NL8001676A - PLASMID DNA AND METHOD FOR OBTAINING IT. - Google Patents

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Plasmid DM and method for obtaining it.

It is known that bacterial plasmids are used for recombinant DM research. For a good overview of developments in this area, please refer to Science, vol. 196, April 1977 · Recombinant DM research on industrially important ant organisms of the genus Streptomyces has been described by

Bibb, M.J., Ward, J.M., and Hopwood, D.A., 1978: "Transformation of plasmid DNA into Streptomyces at high frequency", Nature 271+, 398-1 * 00.

Several Streptomycetes have been found to contain DNA plasmids. See Huber, M.L.B. and Godfrey, 0.

1978: "A general method for lysis of Streptomyces species", Can.

J. Microbiol. 21, 631-632; Schrempf, H., Bufard, H., Hopwood, D.A. and Goebel, W., 1975 * "Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolor A3 (2)", 15 J. Bacterid. 121, 1 + 16-1 + 21; Umezawa, H, 1977: "Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)", Biomedicine 26, 236-21 + 9; and Malik, U.S. 1977: Preparative Method for the isolation of supercoiled DNA from a chloramphenicol producing Streptomycete, 20 J. Antibiotics 30, 897-899. Nevertheless, only one Streptomycete plasmid has so far been isolated and described by Schrempf supra. The presence of other plasmids in the genus Streptomyces can be derived from the results of genetic research described by: 25 (t) Akagawa, H., Okanishi, M. and Umezawa, H., 1975: "A plasmid involved in chloramphenicol production in Streptomyces venezuelae: Evidence from genetic mapping ", J. Gen. Microbiol. 90, 336-31 + 6 8 0 0 1 6 76 2 (2) Freeman, R.F. and Iopwood, D.A., 1978: "Unstable naturally occurring resistance to antibiotics in Streptomyces", J. Gen, Microbiol. 106, 377-381.

5 (3) Friend, E.J., Warren, M. and Hopvood, D.A., 1978: "Genetic evidence for a plasmid controlling fertility in an industrial strain of Streptomyces rimosus", J. Gen. Microbiol. J06, 201-206.

10 (U) Hopvood, D.A. and Wright, H.M. 1973: "A plasmid of Streptomyces coelicolor carrying a chromosomal locus and its inter-specific transfer", J. Gen. Microbiol. 79, 331-3 ^ 2.

(5) Hotta, K., Okami, Y. and Umezava, H. 1977: 15 "Elimination of the ability of a kanamycin-producing strain to biosynthesize deoxystreptamine mine moiety in acriflavine", J. Antibiotics 30, 11U6-11U9 .

(6) Kirby, R., Wright, L.F. and Hopvood, D.A., 1975: 20 "Plasmid-determined antibiotic synthesis and resistance in Streptomyces coelicolor", Nature 25k, 265-267.

(7) Kirby, R. and Hopvood, D.A. 1977: "Genetic determination of methylenomycin synthesis in the SCPI plasmid of Streptomyces coelicolor A3 (2)", J. Gen. Microbiol. 98, 239-252.

(8) Okanishi, M., Ohta, T. and Umezava, H. 1969: "Possible control of formation of aerial mycelium and antibiotic production in Streptomyces at episomic factors", J. Anti biotics 33, 1 * 5-VT.

The microorganism Streptomyces espinosus subsp.acanthus NRRL 11081 has been found to contain a previously unknown plasmid. This plasmid, which was coded p UC7, has a molecular view of about 10-11 megadaltons, is degraded by restriction 800 1 6 76 3 endonuleases according to the fragmentation pattern shown in the diagram and comes in 2-b copies per cell S. espinus subsp.acanthus NRRL 11081 for. The diagram is based on plasmid p UC7 with a molecular weight of about 10.7 megadaltons and a molecular length of about 16.2 kilobases. The restriction sites are indicated as kilobase coordinates relative to the Bgl II restriction site at 0.0 / 16.2 kilobases. The abbreviations used for the restriction endonucleases are: (1) Bgl II, an enzyme from Bacillus globigii; (2) Bam Hl, an enzyme from Bacillus amylolique-10 faciens Hl; (3) Pst I, an enzyme from Providencia stuartii 16 ^; (k)

Kpn I, an enzyme from Klebsiella pneumoniae; (5) Xho I, an enzyme from Xanthomonas holcicola.

