JPS61124386A - Novel plasmid and preparation thereof - Google Patents

Abstract

PURPOSE:To prepare a small-sized multi-copying plasmid, by culturing a bacterial strain belonging to Streptomyces genus, lysing the cultured bacterial cell, and separating the objective plasmid from the lysed product. CONSTITUTION:A bacterial strain belonging to Streptomyces genus (e.g. Streptomyces griseobrunneus) and containing a plasmid (plasmid pBT1 in the above example) is cultured in a medium, the cultured bacterial cell is lysed, and the plasmid (pBT1) is separated from the lysed product. The plasmid (pBT1) has a molecular size of about 5.6k-base, and is characterized by the restriction enzyme map of the figure. Furthermore, a DNA fragment containing an amikacin-resistant gene can be prepared by treating the chromosome DNA of a microorganism belonging to Streptomyces litmocidini and resistant to amikacin with a restriction enzyme BclI.

Description

[Detailed description of the invention]

This article relates to a new plasmid targeting bacteria and its production method. <Prior art> Streptomyces bacteria are antibiotics and anticancer substances. It is known as a useful industrial microorganism that produces clinically important secondary metabolites such as enzyme inhibitors. Therefore, research on genetic modification of these microorganisms is attracting attention as it has the potential to lead to increased productivity of these useful substances and the construction of new substances. However, such research is lagging behind because vectors that can be widely used for Strephtomyces have not been sufficiently developed. One of the reasons for this is that plasmids derived from Strephtomyces are usually latent (cr)'ptiC) and lack a selection marker necessary as a vector. <Problems to be Solved by the Invention> An object of the present invention is to provide a novel small-sized, multi-copy plasmid that can be widely used in Streptomyces genus 1. Another purpose is to provide a recombinant plasmid vector incorporating a gene resistant to antibiotics that inhibits the growth of Streptomyces bacteria as a selection marker. Another object of the present invention is to provide a DNA fragment containing a gene resistant to antibiotics that inhibit the growth of Streptomyces bacteria. Yet another object is to provide methods for producing these novel plasmids, plasmid vectors, and DNA fragments containing antibiotic resistance genes. <Means for Solving the Problems and Effects of the Invention> As a result of searching for various plasmids of Strephtomyces, the present inventors found a small, multi-copy novel plasmid pBT1, and found that this plasmid pBTI K
The present invention has been completed by successfully providing antibiotic resistance genes that inhibit the growth of many Streptomyces bacteria, that is, amikacin resistance genes, sulfomycin resistance genes, and/or novobiocin resistance genes, as selection markers. I came to a conclusion. amikacin, sulfomycin,
Since novobiocin inhibits the growth of many Streptomyces bacteria, plasmids endowed with these resistance genes are useful in that they can be widely used as vectors for Streptomyces bacteria. Preparation of plasmid pBT1 According to the present invention, plasmid pBT1 is prepared by culturing a Streptomyces bacterium containing the plasmid. It can be prepared by lysing cultured bacterial cells and isolating plasmid pBT from the lysate. The microorganism that provides plasmid pBT1 belongs to Streptomyces Ij4, and any microorganism that has plasmid pBT1 may be used. For example, Streptomyces griseobruneus (Str
eptomyces griseobrunneus
) I Sr 5066 (ATCC19762),
Streptomyces griseobrunneus AF46 (Kaikokenboku Yokyu 7ac+s) and the like can be suitably used. The microorganism can be cultured using a method commonly used for culturing Strephtomyces bacteria. As the medium, either a natural medium or a synthetic medium can be used as long as it contains appropriate carbon sources, nitrogen sources, inorganic substances, etc. Cultivation is carried out by liquid culture 2, for example shaking culture, under aerobic conditions. The culture temperature is preferably 1B-32°C, especially 25-3°C. The pH of the medium is 4~
10. It is preferable that the difference is 6 to 8. Although the culture time varies depending on various conditions, 24 to 48 hours is preferable. In addition, Streptomyces griseobruneus l5P50
66 (ATCC 19762) strain possesses multiple plasmids, and in order to efficiently obtain the desired plasmid in such a case, this microorganism should be made into a protogellast and then cultured in a regeneration medium. It is preferable to obtain regenerating protoplasts having a single target plasmid and culture them under the above conditions. The protoplast formation method and the regeneration method from protoplast to hyphae are, for example, agricultural, biological,
Gumistry, Vol. 45, pp. 1271-1273, (19
It can be carried out by a known person & as described in 1981). For example, Streptomyces griseoburnex l5P506
6 (ATCC19762) strain in the presence of glycine and harvested. At this time, the concentration of glycine is preferably a concentration (0.05 to 0.2%) that slightly inhibits bacterial growth. Next, this bacterial cell was treated with sucrose + Mg, Ca
Protoplasts are prepared by dissolving the cell wall by allowing a cell wall lytic enzyme such as lysozyme to act in a buffered medium for protoplast formation containing . This protoplast is then reduced to 0
．． Regeneration methods can be obtained by methods such as culturing in a protoplast regeneration medium with a 5M sucrose concentration. Rapid detection of plasmids in the protoplast regeneration method can be carried out by the known method described in Molecular Op General Genetics, Vol. 185, pp. 223-238 (1982). That is, after the cultured bacterial cells are lysed with lysozyme, an alkaline solution containing sodium dodecyl sulfate (SDS') is added to denature the chromosomal DNA and RNA. Next, a 7-dinol/chloroform solution is added to this lysate, mixed, and centrifuged to remove proteins. The centrifuged supernatant is subjected to acarose gel electrophoresis to detect K. cybrasmid. Note that if protoplasts are regenerated in the presence of an acridine dye such as acri7rabin or acridine orange in the plasmid regeneration medium, it may be possible to efficiently isolate a single plasmid-carrying strain. As a method for regenerating protoplasts obtained in this way, for example, Streptomyces griseobrunneus AF46 (Feikoken Bacteria 4IP
! J7895) strain. In order to isolate the plasmid pBTl from the bacterial cells obtained by the above-mentioned culture, the known method described in, for example, Journal Op Antibiotics, Vol. 36, pp. 99-108 (1983) can be applied. That is, after the bacterial cells are lysed with lysozyme and SDS, 1 table salt is added and cooled on ice to precipitate the SDS. Then, supernatant K IJ bonuclease and pronase obtained from centrifugation are added to digest RNA and proteins. The plasmid is isolated by centrifuging this lysate on a chloride-based chloride density gradient. Plasmid pB71 can be isolated by treating the separated plasmid with inglovir alcohol to remove ethidicumpromide, followed by dialysis. The resulting plasmid pBT1 has a molecular size of about 5.6 kilobases, has the restriction enzyme sensitive sites shown in Table 1 below, and is characterized by the restriction enzyme map shown in FIG. Since the plasmid pBT1 can autonomously replicate in Streptomyces and has cleavage sites for various restriction enzymes, suitable selection markers such as the amikacin (Am1kacin) resistance gene can be added to these sites. Sulfomycin resistance gene and/or Noboviocin
By inserting a resistance gene, a recombinant plasmid useful as a vector can be prepared. Preparation of Plasmid pBT 24 Amikacin is an aminoglycoside antibiotic chemically semisynthesized from kanamycin A that broadly inhibits the growth of Durham-positive and -negative bacteria, including Streptomyces. The amikacin resistance gene used as a selection marker for the vector is chromosome D of a Streptomyces bacterium that is resistant to amikacin.
