NL8001381A - PLASMID DNA AND METHOD FOR OBTAINING IT. - Google Patents

Description

Plasmid DNA and method for obtaining it.

It is known that bacterial plasmids are used for recombinant DNA research. For a good overview of developments in this area, please refer to Science, vol. 196, April 1977. Recombinant DNA testing of industrially important microorganisms of the genus Streptomyces has been described by Bibb, M.J., V7ard, 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, O. 1978: "A general method for lysis of Streptomyces species", Can.

J. Micriobiol. 24, 631-632; Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W., 1975: "Isolation of covalently closed circular deoxyribonucleic acid form Streptomyces coelicolor A3 (2)", J. Bateriol.

15 121, 416-421; Umezawa, H., 1977: "Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compound ^", Biomedicine 26, 236-249; and Malik VS 1977: Preparative method for the isolation of supercoiled DNA from a chloramphenicol producing Streptomycete, J. Antibiotics 30, 897-20 899. Nevertheless, only one Streptomycete plasmid has so far been isolated and described by Schrempf supra. That other plasmids exist in the genus Streptomyces can be deduced from results of genetic studies described by: 1) Akagawa, H., Okanishi, M. and Umezawa, H., 25 1975: "A plasmid involved in chloramphenicol production in Streptomyces venezuelae; Evidence from genetic mapping", J. Gen. Microbiol. 90, 336-346.

(2) Freeman, R.F. and Hopwood, D.A., 1978: 800 1 3 81 J 2 f "Unstable 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 ling 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 coalicolor carrying a chromosomal locus and its inter-specific transfer", J. Gen. Microbiol. 79, 331-342.

(5) Hotaa, K. Okami, Y. and Umezawa, H., 1977: "Elimination of the ability of a kanamycin-producing strain to biosynthesize deoxy-streptamine moiety by 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 celiac-color11

Nature 254, 265-267.

(7) Kirby, R. and Hopwood, D.A., 1977: "Genetic determination of methylenomycin synthesis by the SCPI plasmid of Streptomyces coelicolor 25 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 by episomic factors", J. Antibiotics 33 ^, 45-47 .

The microorganism Streptomyces fradiae NRRL 11446 has been found to contain a previously unknown plasmid. This plasmid, which was coded p UC9, has a molecular weight of approximately 42.4 megadalton, is restricted by endo-nullases BamHI 35 at more than 10 sites, Pst I at more than 15 sites, Xba at 2800 1 3 81 3 sites, Bgl II to> 5 sites, Hind III in 2 sites and Xho fragmented in more than 14 sites, and comes in 2 copies per cell. fradiae nrkl 11446 front.

pUC9 is isolated from Strentomyces fradiae NRRL 11446 5 by fragmenting the mvcelium of a culture, incubating the fragments, harvesting the culture, lumenating the mvcelium and isolating the plasmid from the lvsate. The plasmid is practically pure.

Since p UC9 is degraded or "cut" by various restriction endonucleases, it can be easily recombined and adapted for a number of host vectors, p OC9 can also be used as a cloning vector in recombinant DNA studies in which selected genes are introduced into the plasmid after which the plasmid is introduced into a host bacterium and / or vector.

15 Characteristics of n UC9 42 4

Molecular weight: approx. / Jftegadalton.

Number of copies per cell: 2.

Restriction endonuclease places:

Number of restriction sites Number of restriction sites 20 Enzyme pUC9 Enzyme pUC9

BamHI> 10 Hind III 2

Pst I> 15 Xho I> 14

Xba I 2

Bgl II 5 These values were obtained by fragmenting p UC9 DNA in the presence of excess restriction endonuclease and determining the number of fragments soluble in 0.7 and 1.0% agarose gels.

püC9 can be used to obtain recombinant plasmids that can be introduced into host bacteria. In doing so, the DNA of the circular vector plasmid with restriction endonuclease, for example Hind III or Xba I, is cut open at specific locations, resulting in linear DNA. Between the ends thereof, a piece of foreign non-vector DNA is pasted by cyclization by mixing the linear vector or pieces thereof and mixing the non-linear vector in the presence of an 800 1 3 81 4 polynucleotide ligase.

The foreign DNA obtained with the same enzyme can come from higher micro-organisms. For example, frog DNA encoding ribosomal RNA can be incorporated into pUC9 5. The circular recombinant piasmid then consists of pUC9 and a piece of frog rDNA.

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

p (JC9 derives its significance from its ability to function as a plasmid vector in microorganisms of interest for industrial applications such as Streptomyces. Cloning of DNA from Streptomyces into pUC9 allows the formation of 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 Bacillus do not well expressed 20. Likewise, genes from Gram negative bacteria are poorly expressed or not expressed at all in Gram positive hosts, thereby enhancing the benefit of a Gram positive host fender system and thus the utility of püC9 in such a system underlined.

