JP2524794B2 - Complex plasmid vector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to E. coli and Bacillus subtilis, in particular E. coli recA ⁺ and r.
ECA ^- Usefulness novel composite plasmid vector strain as a cloning vector and host ^- strain, B. subtilis recE ⁺ and recE.

More particularly, the present invention provides plasmid pAM of Streptococcus faecalis (Streptococcus faecalis)
α1 and Escherichia coli vector pACYC1
The present invention relates to a cloning vector composed of a novel derivative of a composite plasmid synthesized from 77, particularly, (a) the tetracycline resistance gene region derived from the plasmid pAMα1 and its replication initiation region, and (b) the ampicillin resistance gene derived from the plasmid vector pACYC177. Region and its replication initiation region, and (c) Sop-α1 (stability of plas which stably holds the plasmid in the Bacillus subtilis recE ^- strain).
mid) region, and (d) both ends with Hind III
A specific polylinker having a recognition cleavage sequence of Eco 47III,
And having the tetracycline resistance gene region,
Bal I, Bss HII, Hpa I, Cla I, Eco RV, Ppu MI, Afl II
I, Bst EII unique region in all regions (one cut sit
e) is located, further, the above ampicillin-resistant gene region, Pvu I Bgl I, Mst I , where, wherein the entire region in the unique recognition cleavage site Apa LI is positioned, Escherichia coli, Bacillus subtilis The present invention relates to a novel composite plasmid vector useful as a cloning vector for both purposes.

Generally, in genetic engineering, in order to transfer a desired foreign gene into a host cell and express its genetic information, it is required to use a vector compatible with the host cell. Regarding the vector in genetic engineering, the host-vector system of Escherichia coli, which has many research cases, has been most developed, but recently, microorganisms other than E. coli, for example,
BACKGROUND ART Actively developing host-vector systems for industrially useful microorganisms such as Bacillus subtilis, antibiotic-producing actinomycetes, and yeasts widely used in the brewing field.

By the way, regarding the essential conditions as a vector,
It is known that (a) a gene sequence required for self-replication and (b) the presence of a restriction cleavage site for recognizing a restriction enzyme for inserting a foreign gene.
For example, in order to enable and facilitate genetic manipulation, (c) diversity of available restriction enzymes and their recognition cleavage sites, (d) high efficiency of trait expression, (e) trait Presence of marker gene required for detection of transformants, (f) compatibility with host cells and host range, (g) stability in host cells, and (h) adaptation to putative biological containment Since the vector is required to have strict properties that satisfy all the various conditions such as sex, the present situation is that there are not many practically useful vector development examples for the host-vector system of Escherichia coli and Bacillus subtilis. Is.

In addition, the most researched and developed gene cloning system at present is a vector system (EK system) for Escherichia coli, which is a gram-negative bacterium. It has become possible to efficiently express both a certain gene derived from Gram-positive bacteria and the like.

On the other hand, as for the system of Gram-positive bacteria, its biological properties are completely different from those of Gram-negative E. coli, so there are more problems to be solved, Particularly, among Gram-positive bacteria, Bacillus subtilis is a useful microorganism having various practical advantages, and therefore, its genetic analysis, and further, high-efficiency cloning system Development is strongly expected.

In view of the present situation, the present inventors have so far examined Gram-negative Escherichia coli whose biological properties are best understood and Gram-positive Bacillus subtilis industrially useful as a production bacterium such as amylase. In order to establish the host-vector system, development research of useful plasmid vector has been accumulated. As a result, the plasmid pAMα1 of Streptococcus faecalis
, And a novel derivative of a composite plasmid synthesized from the Escherichia coli vector pACYC177 has been found to have various properties suitable as a vector in genetic engineering of both Escherichia coli and Bacillus subtilis. ,
For example, JP-A-59-135890, JP-A-59-135891, JP-A-59-227296, JP-A-60-87789, and JP-A-60-149.
We have applied for patents such as 385 and JP-A-60-248184.

Against the background of such technical accumulation, the present inventors have conducted diligent research to develop a more useful plasmid vector, and as a result, without losing the excellent properties of the vectors developed so far, and Have succeeded in developing an excellent shuttle vector capable of utilizing a wider variety of restriction enzymes, and have completed the present invention.