It can be obtained from NA. A specific microorganism that confers amikacin resistance gene is Streptomyces litm.
ocidini) ISP5164 (ATCCI99
14'l#K can be used. To obtain the amikacin resistance gene from this microorganism, 1 conventional genetic recombination techniques can be applied. That is. Zero bacteria were cultured by the method described above, the bacterial bodies were collected, and the cells were cultured using a known method (M
ARMUR method: Journal of Molecular Biology, Vol. 3, pp. 208-218. (1961)] to prepare chromosomal DNA. Then,
This chromosomal DNA is digested with a suitable restriction enzyme such as Bcl i.
Bcl I containing the amikacin resistance gene was completely digested with
Obtain a mixture of fragments. On the other hand, pBT 1 was converted to Bcl I
Digested with [7, Bcl I of chromosomal DNA at this cleavage site]
Connect the pieces. At this time, pBT1 has two Bcl I sensitive sites. So pBT 1 is Bc
Partial digestion with l I yields pBT which has been cut at one of the two Bcl I sensitive sites. Plasmid pBT
1 and the Bcl I fragment of chromosomal DNA is T4D.
This can be done with NA ligase. The desired plasmid vector can be collected from the recombinant plasmid thus obtained as follows. That is,
First, protoplasts of Streptomyces bacteria are transformed with the above recombinant plasmid. Transformation can be carried out using known methods commonly used for Streptomyces bacteria [e.g., Journal of Bacteriology, Vol. 151, 668].
-677, (1982)] can be used. That is, the above recombinant plasmid was introduced into protoplasts of Streptomyces genus bacteria. This protoplast was injected in the presence of amikacin (50 to 200
Pf/wd) to form amikacin-resistant colonies. As the Streptomyces bacteria used for transformation, those that are susceptible to amikacin KIB and have fewer restriction systems are preferred; specifically, Streptomyces lividans (SLreptomyces lividans)
) strain 1326 (ARCC15091) (Japanese Journal of General Microbiology, Vol. 129, pp. 2257-2269 (1983)) can be used. Plasmids from amikacin-resistant colonies are then detected, and pBTI Select a large plasmid and transform Streptomyces lividans again with this plasmid. The recombinant plasmid carrying the amikacin resistance gene gives a high frequency of amikacin resistant colonies. Plasmid pBT 24 #'i approximately 7.4 kilobases. Streptomyces lividans has a molecular size of 13
26 (ARCC15091) strain as a host. It has about 50 copies per chromosomal DNA. Plasmid pBT24 is also characterized by the restriction enzyme sensitive sites shown in Table 1 below and the restriction enzyme map shown in FIG. Plasmid pBT24 is plasmid pBT1
One of the cleavage sites of Bcl I in Streptomyces lithimosidini I SF3164 (ATCC 1991
4) A Bcl I fragment containing the amikacin resistance gene of 1.8 kilobases from strain Yumai has been inserted. This 1.8 kilobase fragment has sensitive sites for the restriction enzymes shown in Table 2 below. and is characterized by the restriction enzyme map shown in FIG. 7A. Within this 1.8 kilobase fragment, there is a 1.2 kilobase Bcl I-
Contained in the Sac II fragment. For more details. The region essential for amikacin resistance expression is located within the 0,7 kilobase fragment shown in Figure 7A, and this fragment is located in the S
It is characterized by having one al I cleavage site. Plasmid pB724 can itself be used as a vector with an amikacin resistance marker and one cloning site such as Bcl I, Xho I. BamHI, Sac II, Bgl II are available. Also, using these cleavage sites, pBT
By adding a new selection marker to 24, a more useful vector can be created. Preparation of Plasmid pB731 Sulfomycin is a lucrative peptide antibiotic that inhibits the growth of Gram-positive bacteria, including Streptomyces [Journal Op Antibiotics, Vol. 22, pp. 12-17 (1969); Tetrahedron Letters, Volume 31, Pages 2791-2794, (1978)
]. Since this organism exhibits cross-resistance to other antibiotics with similar structures, thiostrepton and thiopeptin, vectors with sul7omyciny properties can be used as selective drugs for these antibiotics. It is also possible to use Examples of microorganisms that provide a sulfomycin resistance gene include Streptomyces viridochromogenes subsp. 7omycinii, a sulfomycin-producing bacterium.
ochromogenes 5ubspecies
ulfomycini) MCRL0368 strain (ATC
C29776> strain can be preferably used. The above-mentioned genetic recombination technology can be applied to impart the sulfomycin resistance gene of woody fungi to plasmid pBT24. in this case,
Chromosomal DNA may be partially digested with the restriction enzyme Mbo I. This Mbo I fragment was added to Bcl of plasmid pBT24.
It may be linked to one of the I cleavage sites. With this recombinant plasmid, Streptomyces lividans 1326 (ARC
C15091) strain to obtain colonies that regenerate in the presence of sulfomycin (50-200 uf/nl) and amikacin (50-200 uf/-). Plasmids are extracted from these colonies, and a plasmid whose size is slightly larger than pBT24 and which frequently gives sulfomycin-resistant colonies upon retransformation is selected. One of the plasmids thus obtained is pBT31. Plasmid pBT31 has a molecular size of about 9.2 kilobases and has about 110 copies per chromosomal DNA when Streptomyces lividans 1326 (ARCC15091) strain is used as a host. Plasmid pBT31 is characterized by the restriction enzyme sensitive sites shown in Table 1 below and the restriction enzyme map shown in FIG. Plasmid pBT31 contains one of the Bcl I cleavage sites of plasmid pBT24 with Streptomyces gyridochromognes sapsubicis sulfomycinii MCRLO368 (ATCC 2977
6) The MboI fragment containing the 1.8 kilobase sulfomycin resistance gene of strain Yumai has been inserted. This fragment has sensitive sites for the restriction enzymes shown in Table 2 below. and is characterized by the restriction enzyme map shown in FIG. 7B. An even smaller plasmid can be derived by excising the region essential for the expression of sulfomycin resistance from plasmid pB731 and adding it to plasmid pBT24. That is, plasmid pBT31 was partially digested with Mbo I, and the Mbo I fragment of 1.5 kilobases or less was converted into plasmid pBT24.