Regarding the diagram, it is noted that for the restriction site at about 14.5 kilobase it could not be unambiguously determined whether it applies to Bam MI or Pst I. Furthermore, for reasons of symmetry, it could not be unambiguously determined whether one of the two Κηρ I restriction sites at about 0.7 or at about 3.7 kilobase should be indicated.

The latter ambiguity is expressed with the dashed lines.

20 p UC7 is derived from Streptomyees espinosus subsp.

acanthus NRRL 11081 isolated by fragmenting the mycelium of a culture, incubating the fragments, harvesting the culture, lysing the mycelium and isolating the plasmid from the lysate. The plasmid is practically pure.

Because p UC7 is relatively small and is degraded or "cut" by various restriction endonucleases, it can easily be recombined and adapted for a number of host vectors. p UC7 can also be used as a cloning vector in recombinant DNA studies in which selected genes are built into the plasmid and the plasmid is introduced into a host organism.

Characteristics of p UC7 Molecular weight: about 10-11 megadalton.

Number of copies per cell: 2-k 35 Place restriction endonuclease (see diagram).

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Jt • 1 k

Number of restriction places Number of restriction places

Enzyme püC7 Enzyme pUC7

Bgl I> 7 Bgl II 2

Bam HI H Hpa I O

5 Hpa II much Hind III 2-3

EcoRI 0 Κρη I 2

Pst I 2 Pvu II> 5

Mbo II> 6 Ava I> 8

Xba I 3 Xho I 3 10 Sal I> 7 Sma I a lot

Hinc II a lot Call I a lot

These values were obtained by fragmenting p UC7 DNA in the presence of an excess of restriction endonuclease and determining the number of 15-soluble fragments in 0.7 and 1.0 µg agarose gels.

pUC7 can be used to obtain recombinant plasmids that can be introduced into host bacteria. In addition, the DNA of the circular vectorial plasmid with restriction endonuclease, for example Bgl II or Pst I, is cut open at 20 specific places, whereby linear DNA is created.

Between the ends thereof, a piece of foreign non-vectorial DNA is pasted by cyclization by mixing the linear vector or pieces thereof, and mixing the non-linear vector in the presence of a polynucleotide ligase.

The foreign DNA, obtained with the same enzyme, can come from higher microorganisms. For example, frog-derived DNA encoding ribosomal RNA can be incorporated into pUC7. The circular recombinant plasmid then consists of pUC7 and a piece of frog rDNA.

The recombinant plasmid can be introduced into and expressed in a host organism. Examples of valuable genes are those encoding somatostatin, rat proinsulin and proteases.

pUC7 derives its significance from its ability to function as a plasmid enhancer in microorganisms 800 1 6 76 5 of interest for industrial applications "such as Streptomyces. Cloning of DM from Streptomyces into pUC7" allows for the formation of to increase economically important products from these organisms, such as antibiotics. This proposal can be compared to the cloning of antibiotic genes in Escherichia coli K-12. However, this Gram-negative bacterium has the drawback that genes from certain Gram- positive bacteria such as Baccillus are not well expressed therein. Likewise, genes from Gram negative bacteria are poorly expressed at all or not at all in Gram-positive hosts, thereby enhancing the benefit of a Gram-positive host vector system and thus its utility of pUC7 in such a system.

In addition to the gene expression difficulties outlined above, difficulties may also arise in culturing the microorganism and in the extraction and purity of the product.

