NL8001380A - 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 developments in this area, please refer to Science, vol.

196, April 1977. Recombinant DNA testing of industrially important microorganisms of the genus Streptomvces has been described by Bibb, MJ, Ward, JM, and Hopwood, DA, 1978: "Transformation of Plasmid DNA into Streptomvces 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 Streptomvces species"; Can. J. Microbio. 24, 631-632; Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W., 1975: "Isolation of covalently closed circular deoxyribonucleic acid from Streptomvces coelicolor A3 (2)", J. Bacterid.

15 121, 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, USA, 1977: "Preparative method for the isolation of supercoiled DNA from a chloramphenicol producing Streptoraycete, J. Antiobiotics 20 30." However, only one Streptomycete plasmid has so far been isolated and described by Schrempf supra That other plasmids exist in the genus Streptomvces 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 Streptomvces venenzuelae:

Evidence from genetic mapping ", J. Gen.

Microbiol. 90, 336-346.

(2) Freeman, R.F. and Hopwood, D.A., 1978: 80 0 1 3 80 2 "Unstable naturally occurring resistance to antibiotics in Streptomyces", J. Gen.

Microbiol. 106, 377-381.

(3) Friend, E.J., Warren, M., and HOpwood, D.A., 1978s "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 10 of Streptomyces coellcolor carrying a chromo somal locus and its inter-specific transfer", J. Gen. Microbiol. 79, 331-342.

(5) Botta, K., Okami, Y and Umezawa, H. 1977: "Elimination of the ability of a kanaraycin-producing strain to biosynthesize deoxystreptamine moiety by acriflavine", J. Antibiotics 30, 1146-1149.

(6) Kirby, R., Wright, L.F. and Hopwood., D.A., 1975: "plasmid-determined antibiotic synthe * 20 sis and resistance in Streptomyces coelicolor

Nature 254, 265-267.

(7) Kirby, R. and Hopwood, D.A. 1977: "Genetic determination of methylenomycin synthesis by the SCP1 plasmid of Streptomyces coellcolor 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 fradlae NRRL 11445 has been found to contain a previously unknown plasmid. This plasmid, which was coded p UC8, has a molecular weight of about 45.0 megadaltonf / is restricted by endonuleases BamHl 35 at more than 10 sites, Pst I at more than 10 sites, Xba I at 1 80 0 1 3 80 - Plaats 3 place, Bgl II in 5 places, Hind III in 2 places and Xho I fragmented in more than 13 places.

p UC8 is isolated from Streotomyces fradiae NRRL 11445 by fragmenting the mycelium of a culture, incubating the 5 fragments, harvesting the culture, lysing the mycelium and isolating the plasmid from the lysate. The plasmid is practically pure.

Because p UC8 is degraded or "cut" by various restriction endonucleases, it can easily be recombined and adapted for a number of host vectors. P UC8 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 UC8 15 Molecular weight: about 45.0 megadalton.

Number of copies per cell: -1.

Restriction endonuclease places:

Number of restriction places Number of restriction places

Enzyme pUC8 Enzyme pUC8 20 BamHI> 10 Hind III 2

Pst I> 10 Xho I> 13

Xba I 1

Bgl II 5

These values were obtained by fragmenting βC8 DNA in the presence of excess restriction endonuclease and determining the number of fragments soluble in 0.7 and 1.0% agarose gels.

pUC8 can be used to obtain recombinant Dlasmids that can be introduced into host bacteria. In addition, the DNA of the circular vector plasmid with restriction endonuclease, for example, Hind III or Xba I, is uncut at specific sites, 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 80 0 1 3 80 Γ 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 pUC8.

The circular recombinant plasmid then consists of pUC8 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.

pUC8 derives its meaning from its ability to function as a plasmid vector in microorganisms of importance for industrial applications such as streotomyces. Cloning DNA from Streptomyces into pUC8 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 antibiotic genes in Escherichia coll K-12. However, this Gram-negative bacterium has the drawback that genes from certain Gram-positive bacteria such as Bacillus are not well expressed therein. Likewise, genes from Gram negative bacteria are poorly expressed or not expressed at all in Gram positive hosts. This underlines the advantage of a Gram-positive host vector system and thus the utility of pUC8 in such a system.