Among the recombinant plasmids obtained by ligating to the BamHI cleavage site of , the two smallest plasmids that confer sulfomycin resistance are obtained, pBT37 and pBT38. Both plasmids pBT37 and pBT38 are 8.24
It has a molecular size of kilobases and is characterized by the restriction enzyme sensitive sites shown in Table 1 below and the restriction enzyme maps shown in Figure 4 (pBT37) and Figure 5 (pBT3B). These plasmids are called plasmid pB
Streptomyces viridochromogenes subspisis sulfomycinii at the BamHI cleavage site of T24.
The Mbo I fragment containing the 0.84 kilobase sulfomycin resistance gene of MCRL036 B (ATCC29776') strain Yumai was inserted in opposite directions. This 0.84 kilobase fragment has the restriction enzyme sensitive sites shown in Table 2 below and is characterized by the restriction enzyme map shown in Figure 7C. Plasmid pBT31. pBT37. pBT38 can be used as a vector with amikacin and sulfomycin resistance markers and as a cloning site. For example, Bcl I, Sac II, Bgl II and XhoI can be used. In the case of K pBT 31, BamHI is also available. Novobiocin is an antibiotic that inhibits the growth of Durham-positive and -negative bacteria, including Streptomyces. Microorganisms that confer novobiocin resistance genes include novobiocin-producing bacteria, such as Streptomyces nipex (
Streptomyces n1ve-us) ISP
5088 (ATCC19793) strain can be used. The above-mentioned method can be applied to K for imparting the fungal novobiocin resistance gene to plasmid pBT24. That is, the chromosomal DNA of woody fungi is partially digested with Mbo I, and this Mbo I
The fragment is ligated into one of the Bcl I cleavage sites of pBT24. This recombinant plasmid was used to transform protoplasts of Streptomyces lividans 1326 (ARCC15091) strain, and novobiocin (100-250 M
/rd) and amikacin (50 to 200 μf/−), a plasmid containing the novobiocin resistance gene is selected by the method described above. One of the plasmids thus obtained is pBT46. Plasmid pBT46 has a molecular size of approximately 11.32 kilobases and is derived from Streptomyces lividans 1326.
(ARCC15091) strain as a host. It has about 60 copies per chromosomal DNA. Plasmid pBT46 is characterized by the restriction enzyme sensitive sites shown in Table 1 below and the restriction enzyme map shown in FIG. Region essential for expression of novobiocin resistance #-
j-3,4 kilobase Bcl I-Pvu II fragment. More specifically, the region essential for the expression of novobiocin resistance is located within the 3.3 kilobase fragment shown in Figure 4PJ7, DfC. Plasmid pBT46 can be used as a vector with amikacin and novopiocin resistance markers. Examples of cloning sites include Bcl I,
XhoI. BamHI, C1a I, Sph I, Pvu
U etc. can be used. Number of restriction enzyme cleavage sites Plasmid pBT1. obtained above. pBT24゜pB
T31. pBT37. The number of sensitive sites for restriction enzymes in pBT38 and pBT46 is shown in Table 1 below. Furthermore, the number of sensitive sites for restriction enzymes in the DNA fragments containing antibiotic resistance genes inserted into these plasmids is shown in Table 2 below. Table 1 (Note) NT: Not tested. Table 2 (Note) A: 1.8 kilobase fragment containing the amikacin resistance gene B: 1.8 kilobase fragment containing the sulfomycin resistance gene C: 0.84 kilobase fragment containing the sulfomycin resistance gene Fragment D: 3.92 kilobase fragment containing the novobiocin resistance gene NT: Not tested Example 1 Preparation of plasmid pBT 1 fi+ Preparation of protoplasts Streptomyces griseobruneus 15P5066
(ATCC19762) strain in A medium (glucose 0.
2%, polypeptone 0.3%, bacto yeast extract 0.4%, magnesium sulfate heptahydrate 0.05%
, potassium dihydrogen phosphate 0.2%, potassium dihydrogen phosphate 0.8%), IJcum dodecahydrate 0.8%) 50- were inoculated into a 250-volume triangular 7 flask, and heated to 27°C. Cultured with shaking for 1 day. This culture solution I- was inoculated into a 500-cm triangular Fukusco containing A medium 100-ml containing 0.1% glycine, and cultured with shaking at 27°C for 2 days. This culture solution 8- was collected by centrifugation (8°,000 rotations, 4°C, 10 minutes), and the cells were washed with 17.1% sucrose 8- and collected again. The washed bacterial cells were washed with 8-A buffer (25
Suspend lysozyme (final concentration 1fflf/+d) in mM Tris-HCl, pH 7, 2, 0, 5M sucrose, 5mM magnesium chloride hexahydrate, 5mM lucicum chloride dihydrate, 50mM sodium chloride). The mixture was added and reacted at 27°C for 1 to 2 hours to form protoplasts. (2) Preparation of Prodob 2-Stroke regenerated strain The above protoplast suspension was briefly centrifuged (1,500 rpm, 4°C).
, 5 minutes) to allow unreacted mycelium to settle. Next, this supernatant was centrifuged again (4,500 rpm, 4°C, 5 minutes).
The protoplasts were allowed to settle. After suspending the protoplasts in buffer A again, diluting them appropriately with the same buffer,
The 100 PI was added to B medium [0°025M N-tris(hydroxymethyl)methyl-
2-aminoethanesulfonic acid, pH 7,2,0,5M
Sucrose, glucose 1%, polypeptone 0.
4%, Bacto yeast extract 0.4%, Machnesicum chloride hexahydrate 0.3%, Macnesicum chloride dihydrate 0.02%, agar 1.8% 1 agar plate (inner diameter 88 m
) moved above. After overlaying 4.7 soft agar medium (medium with agar concentration of 0.7 in B regeneration medium) on this plate, 2
Colonies were regenerated from protoplasts by culturing at 7°C for 7 to 10 days. Inoculate this colony into B regeneration medium 2
It was grown for 4-7 days at 7°C. The above regeneration-like plasmid was detected by the following rapid extraction method. That is, this regeneration pattern was observed in A medium containing 0.1 tiglycine.
