NL8001240A - PLASMID DNA AND METHOD FOR OBTAINING IT. - Google Patents

Description

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Plasmid DNA and method for obtaining it.

It is known that bacterial plasmids are used for recombinant DNA research. For a good overview of the development in this field, reference is made to Science, vdL 196, April 1977. Recombinant DNA research on industrially important microorganisms 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 274, 398-400.

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. 24, 631-632; Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W., 1975: "Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolor A3 (2)", 15 J. Bacteriol. 121, 416-421; Umezawa, H, 1977: "Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)", Biomedicine 26, 236-249; 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 been isolated and described by Schrempf supra so far. That even more plasmids exist in the genus Streptomyces can be deduced from results of genetic studies described by: 25 (1) Akagawa, H., Okanishi, M. and Umezawa, H., 1975: "A plasmid involved in chloramphenicol production 800 1 2 40 r * 2 in Streptomyces venezuelae: Evidence from genetic mapping, J. Gen, Microbiol. 90, 336—346.

(2) Freeman, R.F. and Hopwood, D.A., 1978: "Unstable 5 naturally occurring resistance to antibiotics in Streptomyces", J. Gen. Microbiol. 106, 377-381.

(3) Friend, E.J., Warren, M. and Hopwood, D.A., 1978: "Genetic evidence for a Plasmid Control fertility in an industrial strain of

Streptomyces rimosus, "J. Gen. Microbiol. 106, 201-206.

(4) Hopwood, 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, 331-342, (5) Hotta, K., Okami, Y. and Umezawa, H. 1977: "Elimination of the ability of a kanamycin-producing strain to biosynthesize deoxystreptamine moiety at acriflavine", J. Antibiotics 30, 1146-1149.

(6) Kirby, R., Wright, L.F. and Hopwood, D.A., 1975: "Plasmid-determined antibiotic synthesis and resistance in Streptomyces coelicolor", Nature 25 254, 265-267.

(7) Kirby, R. and Hopwood, 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 Umezawa, H. 1969: "Possible control of formation of aerial mycelium and antibiotic production in Streptomyces at episomic factors", J. Antibiotics 33, 45-47.

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The microorganism Streptomyces fradiae NRRL 11443 has been found to contain a previously unknown plasmid.

This plasmid, which was coded p UC1, has a molecular weight of about 47.1 megadalton, is degraded by restriction endonuleases according to the fragmentation pattern shown in the diagram, and occurs in 1 copy per cell S.fradiae NRRL 11443.

The diagram is based on plasmid p UC1 with a molecular weight of approximately 47.1 + 1.2 megadalton and a molecular length of approximately 71.1 kilobases. The restriction sites are indicated as kilobase coordinates relative to the Xba I restriction site at 0.0 / 71.1 kilobases. The abbreviations used for the restriction endonucleases are: (1) Bgl II, an enzyme from Bacillus globigii; (2) Hind III, an enzyme from Haemophilus influenzae and 15 (3) Xba I, an enzyme from Xanthomonas badrii.

p UC1 is isolated from Streptomyces fradiae NRRL 11443 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 20 is practically pure.

Because p UC1 is degraded or "cut" by various restriction endonucleases, it can be easily recombined and adapted for a number of host vectors, p UC1 can also be used as a cloning vector in recombinant DNA studies where selected genes are built into the plasmid the plasmid is introduced into a host bacterium and / or vector.

Characteristics of p UC1

Molecular weight: about 47.1 1.2 mega dalton.

30 Number of copies per cell: 1.

Place restriction endonuclease (see diagram).

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Number of restriction places Number of restriction places

Enzyme pUCl Enzyme pUCl 5 BamHI _> 15 Hind III 2

EcoRI 0 Κρη I> _ 15

Pst I j> 18 Xho I 11

Xba I 2 Sma I> _ 15

Bgl II 5 Call I 6 10

These values were obtained by fragmenting p UC1 DNA in the presence of excess restriction endonuclease and determining the number of fragments soluble in 0.7 and 1.0% agarose gels.