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 pUC9, 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 pUC9 is a streptomycete plasmid 80 0 1 3 81 5, it is ideally suited for these purposes in Strentomyces. Furthermore, since pUC9 is a plasmid from a G ^ am positive microorganism, it can serve as a vector in a number of other microorganisms such as Bacillus and Arthrobacter. 5 Streptomyces fradiae NRRL 11446 can be used in some nutrient medium under submerse aerobic conditions are cultured. 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-10 sources include glucose, brown sugar, sucrose, glycerol, starch, corn starch, lactose, dextrin and molasses. Preferred nitrogen sources include corn soft liquor, yeast, brewers' yeast, autolysed with milk solids, soybean meal, cottonseed meal, corn meal, milk solids, pancreatic digested casein, yeast meal, solid distillers, animal nepton fluids and meat and bone wastes. Combinations of the aforementioned carbon and nitrogen sources are also eligible. Furthermore, since tap water and impurities are used before sterilizing the medium, there is no need to add trace metals such as zinc, magnesium, manganese, cobalt and iron.

The inoculated medium is incubated at a temperature of about 18-50 ° C, preferably 20-37 ° C. The growth of the micro-organism is optimal after about 3-15 days. The medium 25 remains acidic during the growth cycle. The final pH depends partly 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 for inoculating an excessively large layer in the growth of the microorganism. -organism and therefore ineffective use of the equipment. To form a vegetative graft, a nutrient broth is seeded with an appropriately selected amount of culture in a liquid agar, or culture in an inclined tube. The young, active vegetative 800 1 3 81 6 seed 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 microorganisms.

Example - Isolation of pUC9 from a pure culture of Streptomyces fradlae NRHL 11446.

Traces of a pure culture of Streptomyces fradlae NRRL 11446 were inoculated into 10 ml medium containing 1 wt% glucose / 0.4 wt% peptone, 0.4 wt% yeast extract, 0.05 wt% MgSC> 4.7 H 2 O, 0.2 wt% kh 2 PO 4 and 0.4 wt%.

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 rate of 100-250 rpm for about 24-36 hours.

Afterwards, 0.5 ml of the culture was placed in a 50 ml

Erlenmeyer flask mixed with 10 µl of the above medium to which 0.5-2.0 wt / vol% 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 centrifugation O 'at low speed 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 0.05 M NaCl, pH 8.0) to 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 30 KNase 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 800 1 3 81 7 20-30 min was incubated at 37 ° C. The lvsate was then sheared by passing it through a 50 ml syringe without a needle 5-10 times.

The crude stainlessate was then mixed with a salt, for example, cesium sulfate and preferably cesium chloride, and the inserted dye ethidium bromide to obtain a solution with a density of 1.550. The solution was centrifuged to equilibrium at 145,000 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 was 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 buffer (0.015 M NaCl, 0.0015 M Na3C6H507.2H20, dH 7.4) to yield practically pure dUC9.

Characterization of pUC9.

The size of pUC9 was determined by sedimentation in neutral and 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 pUC9 was determined to be 78S. Molecular weight was calculated from the equations of Hudson et al (Hudson, B., Clayton, DA and Vinograd, J., 1968: "Complex mitochondrial DNA", Cold Spring Harbor Syrap, Ouant. Biol. 33, 435-442) and was in good agreement with the determination based on the 30 basic sucrose gradients.

The percentage of plasmid DNA in Strentomyces fradiae NRRL 11446 was determined by labeling the culture with (methyl-3H) thymidine, preparing the crude lysates, and centrifuging samples of the lysates in cesium chloride ethidium bromide density gradients. The gradients were fractionated and the radio-800 1 3 81 8 activity was measured, after which the percentage of plasmid DNA and the number of piasmi decopias 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 5 closed circular duplex DNA: The closed circular DNA in BeLa cells", Proc. Nat. Acad. Sci. US 57, 1514-1520).

Restriction endonuclease fragmentation and agarose gel electrophoresis.

Restriction endonucleases were obtained as trade formulations from Miles laboratories and New England Biolabs which also provided specifications for fragmentation with at least one twofold excess of endonuclease.

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, 15 Sugden, J. and Sambrook, J. , 1973: Detection of two restriction endonuclease activities in Haemophilus parainfluenzas using analytical agarose-ethidium bromide electrophoresis, Biochemistry JL2, 3055-3063). The molecular weights of the fragments were determined relative to the standard migration patterns of bacteriophage DNA 20 fragmented with enzyme EcoRI (Helling, RB, Goodman, HM and Boyer, HW, 1974; Analysis of endonuclease R. EcoRI fragments of DNA from lambdiod bacterophages and other viruses by agarose gel electrophoresis, J. Virology 14, 1235-1244).

All of 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 pUC9 in a pure or practically pure state has a molecular weight of approximately 42.4 megadalton and by the restriction endonucleases BamEI in more than 10 places, Pst I in more than 10 15 places, Xbalop 2 places, Bgl II in 5 places, Hind III in 2 places and Xho I fragmented in more than 14 places. 2. A method of isolating plasmid DNA from a microorganism, characterized in that pure or practically pure pUC9 as defined in claim 1 is isolated from Streotomyces fradiae NRRL 11446 wherein (a) S.fradiae NRRL 11446 is grown to the 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) from the lvsate pure or practically pure pUC9 is isolated. Method according to claim 2, characterized in that the microorganism is cultivated at a temperature of about 32 ° C for about 24-36 hours. 4. A method according to claim 3, characterized in that the fragmented mycelium is incubated in a growth medium containing glycine. 5. Plasmid pUC9 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 3 31