Inoculated into a test tube (inner diameter 21mm) containing
It was shaken back and forth for 3 days. This culture solution]- is centrifuged (1
2,000 rpm, room temperature, 5 minutes) to collect bacteria. Add 300 PI lysozyme solution [TES buffer to 2/-

0,05 M) Lisaminomethane-
Hydrochloric acid, pH 7.4, dissolved in 0.05M ethylenediaminetetophacetate (EDTA), 0.05M sodium chloride] was added and reacted at 37°C for 20 to 30 minutes. Then, add 300 pl of alkaline lysis solution [0
, 5M sodium hydroxide, 2% SDS, 0.0625M
Cyclohexanediaminetetraacetate (CDTA)
) was added and reacted at 37°C for 15 minutes to lyse the bacteria. This lysate K 600 pl of phenol/chloroform solution [phenol 500 f, chloroform 500.
The lower layer of the mixed solution of 0.5-hydroxyquinoline (0.51) was added, stirred for 30 to 60 seconds, and then centrifuged (12,000 rpm, room temperature, 5 minutes) to obtain a supernatant. 10 to 20 μl of this supernatant was used for detection of plus and mido, and the detection was carried out by agarose gel electrophoresis. Streptomyces griseobruneus l by the above method
At least eight types of plasmids were detected in the 5P5066 (ATCC19762) strain. On the other hand, as a result of examining the plasmids of 100 protoplast-regenerating strains of this strain, it was found that one strain, named R37, had only three types of plasmids, including pBTI. (3) Protoplasts were prepared using the acriflavin-treated R37 strain, which allows protoplast regeneration, in the same manner as fl) and (2) above, and colonies were formed again. However, Acry7 Lavin was added to the soft agar to give a final concentration of IPl/-. As a result of examining the plasmids of 10 colonies regenerated in the presence of acriflavine, one regenerated strain having almost a single pBTl gene was obtained. This strain is Streptomyces griseobruneus AF4.
It was named strain 6 (Feikoken Bacillus No. 7895). (4) Isolation of plasmid pBT1 AF46 strain
Inoculate a 250-volume flask containing 0-A medium, and
Rotary shaking culture was carried out at 7°C for 3 days. This culture solution is about 2-0. The cells were inoculated into a 500-capacity Erlenmeyer flask containing 100-mL A medium containing 1% glycine, and cultured with rotational shaking at 27° C. for 2 days. This culture solution was centrifuged (5,000
rotate. The cells were collected, suspended in TES buffer, and centrifuged again (10,000 rpm, 4°C, 20 minutes) to obtain washed cells. This wet bacterial body is
Suspend in TES buffer to give f/20-, and pre-dissolve lysozyme (final concentration IQ/i) and ribonuclease A (final concentration 50q/++sZ, manufactured by Sigma, in 0.8% sodium chloride at 95°C for 10 minutes). (heat-treated) was added, and the bacteria were lysed by reacting at 37°C for 1 hour. Add SDS (final concentration 1.5%) to this lysate and
A clear lysate was obtained by reacting at ℃ for 20 minutes. This lysate was filtered under reduced pressure using HB-cellulose (manufactured by Seikagaku Kogyo Co., Ltd.) as a filter filler. Collect this tear fluid, add sodium chloride (final concentration IM), mix well, cool on ice for 2 hours, and SD
S was allowed to settle. This mixture was centrifuged (6,000
Pronase E (
Final concentration of 1ooμg/-1 Kaken Pharmaceutical Co., Ltd.
The protein was digested by reacting at ℃ for 1 hour. Polyethylene glycol 6000 (final concentration 10%) was added to this reaction solution, mixed well, and left overnight at 4°C. This solution was centrifuged (3,500 rpm, 0°C, 10 minutes) to precipitate the DNA containing the plasmid. This DNA precipitate was dissolved in 4-TES buffer, and 4.25 g of sesicum chloride and ethidium mupromide (final concentration 500 μI/a) were added.
t) After addition and thorough dissolution, equilibrium density gradient centrifugation (60,000 rpm, 20°C, 16 hours) was performed. Ka under UV irradiation! Collect the visible plasmid band from the bottom of a centrifuge tube (approximately 0.5 Wd) by adding 1 liter of isopropyl alcohol (TES buffer 9.4 to cesicum chloride) and 10.7 liter of isopropyl alcohol. The obtained upper layer solution) was added to extract and remove ethidicum gromide. Then the solution containing the plasmid 't 0.5
once with X T E S buffer, once with TE buffer (IOmM
to! J Sue Hydrochloric acid pH 7, 6, 1mM EDTA)
Dialyzed multiple times. Using the method described above, 82 μ9 of plasmid pBT1 was obtained from 500 ml of culture solution. Physical properties of i61 9BT I The sensitive site for restriction enzymes of pBT 1 and the molecular weight of restriction enzyme map 1 were determined by agarose gel electrophoresis of the restriction enzyme digest. Reaction conditions for restriction enzymes were as described in Molecule Cloning, A., Laboratory Manual, Cold Spring Harbor Laboratory Publishing, 1982, pp. 100-101, unless otherwise specified. That is, 0.1 to 1. OPf pBT I was digested with various restriction enzymes, and 115 volumes of a dye solution (xylene cyatool 0.25%, bromophenol glue 0.25%, glycerol 50%) was added to each reaction solution. This solution was added to agarose (Seigm Co., Ltd.) 11 degrees 0.5-2 degrees, Tris-Hocic acid buffer (TBE) [0.08M Trisulfocate, pH 8, 0.087M Honic acid, 0°002ME
DTA], 100-150V. The gel was run for 2 to 5 hours using a plate gel electrophoresis device. Glue after electrophoresis was ethidicumpromide (1 μy/, nt)
After staining by immersing in the solution for 10 minutes, the band emitting fluorescence was photographed under ultraviolet irradiation (305 m) to detect DNA fragments. The molecular weight of each fragment was determined by comparing the migration distance of the DNA fragment of the molecular weight standard. Molecular weight preparations include lambda DNA Hindl [[digested product and PhiX174R]
A Haem digest of FDNA was used. The molecular weight of each fragment is determined by New England Biolapse.