That is, the present invention relates to Escherichia coli and Bacillus subtilis, particularly Escherichia coli recA.
It is intended to provide a novel composite plasmid vector useful as a cloning vector using ⁺ and recA ⁻ strains and Bacillus subtilis recE ⁺ and recE ⁻ strains as hosts. Furthermore, the present invention aims to provide a novel shuttle vector for Escherichia coli and Bacillus subtilis, which has various cloning sites so that various restriction enzymes can be used.

The construction of the composite plasmid vector of the present invention is
(A) Streptococcu
s faecalis ) DS5 (ATCC14508) plasmid pAMα1 derived tetracycline resistance gene region (Tc) and its replication initiation region (Ori-pAMα1), and (b) E. coli vector pACYC177 derived ampicillin resistance gene region (Am
p) and its replication initiation region (Ori-177), (c) the Sop-α1 region which acts to stably hold the plasmid in the Bacillus subtilis recE ⁻ strain, and (d) Hind III at both ends.
And a specific polylinker having a recognition cleavage site for Eco 47III, and representative examples thereof include pHY320PLK and pHY321PLK.

Then, the composite plasmid vector pHY320PLK of the present invention
And the synthetic route of pHY321PLK will be described below with reference to FIG.

Conventionally known composite plasmids pHY340 and pHY300PLK were used as starting materials for the synthesis of the plasmid. These composite plasmids are all composite plasmids synthesized from the Streptococcus faecalis plasmid pAMα1 and the E. coli vector pACYC177, and were developed by the present inventors (Japanese Patent Laid-Open No. 60-87789). No., JP-A-60-248184).

(1) E at the Sac II site of the composite plasmid pHY340 (5.62Kb)
DNA prepared by inserting co RI linker d (pGGAATTCC)
From (5.62 Kb) (Fig. 2C), a region (about 750 bp) flanked by two Eco R1 sites containing the Sop-α1 region was cut out with Eco R1, and the composite plasmid pHY300PLK (4.87 Kb) was excised.
Cloned into the Eco RI site of pHY340PLKE ¹ E ² (5.62K
b) was synthesized (Fig. 2D).

(2) Next, this pHY340PLKE ¹ E ² was partially digested with Eco RI to cut out a DNA having a molecular weight of 3.4 Md, which was treated with T4 DNA polymerase, and then BbeI linker d (pAGGCGCCT)
And treated with T4 ligase to obtain Ec of the pHY340PLKE ¹ E ²
o Insert one BbeI linker at RI ² site to obtain pHY340PLKE ¹ Bb
(5.62 Kb) was synthesized (Fig. 2E).

(3) Since there are two PstI sites in this pHY340PLKE ¹ Bb, the PstI site in the polylinker cannot be used as a cloning site. So pHY340PL
The 5th A in the -CTGCAG- base sequence, which is the PstI ² site of KE ¹ Bb, was subjected to a site-specific base substitution method (Morinaga, Y. et al. Bio.
technology 2 , 636-1984)
pHY340PLK (5.62Kb) was synthesized (Fig. 2F).

(4) Then, cleave pHY340PLK with Eco RI and Eco 47 III.
250 bp between the Eco RI site and the Eco 47 III site was removed, and instead of the removed part, it had an Eco RI cut end at one end and an Eco 47 III cut end at the other end, and Mr between both cut ends was
Insert a specific synthetic DNA with oI site as a linker,
As a result, a novel complex plasmid having a molecular weight of about 3.2 Md and represented by the restriction enzyme digestion map of Fig. 3 was synthesized, and named as pHY320PLK (5.38Kb) (No. 2).
(Figure G).

(5) Furthermore, the Hin of the polylinker part of this pHY320PLK
At the position of 2.36 Kb from the dIII site, one base of -CCTATG-base sequence was replaced by the site-specific base substitution method to -CATATG
-A novel composite plasmid having a modified base sequence and a molecular weight of about 3.2 Md, which is represented by the restriction enzyme digestion map of Fig. 4, was synthesized and named as pHY321PLK (5.38Kb) (Fig. 2H). ).

This novel composite plasmid (pHY321PLK) was compared with the above-mentioned novel composite plasmid (pHY320PLK) by Nde.
It is characteristic in having an I site.