These difficulties could be addressed by introducing a plasmid vector, for example pUC7, into a host that forms the product and cloning the genes for the product in question into the plasmid. Thus, the difficulties with fermentation, extraction and purification would in any case be alleviated. Moreover, in such a system it would not be necessary to clone and multiply all the genes for biosynthesis but only the regulatory genes or genes encoding the enzymes that control the production rate. Since pUC7 is a streptomycete 25 plasmid, it is ideally suited for these purposes

Streptomyces. Furthermore, because pUC7 is a plasmid from a Gram-positive microorganism, it can serve as a vector in a number of other microorganisms such as Bacillus and Arthrobacter.

Streptomyces espinosus subsp. acanthus NRRL 11081, 30 deposited in the permanent collection of the Northern Regional Research Laboratory, Peoria, Illinois, America and taxSmischen in Belgian Patent 861 (-. 1 (-01), can be added in an aqueous nutrient medium under submerged aerobic conditions The nutrient medium may contain a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assigible 800 1 β 75 cfl 6, a miscible nitrogen compound or a protein: Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch , corn starch, lactose, dextrin, and molasses Preferred sources of sdc include corn steep liquor, yeast, brewers' yeast with autolysed solid milk, soybean meal, cottonseed meal, corn meal, solid milk ingredients, pancreatic digested casein, yeast meal, solid distillers, animal peptone liquids and meat and bone waste Combinations of the aforementioned cabbage sources of dust and nitrogen are also eligible. Furthermore, since tap water and impure ingredients are used before sterilizing the medium, there is no need to add spore metals such as zinc, magnesium, manganese, cobalt and iron.

The grafted medium is incubated at a temperature of about 18-50 ° C and preferably 20-37 ° C. After about 3-15 days, the growth of the micro-organism is optimal. The medium remains acidic during the growth cycle. The final pH partly depends on any buffer present and partly on the initial pH of the medium.

When cultivation is carried out in large vessels or tanks, it is preferable to use the vegetative form of the microorganism instead of the spore form for inoculation to avoid excessive growth of the microorganism avoid and thus ineffective use of the equipment. To form a vegetative graft, a broth is seeded with an appropriately selected amount of culture in a liquid agar, or culture in an inclined tube. The young and active vegetative graft is then placed under aseptic conditions in a culture vessel or culture tank. The medium in which the vegetative graft is formed can be the same as that used for the growth of the microorganism.

Example - Isolation of pUC7 from a pure culture of

Streptomyces espinosus subs, acanthus HERL 11081 35 Traces of a pure culture of Streptomyces 800 1 6 76 * 7 w espinos subsp. acanthus KRRL 11081 were inoculated into 10 ml of Difco Antibiotic Medium No. 3 Broth from Difco Labs., Detroit, Michigan containing 0.15% meat extract, 0.15% yeast extract, 0.5% w / pton, 0.1 wt. # Glucose, 0.35 wt. # NaCl, 0.368 wt. # KgHPO4 and 5 0.132 wt. # KHgPO4. The medium was previously sterilized in a 50 ml Erlenmeyer flask. After seeding, the contents of the flask were incubated at 37 ° C in a Gump of New Brunswick rotary shaker at a speed of 100-250 rpm for approximately 36 * 48 hours.