PUC1 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, Hind III or Xba I, is cut open at specific places, resulting in linear DNA. Between the ends thereof, a piece of foreign non-vectorial DNA is pasted by cyclization by mixing the linear vector or pieces thereof, and 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 DNA encoding ribosomal RNA can be incorporated into pUCl. The circular recombinant plasmid then consists of pUCl 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.

pUCl derives its significance from its ability to function as a plasmid vector in micro-organisms of interest for industrial applications such as 800 1 2 40 Μ.

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Streptomyces. Cloning DNA from Streptomyces into pUCl provides the opportunity to enhance the formation of economically important products from these organisms, such as antibiotics. This proposal can be compared to the cloning of genes for 5 antibiotics 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 properly expressed therein. Likewise, genes from Gram negative bacteria are poorly expressed or not expressed at all in Gram positive hosts.

10 This underlines the advantage of a Gram positive host vector system and thus the utility of pUCl 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, for example, introducing a plasmid vector pUCl 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 controlling the production rate. Since pUCl is a streptomycete plasmid, it is ideally suited for these purposes in streptomyces. Furthermore, because pUC1 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 fradiae NRRL 11443 can be grown in an aqueous nutrient medium under submerse aerobic conditions. The nutrient medium may contain a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or a protein.

Preferred carbon sources include glucose, brown 800 1 2 40 6 sugar, sucrose, glycerol, starch, corn starch, lactose, dextrin and molasses. Preferred nitrogen sources include corn steep liquor, yeast, autolysed brewers' yeast with milk solids, soybean meal, cottonseed meal, corn meal, solid milk ingredients, pancreatic digested casein, yeast meal, solid distillers, animal peptone fluids and meat and bone waste. Combinations of the aforementioned carbon and nitrogen sources are also eligible. Furthermore, since tap water and raw materials 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 J8-50 ° C and preferably 20-37 ° C. After about 3-J5 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 the 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 20 for inoculation to prevent excessive growth in 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.

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Yoorheéld - Isolation of pUCJ from a pure culture of Streptomyces fradiae NRRL 11443

Traces of a pure culture of Streptomyces 35 fradiae NRRL 11443 were seeded in 10 ml of medium extracting 1 wt% glucose, 800 1 2 40% 7 0.4 wt% peptone, 0.4 wt% yeast, 0 0.05 wt% MgSo 4 .7H 2 O, 0.2 wt% KH 2 PO 4 and 0.4 wt% I 2 HPO 4.

The medium was previously sterilized in a 50 ml Erlenmeyer flask.

After seeding, the contents of the flask were incubated at 32 ° C in a Gump of New Brunswick rotary shaker at a speed of 100-250 rpm for about 24-36 hours.

Afterwards, 0.5 ml of the culture in a 50 ml Erlenmeyer flask was mixed with 10 ml of the above medium to which 0.5-2.0 w / v% glycine was added. The presence of glycine is not necessary but promotes cell lysis.

Incidentally, the amount of glycine in the medium may vary, but is usually chosen to promote lysis of the cells. The contents of the flask were incubated for an additional 24-36 hours at 32 ° 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 4 ° C followed by decanting the supernatant. After this, the mycelium was suspended in 1.5 ml TES buffer (0.03 M tris (hydroxymethyl) aminomethane (Tris), 0.005 M EDTA and 20 0.05 M NaCl, pH 8.0) at which 20 w / v% sucrose was added.

After adding 0.3 ml of a 5 mg / ml solution of lysozyme and 0.15 ml of a 1 mg / ml RNase in the same buffer, the mixture was incubated at 37 ° C for 30 min with stirring from time to time. After adding 0.6 ml 0.25 M EDTA (pH 8.0) incubation at 37 ° C was continued for a further 15 min and after adding 0.3 ml 5 mg / ml pronase a further 10 min. at 37 ° C.