The novel composite plasmid of the present invention synthesized by the above synthetic pathway is Escherichia coli and Bacillus subtilis, especially Escherichia coli recA ^+.
Strains and recA ^- strains, Bacillus subtilis recE ⁺ strains and recE ^- strains have the characteristic that they are stably maintained for a long period of time, and the transformation efficiencies of both strains are almost the same. Therefore, it is useful as a shuttle vector for Escherichia coli and Bacillus subtilis.

 The composite plasmid vector of the present invention has the DNA on its DNA.
The region resistant to tracycline and ampicillin
When present in E. coli, the host cell
, With the property of conferring resistance traits to both drugs
There is. This property is due to recombination incorporating the desired foreign gene.
To obtain strains carrying the plasmid, that is, transformants
Selection marker for detecting and selecting
Contribute to the role of the car. In addition, this composite plasmid vector
In the tetracycline resistance gene region ofBal
I,BssHII,HpaI,ClaI,EcoRV,PpuMI,AflIII,Bs
tThe only recognition cleavage site is located in the entire region of EII and its
In the picillin resistance gene region,BglI,PvuI,MstI,A
paThe only recognition cleavage site is located in the entire region of LI
The restriction enzyme corresponding to the recognition and cleavage site
When the complex plasmid vector of the invention is specifically cleaved
Generated multiple unwanted cleavage sites in the vector.
Each of these restriction enzymes without fragmentation
Any of the recognition cleavage sites in the
It can be used as an insertion point.

In addition, this composite plasmid vector contains Hind
By inserting specific polylinker with recognition cleavage site III and Eco 47III, since numerous cloning sites are added, there is a feature that cloning by using various restriction enzymes can be implemented.

As summarized in Table 1, 30 restriction enzyme cleavage sites are available as cloning sites for pHY320PLK, while 3 for pHY321PLK.
One restriction enzyme cleavage site can be used as a cloning site, and both have a wide range of applications.

As described above, since it is an urgent industrial demand to establish a useful host-vector system for Bacillus subtilis today, Escherichia coli, which is a representative species of Gram-negative bacteria, and Gram-positive bacteria, A novel low-molecular-weight composite plasmid vector that grows using both of the representative bacterial species, Bacillus subtilis, as a host and is stably retained for a long period of time is particularly suitable for industrial use of a gene cloning system for Gram-positive bacteria. Its utility value is extremely high in that it is significant from the viewpoint of realization, and that it has various cloning sites.

In addition, other Gram-positive bacteria such as Lactobacillus , Bifidobacterium ( Bif
It is promising in that it has the potential to be used extensively in the direction of gene analysis and molecular breeding of other microorganisms such as idobacterium ).

 Then, the Example of this invention is described in detail below.

<Example 1> (1) Insertion of Eco RI linker into SacII site of pHY340 1 μg of pHY340 DNA (see JP-A-60-87789) was added to SacI.
After cleavage with I and treatment of the ends with T4 polymerase, Eco RI
Linker d (pGGAATTCC) (Takara Shuzo) was added, and treated with T4 ligase by a conventional method to ligate the plasmid, and an Eco RI linker was inserted into the SacII cleavage site in pHY340 DNA.

(2) Transformation of Escherichia coli 150 μl of plasmid DNA obtained by the treatment of (1) above
To (about 0.5 μg), 150 μl of Escherichia coli competent cells were added, left at 0 ° C. for 10 minutes, 0.7 ml of L-broth was added, and the mixture was incubated at 37 ° C. for 1 hour. Then, this culture solution was applied to the surface of an agar medium which was prepared by adding 1.5% agar and 20 µg / ml tetracycline to L-broth, and cultured overnight at 37 ° C. Twelve strains of transformants were collected from the colonies formed on this agar medium, and the size of the plasmid contained therein was examined.

