys His Gly Asp Gly Phe Val Thr Val Ile Val             175 180 185 GAT ATG ACC GGG CAT CGG TAT GAC TCA CGG TGT CCT GCC CGG TTA TTA 746 Asp Met Thr Gly His Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu         190 195 200 GAT GTC GTC CCA GGT CGT AGT GCT GAT GCT TTA CGG TCT TGG CTT GGC 794 Asp Val Val Pro Gly Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly     205 210 215 TCC CGC GGT GAA CAG TTC CGC AAT CAG ATA CGG ATC GTG TCC ATG GAT 842 Ser Arg Gly Glu Gln Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp 220 225 230 235 GGA TTC CAA GGC TAC GCC ACA GCA AGT AAA GAA CTC ATT CCT TCT GCT 890 Gly Phe Gln Gly Tyr Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala                 240 245 250 CGT CGC GTG ATG GAT CCA TTC CAT GTT GTG CGG CTT GCT GGT GAC AAG 938 Arg Arg Val Met Asp Pro Phe His Val Val Arg Leu Ala Gly Asp Lys             255 260 265 CTC ACC GCC TGC CGG CAA CGC CTC CAG CGG GAG AAA TAC CAG CGT CGT 986 Leu Thr Ala Cys Arg Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg         270 275 280 GGT TTA AGC CAG GAT CCG TTG TAT AAA AAC CGG AAG GCT TTG TTG ACC 1034 Gly Leu Ser Gln Asp Pro Leu Tyr Lys Asn Arg Lys Ala Leu Leu Thr     285 290 295 ACG CAC AAG TGG TTG AGT CCT CGT CAG CAA GAA AGC TTG GAG CAG TTG 1082 Thr His Lys Trp Leu Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu 300 305 310 315 TGG GCG TAT GAC AAA GAC TAC GGG GTG TTA AAG CTT GCG TGG CTT GCG 1130 Trp Ala Tyr Asp Lys Asp Tyr Gly Val Leu Lys Leu Ala Trp Leu Ala                 320 325 330 TAT CAG GCG ATT ATT GAT TGT TAT CAG ATG GGT AAT AAG CGT GAA GCG 1178 Tyr Gln Ala Ile Ile Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala             335 340 345 AAA AAG AAA ATG CGG ACC ATT ATT GAT CAG CTT CGG GTG TTG AAG GGG 1226 Lys Lys Lys Met Arg Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly         350 355 360 CCG AAT AAG GAA CTC GCG CAG TTG GGT CGT AGT TTG TTT AAA CGA CTT 1274 Pro Asn Lys Glu Leu Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu     365 370 375 GGT GAT GTG TTG GCG TAT TTC GAT GTT GGT GTC TCC AAC GGT CCG GTC 1322 Gly Asp Val Leu Ala Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val 380 385 390 395 GAA GCG ATC AAC GGA CGG TTG GAG CAT TTG CGT GGG ATT GCT CTA GGT 1370 Glu Ala Ile Asn Gly Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly                 400 405 410 TTC CGT AAT TTG AAC CAC TAC ATT CTG CGG TGC CTT ATC CAT TCA GGG 1418 Phe Arg Asn Leu Asn His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly             415 420 425 CAG TTG GTC CAT AAG ATC AAT GCA CTC TAA AA CAGGAAGAGC CCCTTTTGC 1469 Gln Leu Val His Lys Ile Asn Ala Leu Stop         430 435 SEQ ID NO: 2 Array length: 29 Sequence type: nucleic acid Number of chains: single strand Topology: linear Sequence type: Other nucleic acid Synthetic DNA Array CCAGGATCCG ATCCTTTTTA ACCCATCAC 29 SEQ ID NO: 3 Array length: 29 Sequence type: nucleic acid Number of chains: single strand Topology: linear Sequence type: Other nucleic acid Synthetic DNA Array CCCGGATCCA AAAGGTTAGG AATACGGTT 29

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a plasmid p used for isolating the inserted sequence of the present invention.
CRY30-sacB restriction enzyme map. FIG. 2 is a strategy map for restriction enzyme cleavage point mapping and nucleotide sequence determination of the inserted sequence of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Tsukuba Research Laboratory, Mitsubishi Yuka Co., Ltd. (58) Field surveyed (Int. Cl. ⁷ , DB Name) C12N 15/00-15/90 PubMed, MEDLINE (STN) BIOSIS / WPI (DIALOG) EUROPAT (QUESTEL) GenBank / DDBJ / EMBL / GeneSeq

Claims (1)

(57) [Claim 1] An insertion sequence having the sequence shown in SEQ ID NO: 1.