land Biolabs) company catalog (1983~
1984), pages 35-36. pB
The molecular weight and restriction enzyme map of T1 were determined from the molecular weights of fragments generated by single or double digestion with each restriction enzyme. As a result, the physical properties of plasmid pB71 were as follows. Molecular size: Approximately 5.6 kilobases Sensitive site for restriction enzymes: As shown in the above table. Restriction enzyme map: As shown in Figure 1 (6) Copy number of plasmid pBT I Streptomyces lividans 1326 (ARCC 15091) strain harboring plasmid pBT I was mixed with 10 Pf/- in 10 tissues of sucrose YEME medium (sucrose 1
0chi, glucose 0.5%. Yeast extract 0.3%, polypeptone 0.5%,
27 in a test tube containing malto extract 0.3%, glycine 0.5%, 5mM magnesium chloride hexahydrate) 4-
The cells were cultured at ℃ for 3 days. The copy number of the plasmid in this cultured bacterial cell was measured by the method described in Molecular and General Genetics, Vol. 185, pp. 223-238 (1982). That is. Protoplasts were prepared from the cultured cells and SDS T
The dissolved Wffl was treated with phenol/chloroform (]:1) to remove protein. Next, this lysate was diluted with 4% sucrose, treated with ribonuclease A, and subjected to agarose electrophoresis to separate chromosomal DNA and plasmid. This agarose gel was stained with ethidicumpromide and then decolorized. Spectrophotometer the negative film of this photograph. Measurement was performed at a wavelength of 640 nm, and the ratio of chromosomal DNA to plasmid basid was determined. At this time, since the cutting treatment of the plasmid with ultraviolet rays was omitted, the value of the plasmid obtained by the above method was not included in the reference 1 [Plasmid. Volume 9, 182-] Page 90 (1983)]
The numerical value is corrected by multiplying by 1.36, and the molecular size of chromosomal DNA is 104 kilobases [Applied Microbiology, Vol. 30, pp. 324-326 (1975)], and the molecular size of pBT is 5. Calculated based on 6 kilobases. There are 70 copies of plasmid pBTI per chromosome! I used it for l'lJ. Example 2 Production method of plasmid pBT 24+11 Chromosomal DNA having amikacin resistance gene
Fragment of Streptomyces lithimosidini - ISP5164
(ATCC] 9914) strain was resistant to amikacin, with a minimum inhibitory concentration (MIC) of 100 μf/− when using fdB regeneration medium. 14 of Example 1) (
! - Cultivation, collection, and lysis were carried out in the same manner, and chromosomal DNA was prepared from the lysate using the MARMUR method [Journal of Molecular Biology, Vol. 3, pp. 208-218 (1961)]. Next, this chromosomal DNA was digested with the restriction enzyme Bcl■. The composition of the reaction solution used at this time was 25 pl of chromosomal DNA (containing 10 py DNA),
BclI 3μ1 (30 units) 1M buffer [0.5M sodium chloride, 0.1M) Lis-HCl pH 7.5, 0.1
The total volume was made up to 50 μl with M magnesium chloride hexahydrate, 15 l of 10 mM dithiothreitol (DTT), and 17 tL of TE buffer. The reaction was carried out at 50°C for 2 hours to ensure complete digestion. The reaction was stopped by adding an equal amount of phenol (saturated with TE buffer) to the reaction solution and mixing. (2) Connection of chromosomal DNA fragment and plasmid pBT] Since plasmid pBT I has two Bcl I cleavage sites, the cleavage with Bcl I was performed by partial digestion. That is, one reaction solution was composed of pBT] 80μ1 (5pyDN
A), Bcll 1tLl (1 unit) Add 10μl of 1M buffer and 9μl of TE buffer to make the entire view 100μl
It was set as l. The reaction conditions were 50°C for 1 hour. The reaction is stopped in the same manner as fl) above. Next, this phenol-treated reaction solution was centrifuged (12,000 rpm, room temperature, 5 minutes) to obtain a supernatant containing the Bcl I digest of pBTJ, and the above-mentioned chromosome obtained by phenol extraction was obtained in the same manner. The supernatant containing the Bcl I digest of DNA was mixed. This mixture was extracted with phenol again, and the supernatant was extracted twice with twice the amount of ether to remove phenol. A trace amount of ether contained in the water bath solution was removed by treatment at 70° C. for 10 minutes. To this mixture, add 1/1 volume of 3M sodium acetate (pH 5,0) and double volume of cold ethanol,
After cooling at -80°C for 30 minutes, the DNA was precipitated by centrifugation (12,000 rpm, 0°C, 10 minutes). After washing the precipitate with cold ethanol, the ethanol was removed under reduced pressure and the precipitate was dissolved in 90 μl of TE buffer. The composition of the DNA ligation reaction solution is Bcl I of the above DNA.
Digest: 90Pl, Ligase buffer (0.5M Tris-HCl pH 7,8,0,2MDTT, 0.1M magnesium chloride hexahydrate) 101Ll, 70mM A
1 μl of TP solution and T4 DNA! J gauze 1μ
I! (manufactured by Takara Shuzo Co., Ltd., 0.1 unit), one reaction is 2 at 16℃
Do time. This reaction solution was left at 4°C overnight. 50μ of this reaction solution
l was used for the following transformation. (3) Transformation The host for transformation is Streptomyces lividans 13.
26 (ARCC15091 strain was used. The MIC for amikacin in woody fungi was 5 μy in B regeneration medium.
It was /ffl/. Strain 1326 was inoculated into a 250-volume Erlenmeyer flask containing 50 rnl of 34-tissue monoclose YEME medium, and cultured in a rotary root plate at 27°C for 4 days. This culture solution 1- was inoculated into the same medium and cultured for an additional 4 days under the same conditions. After collecting this culture solution, protoplasts were prepared according to the method of Example 10 (11).The protoplast suspension was then centrifuged to precipitate the protoplasts according to the method (2) of Example 1. to 1
-〇B buffer (25mM Tris-HCl, pH 7.2,
0.5M sucrose, 10mM magnesium chloride.
Hexahydrate, 20mM Lucicum chloride dihydrate, 50m
M sodium chloride) was added and suspended. The reaction composition for transformation was the above protoplast suspension (
5 x 10 cells/d) 100 μl, 50 P/g of the above (2) glue-gauze-reacted DNA solution, 40% PEG2, O
After thoroughly mixing 400 µl of this reaction solution with 200 pl of an aqueous solution of OO and 50 µl of B buffer, the mixture was cooled on ice for 10 minutes and then heat-treated at 42°C for 1 minute to incorporate the DNA into protoplasts. 600 μl of B buffer solution was added to this reaction solution, and the total volume was 1-closed. (4) Isolation of amikacin-resistant colonies The transformed protoplast suspension obtained in (3) above was spread on the surface of an agar plate containing 100μ/B regeneration medium using a glass spreader. After overnight incubation at 27°C. A 4-ml soft agar medium containing 50 μ9/− of amikacin was overlaid and cultured at 27° C. for 3 days. As a result, 23 amikacin-resistant colonies were obtained. 5 of these colonies
The bacteria were plated on an agar slant of B regeneration medium containing 0 μg/− of amikacin, and grown by culturing at 27° C. for 5 to 7 days. (5) Screening for plasmid having amikacin resistance gene The amikacin resistant strain obtained in (4) above was inoculated into a test tube containing 4-10% sucrose YEME medium. Amikacin was added to this to a final concentration of 5 μg/−, and cultured at 27° C. for 3 to 5 days. Plasmids were detected from cultured bacterial cells by the rapid method described in Example 1 (2). As a result, it was found that three strains had plasmids larger in size than plasmid pBT1, and the smallest plasmid among them was named pBT24. In order to confirm that plasmid pB724 has an amikacin resistance gene, Streptomyces lividans was isolated using plasmid pBT24 using the method of Example 2 (3).