(3) Extraction of plasmid, measurement of molecular weight and confirmation of Eco RI cleavage site 20 μg / ml of the transformant obtained by the treatment of (2) above
After culturing overnight with 3 ml of tetracycline-added L-broth, the cultivated bacteria were collected. Then 0.1 mg / ml
Lysozyme-5 μg / ml Dissolve in 0.5 ml of RNase and add 0.2m to it.
l of 0.2% SDS solution was added and reacted at room temperature for 1-2 minutes,
Further, 16 μl of 5M NaCl was mixed and allowed to stand at 0 ° C. for 10 minutes, and then this solution was subjected to centrifugation at 20,000 rpm 0 ° C. for 10 minutes. To the supernatant separated by the above centrifugal separation treatment, buffer A (0.1 M Tris, pH 8.0), 0.1% 8-hydroxyquinoline, and 0.2% β-mercaptoethanol saturated phenol were added in equal amounts, Shake this well. Furthermore, this was centrifuged at 15,000 rpm at room temperature for 3 minutes, an aqueous layer containing DNA was collected, and an equal amount of phenol was added to perform a second DNA extraction. Then, centrifuge again to collect the aqueous layer containing DNA, add twice the amount of cold ethanol, leave at -20 ° C for 60 minutes, then
DNA was collected as a precipitate by centrifugation at 0 ° C for 10 minutes. The collected DNA was washed twice with cold ethanol and dried, and 50 μl of TE buffer (10 mM Tris
It was dissolved in -0.1 mM EDTA (pH 7.4)).

10 μl of each of the DNA preparations obtained here was electrophoresed in a 0.7% agarose gel to measure its molecular weight. As a result, the molecular weights of the 12 transformants were all about 3.2 Md.

Next, from among the transformants of these 12 strains,
In order to select a desired strain that has a SacII cleavage site deleted and has an Eco RI linker inserted,
Extract DNA from each strain and select 1 from each
As a result of treating 0 μl with Eco RI, it was confirmed that 4 strains had DNA having an Eco RI cleavage site ( Eco RI linker). 1 DNA each carried by these 4 strains
Attempts to treat 0 μl with SacII did not cleave either. Therefore, the obtained plasmid was
Concluded that Eco RI linkers are those formed by inserted into SacII cleavage site, which was designated as pHY340E ¹ E ² (Fig. 2 C).

(4) pHY340E ¹ to pHY340E ¹ E ² DNA extraction 100μg of Sop-[alpha] 1 region of the E ² was cleaved with Eco RI, the cut piece of 0.7% low melting point agarose gel (IBI, Inc., including 1μg1ml ethidium bromide) Electrical Electrophoresis, 750bp D
The NA band was cut out. After adding an equal amount of migration buffer to this and liquefying at 65 ° C, in connection with the treatment of (3) above,
The indicated phenol extraction method and ethanol precipitation method were applied to recover DNA, which was dissolved in 10 μl of TE buffer.

(5) Cleavage of pHY300PLK 100 μg of pHY300PLK DNA (see JP-A-60-248184)
After digestion with Eco RI, the cleaved pieces were treated with alkaline phosphatase, and the DNA was recovered by the phenol extraction method and ethanol precipitation method shown in connection with the treatment of (3) above, and 10 μl of TE buffer was used to recover the DNA. It dissolved in the liquid.

(6) Insertion of Sop-α1 region into Eco RI cleavage site of pHY300PLK 1 μl of the DNA prepared by the treatment of (4) above and 1 μl of the DNA prepared by the treatment of (5) above were mixed to prepare T4 DNA.
After ligating these with ligase, E. coli was transformed by the treatment of (2) using the ligated DNA.
The 12 transformants thus obtained were subjected to the treatment of (3) above to extract plasmid DNA, and the molecular weight of the extracted DNA was measured. As a result, it was confirmed that all were about 3.4 Md.

(7) Determination of the directionality of the Sop-α1 region with respect to pHY300PLK Regarding the DNA having the Sop-α1 region obtained by the treatment of (6) above, the DNA of the Sop-α1 region with respect to pHY300PLK was
Since there is a possibility that the region will be inserted in both the forward and reverse directions, in order to determine this directionality, one specific restriction enzyme recognition site is selected for each of the inside and outside of the Sop-α1 region, By measuring the molecular weight of the DNA excised by two restriction enzymes corresponding to these recognition sites, the products inserted in the forward and reverse directions were discriminated. That is, Sop-
Eco 47III was selected inside the α1 region and Bst EII was selected outside the region, and the Sop-α1 activity was measured using pHY340 as a control.