Afterwards, the cell suspension was lysed by adding 3.0 ml of 2 wt% sarkosyl containing TES buffer and incubated at 37 ° C for 20-30 min. The lysate was then sheared by passing it 5-10 times through a 50 ml syringe without a needle.

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 145,000 x g (isopynic density 800 1 2 40 8 gradient). The covalently closed circular plasmid DNA was observable as a faint fluorescent band under the intensely fluorescent band of linear chromosomal 5 and plasmid DNAs in the centrifuge tube under long wave ultraviolet radiation (320 ma).

The plasmid DNA was removed from the isopian gradients and the ethidium bromide is extracted by treating twice with 1/3 volume of isopropanol after which the aqueous phase was dialyzed against a buffer such as a 0.1 X SSC 10 buffer (0.015 M NaCl, 0.0015 M Na ^ gH ^^ 0, pH - 7.4) to yield practically pure pUCl.

Characterization of pCIJl

The size of pUC1 was determined by sedimentation and in neutral / basic sucrose gradients using an internal marker plasmid DNA with a molecular weight of about 28.0 megadaltons and a corresponding sedimentation value of about 58S. From the neutral sucrose gradients, the sedimentation value of the isolated pUCl was determined to be 76S.

Molecular weight was calculated from the equations of Hudson et al. ("Complex mitochondrial DNA", Cold Spring Harbor

Symp. Quant, Biol. 33, 435-442) and was in good agreement with the determination based on the basic sucrose gradients.

Molecular weight of pUC1 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, eds.) Vol. 12B , biz. 361-377,

Academic Press, New York),

For the average contour length, a value of 24.0 + 0.6 µm was found and a corresponding molecular weight of 47.1 1.2 megadaltons.

The percentage of plasmid DNA in Streptomyces fradiae NRRL 11443 was determined by labeling the culture with (methyl-) thymidine, preparing the crude lysates, and centrifuging samples of the 35 lysates in cesium chloride ethidium 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, 5 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, 1514-1520).

Restriction endonuclease fragmentation and agarose gel electrophoresis.

JO 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 according to two methods. According to the first x, the plasmid DNA is 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 an 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 or deoxyribonucleic acid, J. Bacteriol.

25 354-362).

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, ie a non-specific cleavage of DNA does not occur after changing the ionic strength of the fragmentation used mixture.

The fragmented samples were applied to 0.7 and 1 wt% agarose gels and subjected to electro-800 for 2 hours 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, 5 Biochemistry 12, 305-3063). The molecular weights of the fragments were determined relative to the standard migration patterns of bacteriophage λ DNA fragmented with enzyme EcoRI (Helling, RB, Goodman, HM and Boyer, HW 1974: Analysis of endonuclease R. EcoRX fragments of DNA from lambdiod bacteriophages 10 and other viruses agarose gel electrophoresis, J. Virology 14, 1235-1244).

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

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Claims (6)

1. Plasmid DNA, characterized in that plasmid DNA with the code pUC1 in pure or practically pure state has a molecular weight of approximately 47.1 ± 1.2 megadalton and a restriction endonuclease fragmentation pattern as shown in the diagram. 2. A method of isolating plasmid DNA from a microorganism, characterized by pure or practically pure pUCl as defined in claim 1 is isolated from Streptomyces fradiae NRRL I1443 wherein (a) S.fradiae NRRL 11443 is grown to mycelium has grown sufficiently, (b) the mycelium is fragmented, (c) the fragmented mycelium is incubated, (d) the culture is harvested, (e) the harvested mycelium is lysed, and (f) pure from the lysate or practically 20 pure pUCl is isolated. The method according to claim 3, characterized in that the microorganism is cultivated at a temperature of about 32 ° C for about 24-48 hours. A method according to claim 3, characterized in that the fragmented mycelium is incubated in a growth medium containing glycine. 5. Plasmid pUCl available by the method as described in the description and the example. 6. Products and methods as described in the description and the example. 800 1 2 40