326 (ARCC1509]) strain was transformed. That is, 400 ml of aqueous solution containing the plasmid obtained by the above-mentioned rapid method.
μl was treated with phenol again, extracted with ether, precipitated with ethanol, and dissolved in 400 μl of TE buffer. Add ribonuclease A (final concentration 50 pp) to this solution.
/d), and after reacting at 37°C for 15 minutes, this 10μI!
(About 0.1μ (lDNA) was used for transformation. As a result, plasmid pBT 24!''i was frequently given amikacin-resistant transformants. Plasmid pB724 obtained in (6) below was used. When I was there 1.]μfDN
A pH 724 gave 2.7 x 10 7 transformants. (6) Isolation of plasmid pB724 The Streptomyces lividans 1326 (ARCC) 5091 strain carrying the above plasmid pBT 24 was used as described in (3) of Example 2.
Example 1 (4) was cultured using the method described in Example 1.
The plasmid was isolated using the method described in ). As a result, 1
70 ILy of pBT24 was obtained from the culture solution of 00-. (7) Physical properties of plasmid pBT24 (5
) As a result of conducting a restriction enzyme cleavage experiment in the same manner as
The physical properties of plasmid pBT24 were as follows. Molecular size: approximately 7.4 kilobases Restriction enzyme sensitive site: As shown in Table 1 above. Restriction enzyme map: As shown in Figure 2, Stregutomyces ritimosidini-ISP5164 (ATCC199
14) Contains a fragment (loco portion) containing the strain-derived amikacin resistance gene. In addition, using the method of Example 1 (6), plasmid pBT
The copy number of Streptomyces lividans 1326 (ARCCI5) carrying pBT24 was measured.
091) strain was found to exist in 50 copies per chromosome. Furthermore, the physical properties of the Bcl2 fragment containing the amikacin resistance gene are as follows. Molecular weight: approximately 1.8 kilobases Restriction enzyme sensitive site: As shown in Table 2 above. Restriction enzyme map: As shown in Figure 7A, the amikacin resistance gene (akr) is located within the underlined region (0.7 kilobases). Example 3 Production method of plasmid pBT31 +l) Preparation of chromosomal DNA fragment having sulfomycin resistance gene Streptomyces viridochromogenes subspisis sul7omycinii MCRL 0368 (ATCC
29776) strain shows good growth in B regeneration medium containing 100 PI/- sulfomycin. Chromosomal DNA was prepared from this bacterium according to method +i+ of Example 2. Cutting chromosomal DNA with restriction enzyme tiMboI
by partial digestion of That is, the reaction solution composition is chromosomal DNA
70μt (101Lf DNA), MboI 2μl
Reaction 1 -t37℃
I went there for an hour. This reaction solution was mixed with 7 enol and 1 ether.
．． Treat with ethanol to precipitate the DNA. This DNA was dissolved in 50 μl of TE buffer. (2) Linkage of chromosomal DNA fragment and plasmid pBT24 Since plasmid pBT24 has three BclI cleavage sites, the cleavage of pBT24 with Bcli was by partial digestion. That is, the reaction solution composition was 65 μl of pBT24.
(6Pf DNA), BclI 3μl (3 units)
. M buffer 10/&l and TE buffer 22P/(total volume 1
00 μl). The reaction was carried out at 50°C for 1 hour. After stopping the reaction and precipitating the DNA in the same manner as above, the DNA was dissolved in 20 μl of TE buffer. D
The composition of the NA ligation reaction solution was 25p/(approximately 5 μp DNA) of Mbo I digested chromosomal DNA and Mbo I digest of pBT24.
6 plC of I digest (approximately 1.8 iff DNA), 5 μt of ligase buffer, 70 mM-ATP I
Pi, T4 DNA ligase 1μ/(0,1 unit) and TE buffer 12μI (total volume 50μl. The reaction was carried out in the same manner as in Example 2 (2), and the total amount of this reaction solution was used for the following transformation. (3) Transformation and isolation of sulfomycin-resistant colonies Streptomyces lividans 13 was used as the host for transformation.
26 (ARCC15091) strain was used. Wood fungi cannot be produced on B regeneration medium containing 10 μp/Pl sulfomycin. The method of transformation is as described in Example 2 (3)
The sulfomycin-resistant colonies were isolated according to the method described in Example 2 (4). however. 50 py/- amikacin and 100 tsy/- sulfomycin were added to the soft agar medium used for colony regeneration. As a result, 6 sulfomycin-resistant colonies were obtained. This colony was picked on an agar slant of B regeneration medium containing 50 tby/-=1 sulfomycin, and
It was grown for 5-7 days at 7°C. (4) Screening for plasmid having sulfomycin resistance gene Plasmid '5r of sulfomycin resistant strain obtained above
The test was conducted in the same manner as in Example 2 (5). However, the strain was cultured in the presence of 5Otstl- of sulfomycin. As a result, 3 strains were found to have plasmids larger than pBT24'f4I. The smallest plasmid among them was named pBT31. Using the method of Example 2 (6), plasmid pBT3
] in Streptomyces lividans 1326 (ARC
When the strain C15091) was transformed, sul7omycinidic transformants were obtained with high frequency. Furthermore, when the plasmid pBT 31 obtained in (6) below was used, 1 μf of pBT 31 gave 1.5×10′ transformed strains. (5) Isolation of plasmid pBT31 Streptomyces lividans 1 carrying the above plasmid pBT31
326 (ARCCI 5091) strain of Example 2 (6
The plasmid was isolated by the method described in ). However, culture is 50
It was carried out in the presence of μf/-sulfomycin. the result,
47.9 μda of pBT 31 was obtained from the culture solution of 00-. (6) Physical properties and copy of plasmid pB731 As a result of a restriction enzyme cleavage experiment conducted in the same manner as in (5) of Example 1, the physical properties of plasmid pBT 3 ] were as follows. Molecular size: approximately 9.2 kilobases Restriction enzyme sensitive site: As shown in Table 1 above. Restriction enzyme map: The fragment shown in Figure 3 and containing the sulfomycin resistance gene derived from Streptomyces viridochromogenes subspisis sul7omycinii strain MCRLO368 (ATCC29776) (wm portion)
including. In addition, using the method of Example 1 (6), plasmid pBT
The copy number of Streptomyces lividans 1326 (ARCC15) carrying pBT31 was measured.