By the way, pHY340 with Sop-α1 activity is Bacillus subtilis
(B. subtilis ) recE ^- strain shows good colony growth, and the plasmid is stably retained, but pHY300PLK, which does not have the Sop-α1 region, shows that transformants of the Bacillus subtilis recE ^- strain It has already been confirmed that the growth is poor and the plasmid is extremely unstable. Therefore, among the composite plasmids obtained by cloning Sop-α1 into pHY340PLK, pHY340PLKE ¹ E ^{2 in} which Sop-α1 was inserted in the same direction as pHY340 and p in the reverse insertion in Sop-α1 were inserted.
Both in the individually Bacillus subtilis with HY340PLKE ² E ¹ (Bacillussubti
When competent cells of lis 168 YIT 6007) (see Japanese Patent Laid-Open No. 60-120980) were transformed, a significant difference was observed between the size of the transformant colonies and the transformation efficiency.

Incidentally, with respect to pHY340PLKE ¹ E ² in which the Sop-α1 region was inserted in the same direction as pHY340, colony development was good and transformation efficiency similar to that of pHY340 was observed, but in contrast to this, Sop- pHY with the α1 region inserted in the opposite direction
Regarding 340PLKE ² E ¹ , colony development and transformation efficiency were poor.

(8) Deletion of Eco RI ² site in pHY340PLKE ¹ E ² and Bbe I
Insertion of Linker 5 μg of pHY340PLKE ¹ E ² DNA was partially digested with Eco RI, and the partially digested DNA fragment was subjected to low melting point agarose gel electrophoresis to cut out a DNA band having a molecular weight of about 3.4 Md to recover the DNA fragment. This DNA fragment was treated with T4 DNA polymerase, Bbe I linker d (pAGGCGCCT) was added, and T4 ligase treatment was further performed to ligate the Bbe I linkers to form a circular D
NA.

(9) Transformation of Escherichia coli 0.5 μg of the DNA obtained by the treatment of the above (8) was subjected to the same treatment as the treatment of the above (2) to transform Escherichia coli.

(10) Extraction of plasmid, measurement of molecular weight and confirmation of Bbe I site 12 transformants were subjected to the same treatment as the treatment of (3) above, the plasmid was extracted and the molecular weight was measured. , All were about 3.4 Md. Next, when these plasmid DNAs were treated with Bbe I, it was confirmed that 5 strains possessed a plasmid in which the Eco RI cleavage site was reduced to 1 due to the insertion of the Bbe I linker. .

(11) Determination of Bbe I linker insertion site It was obtained by the process of (10) above so as to delete the Eco RI ² cleavage site of pHY340PLKE ¹ E ² and select the clone having the Bbe I linker insertion site at that site. About 5 strains,
The plasmid DNA containing the Bbe I linker was inserted into Bbe I and E
It was cleaved with co RV, and the presence or absence of the Bbe I cleavage site in the plasmid DNA was examined based on the size of the DNA fragment produced thereby. About 3 out of 5 strains, the Eco RI ² cleavage site in their plasmid DNA disappeared and B at the same position
Those obtained with the be I cleavage site were obtained. Of these plasmid DNAs that have acquired the Bbe I cleavage site, the plasmid DNA with only one Bbe I linker inserted into
It was named Y340PLKE ¹ Bb (5.62 Kb) (Fig. 2E).

(12) Elimination of Pst I ² site by site-specific base substitution method pHY340PLKE ¹ Bb prepared by the process of (11) above
Since there are two Pst I cleavage sites in total, the Pst I site in the polylinker cannot be used as a cloning site. Therefore, in order to delete the Pst I ² site located in the ampicillin resistance gene region of pHY340PLKE ¹ Bb, the site is treated as follows.
By applying a base substitution that does not accompany the amino acid substitution of the ampicillin resistance gene to the Pst I ² cleavage site, care was taken to maintain the function of the resistance gene as a gene marker. That is, the fifth of A when substituted C Pst I cleavage site of -CTGCAG- of the base sequence of Pst I cleavage site is deleted, this case, does not involve an amino acid substitution. Therefore, such a selective base substitution of A → C was carried out by the following procedure by employing the site-specific base substitution method.