Afterwards, the mycelium broth suspension was homogenized under 10 sterile conditions and then mixed in a 125 ml Erlenmeyer flask with 10 ml of the above medium to which 68% w / v sucrose and 1% w / v glycine were added. The presence of sucrose and glycine is not necessary but promotes cell lysis. The amount of sucrose and glycine in the medium may vary otherwise, but is usually chosen to promote the lysis of the cells. The contents of the flask were incubated for an additional 36-8 hours at 37 ° C in a Gump rotary shaker. Afterwards, the mycelium was separated from the culture fluid by low speed centrifugation such as 6000 x g for 15 min at U ° C followed by decanting the supernatant. After this, the mycelium was suspended in 1.5 ml isotonic TES buffer (0.03 M tris (hydroxymethyl) aminomethane (Tris), 0.005 M EDTA and 0.05 M NaCl, pH 8.0) at which 20 w / v. sucrose was added. After adding 1.5 ml of a 5 mg / ml solution of lysozyme in the same buffer, the mixture was incubated at 37 ° C for 30 min with intermediate stirring. After adding 1.5 ml 0.25 M EDTA (pH 8.0) incubation at 37 ° C was continued for another 15 min. Afterwards, the cell suspension was lysed by adding 2.5 ml of a lytic mixture (1.0 w / v # 30 Brij-58 (a detergent from Pierce Chem. Co., Rockford, Illinois),

0.1 + w / v vol deoxycholic acid, 0.05 M Tris (pH 8.0) and 0.06 M EDTA) and incubated at 37 ° C for 20 min. The lysate was then sheared by passing it through a 10 ml pipette 5-10 times. The crude lysate was then digested with ribonuclease (1H0 µg / ml) and prionase (300 yug / ml) for 20 min at 37 ° C. The cell lysozyme-EDTA

However, 800 1 6 76 * 8 mixture may also be digested first with rihonuclease and pronase before being lysed with a lytic agent such as a 2 wt% solution of sodium dodecyl sulfate in water.

The crude lysate was then mixed with a salt, for example, cesium sulfate and preferably cesium chloride, and the inserted dye ethidium bromide to obtain a 1.550 density solution. The solution was centrifuged to equilibrium at 11 * 5 * 000 x g (isopynic density gradient).

The covalently closed circular plasmid DNA was observable in the centrifuge tube under long-wave ultraviolet radiation (320 nm) as a faint fluorescent band under the intensely fluorescent band of linear chromosomal and plasmid DNAs.

The plasmid DNA was removed from the isopian gradients and the ethidium bromide is extracted by treating twice with 1/3 volume of isopropanol and then dialyzing the aqueous phase against a buffer such as a 0.1 X SSC buffer (0.015 M NaCl, 0.0015 M Na 0 ^ 0 ^ .2 ^ 0 pH »7.0 * 0 to yield practically pure pUC7.

Characterization of pUC7 The size of pUC7 was determined by sedimentation in neutral and basic sucrose gradients using an internal marker plasmid DNA with a molecular weight of about 9.0 megadaltons and a corresponding sedimentation value of about 3 µS. From the neutral sucrose gradients, the sedimentation value of the isolated pUC7 was determined to be 35-37 S. Molecular weight was calculated from the equations of Hudson et al (Hudson, B, Clayton, DA and Vinograd, J., 1978) Complex mitochondrial DNA, Cold Spring Harbor Symp. Quant, Biol. 33, 1 * 35- ^ 2) and was in good agreement with the determination based on the 30 basic sucrose gradients.

The molecular weight of pUC7 was also determined by electron microscopy of individual DNA molecules (Kleinschmidt, AK (1968): Monolayer techniques in electron microscopy of nucleic acid molecules, "Method in Enzymology" (SP Colowick and NO Kaplan 35 eds.) Vol. 12B, biz. 361-377, Academic Press, New York).

800 1 6 76 TTr 9

For the mean contour length, a value of 5. ^ 3-10 ~ ^ m was found and a corresponding molecular weight of 10.7 megadaltons.

The percentage of plasmid DNA in Streptomyces espinosus 5 subsp. acanthus NRRL 11081 was determined by labeling the culture with (methyl-H) -thymidine, preparing the crude lysates, and centrifuging samples of the lysates in cesium chloride ethium bromide density gradients. The gradients were fractionated and the radioactivity was measured, after which the percentage of plasmid DNA and the number of plasmid copies were calculated from the percentage of radioactivity in the plasmid band (Radloff, R., Bauer, W. and Vinograd, J. 1967: "A dye -buoyant density method for detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells ", Proc. Nat. Acad. Sci. USA 57, 151 ^ -1520).