[09]) strain was found to have 110 copies per chromosome. Furthermore, the physical properties of the Mbo I fragment containing the sulfomycin resistance gene were as follows. Molecular size: approximately 1.8 kilobases Restriction enzyme sensitive site for experiment: As shown in Table 2 above. Restriction enzyme map: As shown in Figure 7B, the sulfomycin resistance gene (sfr) is located within the underlined region (0.84 kilobases). Example 4 Production method of plasmids pBT 37 and pBT 38 [1]
The Mbo I fragment of plasmid pBT31 and the plasmid pBT24 were ligated.
partial digestion (37° C., 80 minutes). This digest was subjected to agarose electrophoresis to separate Mbo 1 fragments of 1.5 kilobases or less using the DEAE cellulose paper method described in Analytical Biochemistry, Vol. 112, pp. 295-298 (1981) E. On the other hand, plasmid pBT24 was completely digested with BamHi and
．． 5 kilobases f) ligated with the Mbo I fragment. fil shape! Transformation, isolation of sulfomycin-resistant colonies, and screening of plasmids The transformation host was Streptomyces lividans 1326 (ARC
C]5091) strain was used. Transformation was carried out according to the method of Example 3 (3), and a large number of sulfomycin-resistant colonies were obtained. Example 3 (4)
According to the method described above, plasmids of 100 resistant colonies were examined and four strains having the smallest plasmid size were selected. All of these plasmids frequently
It gave me a sense of humor. In addition, from the plasmid preparation and restriction enzyme experiment described below,
All of these plasmids have a molecular size of 8.24 kilobases, and the Mbo I fragment containing a 0.84 kilobase sulfomycin resistance gene is inserted into the BamHI cleavage site of plasmid pBT24 in opposite directions. There was found. Therefore, we named one strain pBT37 (3 strains) and the other pBT38 (1 strain). (3) Isolation of plasmids pBT37 and pBT3B Plasmids were isolated by the method described in Example 2 (6). As a result, three strains of Streptomyces lividans 1326 carrying plasmid pBT 37 were obtained from a culture medium containing 100 ffI/310 μy, 33.8 μy, and 75 μy, respectively.
．． 5μf of plasmid was given. On the other hand, puxmid pBT
One strain with 38 gave 40 μf of plasmid from a 100-culture. (4) Physical properties of plasmids pBT37 and pBT38 As a result of a restriction enzyme cleavage experiment conducted in the same manner as in (6) of Example 1, the physical properties of the restriction enzymes of plasmids pBT37 and pBT38 were as follows. Molecular size: 8.24 kg Restriction enzyme sensitive site: As shown in Table 1 above Restriction enzyme map: Figure 4 (pBT37), Figure 5 (pBT
38) As shown in K, Streptomyces guiridochromognes sapspisis sulfomycinii - MC
Fragment containing the sulfomycin resistance gene of RL0368 (ATCC29776) & Yuffi (part 11111)
including. Further, the physical properties of the MboI fragment containing the essential region of the sulfomycin resistance gene excised from plasmid pBT31 were as follows. Molecular size: 0.84 kilobases Restriction enzyme sensitive site: As shown in Table 2 above. Restriction enzyme map: The map shown in Figure 7C shows the essential region of the sulfomycin resistance gene (sfr). Example 5 Production method of plasmid pBT 46 (1) Chromosomal DNA having novobiocin resistance gene
Preparation of fragment Streptomyces nivecus 15P508B (ATCC
19793) was B containing 100 μl/- of 7bopiocin.
Shows good growth on regeneration medium. Chromosomal DNA was prepared from one bacterium in the same manner as in Example 3 (1), and this was transformed into Mbo
It was partially digested with I. (2) Ligation of chromosomal DNA fragment and plasmid pBT24 In the same manner as in Example 3 (2), plasmid pBT24 was
was partially digested with Bcl I, and this Bcl I fragment was ligated with the Mbo I fragment of chromosomal DNA. +3+ Transformation and Isolation of Novobiocin-Resistant Colonies As described above, Streb) E. Ses lividans strain 1326 (ARCC15091) was used as the host for transformation. This bacterium cannot grow in B regeneration medium containing 10 tn/- of novobiocin. Transformation was performed by the method described in Example 3 (3), and novobiocin-resistant colonies were regenerated. However, 50 μf/- amikacin and 100 Ptt/nd 7-pobiocin were added to the soft agar medium used for regeneration. As a result, a large number of resistant colonies were obtained. (4) Screening for plasmids having novobiocin resistance gene The plasmids of the 50 novobiocin resistant strains obtained above were examined in the same manner as in Example 2 (6). However, the experiment was carried out in the presence of novobiocin at 10 .mu.f/- per strain culture. As a result, it was found that three strains had a plasmid larger than plasmid pBT24. The smallest plasmid among them was named pBT46. Using the method of Example 2 (5), plasmid pBT46
Streptomyces lividans 1326 (ARCC1
When the 5091) strain was transformed, transformants resistant to novobiocin were obtained with high frequency. or. When using plasmid pB746 obtained in (6) below. ]μf pBT46 gave 3×10′ transformants. (6) Isolation of plasmid pB746 Streptomyces lividans 1 carrying the above plasmid pBT46
Example 2 (6) from 326 (ARCC] 5091) strain
The plasmid was isolated by the method described in . However, culture II'
The test was carried out in the presence of novobiocin at 10 μf/−. As a result, 50 μf of pBT46 was obtained from the 100-ml culture solution. (6) Physical properties of plasmid pBT46 and copying As a result of a restriction enzyme cleavage experiment conducted in the same manner as in Example 1 (6), the physical properties of plasmid pBT46 were as follows. Molecular size: approximately 11.32 kilobases Restriction enzyme sensitive site: As shown in Table 1 above. Restriction enzyme map: As shown in Figure 6, Strephtomyces nivec 7. ISP5088 (ATCC19793
) Contains a fragment (11 parts) containing the novobiocin resistance gene derived from m. In addition, using the method of Example 1 (6), plasmid pBT
The copy number of Streptomyces lividans 1326 (ARCC15) carrying pB746 was measured.
091) strain was found to have 60 copies per chromosome. Furthermore, the physical properties of the Bcl I fragment containing the novobiocin resistance gene were as follows. Molecular size: approximately 3.92 kilobases Two sensitive sites for restriction enzymes As shown in Table 2 above. Restriction enzyme map: As shown in Figure 7, the novobiocin resistance gene (nbr) is located within the underlined region (3.3 kilobases).

[Brief explanation of drawings]

Figures 1 to 6 show guxmid pBTI, respectively. pBT24. pBT31. pBT37. pBT38
and a restriction enzyme map of pBT46. Figure 7 shows a restriction enzyme map of a DNA fragment containing an antibiotic resistance gene.
A is a 1.8 kilohese fragment containing the amikacin resistance gene, B and C are 1 containing the sulfomycin resistance gene.