First, 5'-ATG of a primer DNA consisting of 15 bases
DNA produced by synthesizing the nucleotide sequence CCTGCCGCAATG-3 '
If, as it was alkaline phosphatase treated is cleaved in the pHY340PLKE ¹ BbDNA Eco RV, the pHY340PLKE ¹ Bb Pvu
Partially deleted DNA obtained by digestion with I and Bgl I
And equimolar amounts of each, and add 100 to this mixture.
A heat denaturation treatment was performed at 3 ° C for 3 minutes. Then, this 30
Incubate at 30 ° C for 30 minutes, then leave at 4 ° C for 30 minutes, 0 ° C
After cooling for 10 minutes, a hybrid DNA of the vector DNA and the synthetic DNA primer was prepared. Then this hybrid DNA
Was introduced into competent cells of Escherichia coli by the treatment of the above (2) to prepare a transformant. This transformant is subjected to a process (3), plasmid DNA was extracted from the transformants, and a portion thereof with Pst I treatment, confirmed the presence of the Pst I ² cleavage site at the plasmid DNA in did. As a result, it was confirmed that 2 out of 24 strains carried the plasmid DNA in which the Pst I ² site had disappeared. Then, one of the plasmid DNAs held therein is pHY340PLK.
It was named (5.62 Kb) (Fig. 2F).

(13) PHY340PLK the Eco RI- Eco 47III portion PHY340PLK DNA to Eco RI created in the process of removing (12) the
And Eco 47III, and the molecular weight of about
3.2 Md DNA was extracted. On the other hand, a synthetic DNA having a nucleotide sequence equivalent to that of each of the cut ends of Eco RI and Eco 47III and having a Mro I recognition sequence between them was adopted as a linker, and this was mixed with 3.2 Md of DNA described above. The DNA and the linker were ligated by treatment with T4 ligase. Then, by the treatment of (2) above, Escherichia coli was transformed with the reaction solution containing this linker-ligated DNA. (3)
As a result of extracting the plasmid DNA from the 6 transformants by the above treatment and measuring the molecular weight thereof, all were about 3.2 Md. In addition, part of the DNA obtained here (about 0.5 μg)
When treated with Eco RI, Mrol , Eco 47III , etc., it was confirmed that each restriction enzyme possesses one cleavage site. And, the plasmid DNA is pHY340P
It was named LK (5.38 Kb) (Fig. 2G, Fig. 3).

(14) Transformation of Escherichia coli and Bacillus subtilis Plasmid DNA prepared by the treatment of (13) above
Is a shuttle vector, and to confirm that it has the Sop-α1 region, the plasmid DNA
Was transformed with E. coli and Bacillus subtilis as a host. In this case, the above-mentioned pHY340 and pHH300PLK were used as controls.

i) Transformation of Escherichia coli DNA (1 μg) obtained by the treatment of the above (13)
And plasmids pHY340 (1 μg) and pHY300PLK
(1 μg), 100 μl of E. coli competent cells was added to 5 μl of each, and after adsorbing at 0 ° C. for 15 minutes, 0.9 ml of L-broth was added and the cells were cultured at 37 ° C. for 1 hour. did. Then, this culture solution was added to L-broth to 1.
The mixture was applied to the surface of an agar medium containing 5% agar and 20 μg / ml tetracycline, and cultured overnight at 37 ° C. The growth status of the transformant in this case was as shown in Table 2.

ii) Transformation of Bacillus subtilis DNA (1 μg) obtained by the treatment of (13) above
And plasmid pHY340 (1 μg) and pHY300PLK
To each 10 μl of (1 μg), 500 μl of Bacillus subtilis recE ⁺ (ISW1214), recE ⁻ (YIT60) was added.
Add competent cells from 07), adsorb at 37 ° C for 30 minutes, then 50
The cells were shake-cultured by adding 0 μl of L-broth.
Then, this culture solution was applied to the same agar medium as described above in i) and cultured at 37 ° C. overnight. The growth status of the transformants in this case was as shown in Table 2 above.

(15) Stability of the vector in Bacillus subtilis recE ⁺ and recE ⁻ strains The plasmid pHY320PL obtained by the treatment of (13) above
K is, Bacillus subtilis recE ^- in order to ensure that it is stably held in stock, taken PHY320PLK DNA from E. coli transformants and used to Bacillus subtilis recE ⁺ strain (ISE1214) and recE ^- (YIT
6007) was transformed.