15 Restriction of endonuclease fragmentation and agarose gel electrophoresis.

Restriction endonucleases were obtained as trade preparations from Miles Laboratories and New England Biolabs which also provided the specifications for fragmentation with at least a 2-fold excess of endonuclease. In some assays, the plasmid DNA was fragmented with more than one endonuclease. The assays were performed by two methods. According to the first, the plasmid DNA was first fragmented with the enzyme that needs a lower ionic strength and then with the enzyme that needs a higher ionic strength after adding an equal volume of 2X buffer of the second enzyme. According to the second method, restriction fragments of one enzyme were isolated from a preparative agarose gel as described by Tanaka and Weisblum (Tanaka, T., and Weisblum, B. 1975: Construction of a colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid, J. Bacteriol. 121, 35 ^ -3 ^ 2).

The isolated restriction fragments were concentrated by ethanol precipitation and then fragmented with other restriction enzymes. Duplicate fragmentations were compared with single fragmentations to ensure that no abnormal restriction patterns were obtained, that 80 0 1 6 76 'vy * 10 vil say that a nonspecific DNA cleavage does not occur after alteration of the ionic strength of the mixture used for fragmentation.

The fragmented samples were applied to 0.7 and 1 wt% agarose gels and electrophoresed at a constant voltage of 10-15 V / cm gel height for 2 hours (Sharp, PA, Sugden, J. and Sambrook, J. , 1973: Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis, Biochemistry 12, 305-3063). The molecular weights of the fragments were determined relative to the standard migration patterns of bacteriophage λ DNA fragmented with Hind III (Murray, K. and Murray, NE 1975: Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease III of Haemophilus influenzae and restriction endonuclease 15 of Escherichia coli J. Mol. Biol. 98, 551-56¾) or EcoRI (Helling, RB Goodman, HM and Boyer, HW 197 ^: Analysis of endonuclease R. EcoRI fragments of DNA form lambdiod bacteriophages and other viruses in agarose -gel electrophoresis, J. Virology 1¾ ^ 1235-12¾¾).

All of the tests and assays described were performed according to the specifications in the NIH Guidelines.

800 1 6 76

Claims (7)

1. Plasmid DNA, characterized in that plasmid DNA with the code pUC7 in pure or practically pure state has a molecular weight "of about 10-11 megadalton and a restriction endonuclease fragmentation pattern as shown in the diagram. A method of isolating plasmid DNA from a microorganism, characterized by pure or substantially pure pUC7 as defined in claim 1 is isolated from Strepto-myces espinosus subsp. acanthus NRR1 11081 wherein (a) S.espinosus subs, acanthus NRRL 11081 is grown until the mycelium has grown sufficiently, (b) the mycelium is fragmented, (c) the fragmented mycelium is incubated, (d) the culture harvested, (e) the harvested mycelium is lysed, and 15 (f) isolated from the lysate is pure or substantially pure pUC7. The method according to claim 3, characterized in that the microorganism is cultivated at a temperature of about 37 ° C for about 36-48 hours. The method according to claim 3, characterized in that the fragmented mycelium is incubated in a growth medium containing sucrose and glycine. 5. Plasmid pUC7 available by the method as described and the description and example. 6. Products and methods as described in the description and the example. 800 1 6 76. \ (14.5) Bam HI (15.5) Pst Jj® * * Bgl II (0 / 16.2), Κρη I (0.7) / \ Xho I (1.9) / Α ^ Κρη I (2.2) “O- Xho I (2.4) pUC7 (10.1) Xho I -V / 'Kpn I (3.7) (9.7) Bam Hry \ / N \' HI (4.6) (8.5) Bam Hl ^ "^ --- ^ Bgl II (5.3 ) 80. T 6 76 Th · jρjoha Co * p * ny # t · Kaluaxoo, Michigan, USA St.Aerlka