．． D indicates the 8 and 084 kilobase fragments, and D indicates the 3.92 kilobase fragment containing the novobiocin resistance gene, respectively. '141 Figure sph I (3,00) Atsushi 2 Mouth 3 Figure 'lA40 Atsushi 5th page 6th nl) r (J-jkt) 1 Voluntary procedure amendment 1127, 1985 1, Llj display Showa! 1959 patent application No. 2. +'2.03'f No. 2,
Name of the invention vi Akira Utomi 1. -Relationship with the amended person's case Patent applicant 3-21 Doshomachi, Higashi-ku, Osaka-shi, Osaka (541)
295) Tanabe Pharmaceutical Co., Ltd. Representative Matsubara Part 4, Agent 3-16-89 KaQ, Yodogawa-ku, Osaka-shi, Osaka Prefecture (532
) 5. Number of inventions increased by amendment 6. Contents of amendment subject to amendment 1. "Equilibrium density gradient centrifugation" in the 8th line from the bottom of No. 12 of the specification is corrected to "equilibrium density gradient centrifugation." 2. Correct rNobobiocinJ on page 13, line 7 of the specification to 1" NovobiocinJ. 3. Correct [・Lividans 1326 (A RCC15091) strain] on page 34, line 8 of the specification to ". Renaeus AF46 (m1 research group 789
No. 5) Stock”.

Claims (1)

[Scope of Claims] 1. having a molecular size of about 5.6 kilobases;
Plasmid pBT1 characterized by the restriction enzyme map shown in the figure. 2, has a molecular size of about 5.6 kilobases, and has a first
Plasmid p characterized by the restriction enzyme map shown in the figure
DN containing an amikacin resistance gene, a sulfomycin resistance gene, and/or a novopiocin resistance gene in BT1
A recombinant plasmid incorporating the A fragment. 3. The set according to claim 2, which is plasmid pBT24, which carries an amikacin resistance gene, has a molecular size of about 7.4 kilobases, and is characterized by the restriction enzyme map shown in FIG. Replacement plasmid. 4. The claim is plasmid pBT31, which carries an amikacin resistance gene and a sulfomycin resistance gene, has a molecular size of approximately 9.2 kilobases, and is characterized by the restriction enzyme map shown in FIG. Recombinant plasmid according to Section 2. 5. The claim is plasmid pBT37, which carries an amikacin resistance gene and a sulfomycin resistance gene, has a molecular size of approximately 8.24 kilobases, and is characterized by the restriction enzyme map shown in FIG. Recombinant plasmid according to Section 2. 6. The claim is plasmid pBT38, which carries an amikacin resistance gene and a sulfomycin resistance gene, has a molecular size of approximately 8.24 kilobases, and is characterized by the restriction enzyme map shown in FIG. Recombinant plasmid according to Section 2. 7. Claim 2, which is a plasmid pBT46, which carries an amikacin resistance gene and a novopiocin resistance gene, has a molecular size of about 11.32 kilobases, and is characterized by the restriction enzyme map shown in FIG. Recombinant plasmid as described in Section. 8. A DNA fragment that carries an amikacin resistance gene, has a molecular size of about 1.8 kilobases, and is characterized by the restriction enzyme map shown in FIG. 7A. 9. A DNA fragment that carries a sulfomycin resistance gene, has a molecular size of about 1.8 kilobases, and is characterized by the restriction enzyme map shown in FIG. 7B. 10, possesses sulfomycin resistance gene, approximately 0.8
A DNA fragment having a molecular size of 4 kilobases and characterized by the restriction enzyme map shown in Figure 7C. 11. A DNA fragment that carries a novobiocin resistance gene, has a molecular size of about 3.92 kilobases, and is characterized by the restriction enzyme map shown in FIG. 7D. 12. Streptomyces griseobruneus (Str
eptomyces griseobrunneus)
1. A method for producing plasmid pBT1, which belongs to the following methods and is characterized by culturing a microorganism containing plasmid pBT1 in a medium, lysing the cultured cells, and then isolating plasmid pBT1 from the lysate. 13. A DNA fragment carrying the amikacin resistance gene, having a molecular size of approximately 1.8 kilobases, and characterized by the restriction enzyme map shown in Figure 7A, was added to the plasmid pB.
A method for producing plasmid pBT24, which is characterized by integrating into T1. 14, possesses sulfomycin resistance gene, approximately 1.8
Plasmid pB, which is characterized by incorporating into plasmid pBT24 a DNA fragment having a kilobase molecular size and characterized by the restriction enzyme map shown in FIG. 7B.
Manufacturing method of T31. 15, contains the essential region of the sulfomycin resistance gene, has a molecular size of approximately 0.84 kilobases, and has the seventh
A method for producing plasmids pBT37 and pBT38, which comprises incorporating a DNA fragment characterized by the restriction enzyme map shown in Figure C into plasmid pBT24. 16. A plasmid characterized by incorporating into plasmid pBT24 a DNA fragment that carries a novobiocin resistance gene, has a molecular size of approximately 3.92 kilobases, and is characterized by the restriction enzyme map shown in FIG. 7D. pBT
46 manufacturing methods. 17. Streptomyces lithimosidini (Str
The chromosomal DNA of a microorganism belonging to the genus Eptomyces litmocidini and resistant to amikacin was treated with the restriction enzyme BclI to obtain a microorganism that possesses an amikacin resistance gene, has a molecular size of approximately 1.8 kilobases, and is shown in Figure 7A. A method for producing a DNA fragment, which comprises cutting out and collecting a DNA fragment characterized by a restriction enzyme map. 18. Streptomyces viridochromogenes (St
reptomyces viridochromoge
The restriction enzyme MboI is applied to the chromosomal DNA of a microorganism that belongs to the genus nes) and is resistant to sulfomycin, and the chromosomal DNA of a microorganism that is resistant to sulfomycin and has a sulfomycin resistance gene and has a molecular size of approximately 1.8 kilobases is obtained. A method for producing a DNA fragment, which comprises cutting out and collecting a DNA fragment characterized by the restriction enzyme map shown in B. 19. Streptomyces niveus (Strepto-
Myces niveus), the chromosomal DNA of a microorganism resistant to novobiocin was treated with the restriction enzyme MbbI to obtain a microorganism that possesses a novobiocin resistance gene, has a molecular size of approximately 3.9 kilobases, and is shown in Figure 7D. A method for producing a DNA fragment, which comprises cutting out and collecting a DNA fragment characterized by a restriction enzyme map. 20. Plasmid pBT31 was treated with the restriction enzyme MboI to extract the essential region of the sulfomycin resistance gene, which has a molecular size of about 0.84 kilobases, and the seventh
DNA characterized by cutting out and collecting a DNA fragment characterized by the restriction enzyme map shown in Figure C
How to make fragments.