At that time, as a control, pHY340 and pHY300PLK were similarly transformed. Then, each of the three strains of transformants obtained therein was cultured in L-broth at 37 ° C. for a period corresponding to 100 cell divisions. Next, the proportion of plasmid-carrying bacteria was examined for each culture. The results are shown in Table 3. As is clear from the table,
PHY300PLK, which does not have the Sop-α1 region, is stable in the Bacillus subtilis recE ⁺ strain, but remarkably unstable in the recE ⁻ strain, and after a period corresponding to 100 cell divisions, most cells should possess it. The lasmid (pHY300PLK) had been lost. In contrast, it was confirmed that, with regard to pHY320PLK and pHY340, each of the plasmids was stably maintained in the Bacillus subtilis recE ⁻ strain.

Escherichia coli as a transformant described in (14) i) above has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology, under the accession number Microindustrial Research Institute No. 9829.

<Example 2> (1) Expansion of NdeI site in pHY320PLK Regarding the pHY320PLK of Example 1, in order to further expand a single cleavage site with a restriction enzyme, HindIII of polylinker was added.
A specific base in the -CCTATG-base sequence located at 2.36 Kb from the site was replaced by the site-specific base substitution method described above to expand the Nde I site. That is, of the primer DNA,
5'-TTAATCTCATATGGTGG-3 'base sequence and pHY
320PLK was cleaved with HindIII and treated with phosphatase, and pHY320PLK was cleaved with Nsi I and Hpa I to a DNA fragment having a molecular weight of about 2.7 Md, which was treated in the same manner as in Example 1 (12) to give a hybrid DNA. Of the Nde I cleavage site
A plasmid having a base sequence of CATATG- was selected and named as pHY321PLK (5.38Kb). This pHY321PLK is
It has a molecular weight of about 3.2 Md and has acquired a unique Nde I cleavage site. And in such pHY321PLK, pHY32
This means that the base located in the replication initiation region of 0PLK in Bacillus subtilis was replaced at one position, but the base sequence after the replacement was designed so as not to affect the amino acid represented by it. As a result of the transformation experiment, it was confirmed that the activity of the genetic information for replication initiation was not lost at all, which is fully supported.

[Brief description of drawings]

FIG. 1 is a restriction enzyme digestion map for identifying the composite plasmid vector according to the present invention. FIG. 2 is a set of schematic restriction enzyme digestion maps showing the synthetic pathways of pHY320PLK and pHY321PLK. (A) and (B) relate to composite plasmids pHY340 and pHY300PLK as starting materials, (C) (D). ) (E) and (F) are synthetic intermediates pHY340E ¹ E ² and pHY340PLK, respectively.
For E ¹ E ² , pHY340PLKE ¹ Bb and pHY340PLK,
And, (G) and (H) relate to the composite plasmid vectors pHY320PLK and pHY321PLK according to the present invention. 3 and 4 show the composite plasmid pHY320 of the present invention.
It is a detailed restriction enzyme cutting map of PLK and pHY321PLK.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1: 125) C12R 1:46 1:19) (C12N 15/00 A C12R 1: 125)

Claims (2)

(57) [Claims] 1. A tetracycline resistance gene region derived from a plasmid pAMα1 of Streptococcus faecalis and its replication initiation region, and (b) an ampicillin resistance gene region derived from a plasmid vector pACYC177 of Escherichia coli and Its replication initiation region, (c) the Sop-α1 region that stably holds the plasmid in the Bacillus subtilis recE ⁻ strain, and
(D) It is composed of a polylinker having Hind III and Eco47 III recognition cleavage sites at both ends, has a molecular weight of about 3.2 Md, and has a -TCCGGA-base sequence structure at a position 41b from the Hind III site, A composite plasmid vector (pHY320PLK) having a -CCTATG-base sequence structure at a position of 2.36 Kb from the Hind III site and characterized by the following restriction enzyme cleavage map (I). 2. A tetracycline resistance gene region derived from a plasmid pAMα1 of Streptococcus faecalis and its replication initiation region, and (b) an ampicillin resistance gene region derived from a plasmid vector pACYC177 of Escherichia coli and Its replication initiation region, (c) the Sop-α1 region that stably holds the plasmid in the Bacillus subtilis recE ⁻ strain, and
(D) It is composed of a polylinker having Hind III and Eco47 III recognition cleavage sites at both ends, has a molecular weight of about 3.2 Md, and has a -TCCGGA-base sequence structure at a position 41b from the Hind III site, A composite plasmid vector (pHY321PLK) having a -CTATG-base sequence structure at a position of 2.36 Kb from the Hind III site and characterized by the following restriction enzyme cleavage map (II).