In addition to gene expression difficulties outlined above, difficulties may also arise in culturing the microorganism and in extraction and purity of the product. These difficulties could be addressed by introducing a plasmid vector, for example pUC8, 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 800 1 3 80 5 4

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for the enzymes that control the production rate. Since pUC8 is a streptomycete plasmid, it lends itself to Streptomyces for these purposes. Furthermore, because pUC8 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 NRKL 11445 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 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, milk solids, pancreatic digested vasein, yeast meal, solid distillers, animal peptone liquids and meat and bone waste. Combinations of the aforementioned carbon and nitrogen sources 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 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 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 for inoculation in order to have an excessive layer in the growth of the micro-organism. -organism and therefore ineffective use of the equipment. To form a vegetative graft, a nutrient broth is inoculated with an appropriately selected amount of culture in a liquid agar, or culture in an inclined tube. The young, 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 pUC8 from a pure culture of Streptomyces fradiae NRRL 11445.

Traces of a pure culture of Streptomyces 10 fradiae NRRL 11445 were seeded in 10 ml of medium containing 1 wt% glucose, 0.4 wt% peptone, 0.4 wt% yeast extract, 0.05 wt% MgSO 4. 7H 2 O, 0.2 wt% kh 2 PO 4 and 0.4 wt% K 2 HPC> 4.

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

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

Afterwards, 0.5 ml of the culture was mixed in a 50 ml Erlenmeyer flask 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. After completion, 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 0.05 M NaCl, 30 pH 8.0) at 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 another 15 min. And after adding 0.3 ml 5 mg / ml ponase another 800 1 3 80

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After 10 minutes 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 minutes. The lvsate was then sheared by passing it 5-10 times through a 50 ml syringe without needle 5.

The crude stainlessate was then mixed with a salt, for example, cesium sulfate and preferably cesium chloride, and the imported dye ethidium bromide to obtain a solution having a density of 1.550. The solution was centrifuged at equilibrium at 145,000 st 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 after which the aqueous phase was dialysed against a buffer such as a 0.1 X SSC buffer (0.015 M NaCl, 0.05 M 0015 M Na ^ HgO ^ HjO, pH 7.4) yielding practically 20 pure pUC8.

Characterization of pUC8.

The size of pUC8 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 pUC8 was determined at 76S. The molecular weight was calculated from the equations of Hudson et al (Hudson, B., Clayton, DA and Vinograd, J, 1968; "Complex mitochondrial DNA", 30 Cold Spring Harbor Symp. Quant. Biol. 33, 435-442) and was in good agreement with the determination based on the basic sucrose gradients.

The percentage of plasmid DNA in StrePtomvces fradiae NRRL 11445 was determined by marking the culture with 35 (methyl-3H) thymidine, preparing the crude lysates, and centrifuging samples 80 0 1 3 80> 8 ⁄ of the lysates in cesium chloride ethidium bromide density gradients. The gradients were fractionated and the radioactivity was measured, after which the percentage of plasmid DMA 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 HèLa cells ", Proc. Nat. Acad. Sci. PSA 57, 1514-1520).

Restriction endonucelase fragmentation and agarose gel electrophoresis.

Restriction endonucleases were obtained as trading preparations from Miles Laboratories and Mew England Biolabs which also provided the specifications for fragmentation with at least a two-fold excess of endonuclease.

The fragmented samples were applied to 0.7 and 1 wt% agarose gels and electrophoresed for 2 hours at a constant voltage of 10-15 v / cm gel height (Sharp, PA, Sugden, J. and Sambrook, J ., 1973: Detection of two restriction endonuclease activities in Haemophilus paralnfluen2ae using analytical agarose-ethidium bromide electrophoresis, Biochemistry 12, 3055-20 3063). The molecular weights of the fragments were determined relative to the standard migration patterns of bacteriophage DNA fragmented with enzyme EcoRl (Helling, RB, Goodman, HM and Boyer, HW, 1974: Analysis of endonuclease R. EcoRl fragments of DNA from lambdiod bacteriophages and other viruses by agarose gel 25 electrophorsis, 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 pUC8 in a pure or practically pure state has a molecular weight of approximately 45.0 megadalton and, by the restriction endonucleases BamH I in more than 10 places, Pst I in more then 10 sites, Xba I in 1 olate, Bgl II in 5 sites, Hind III in 2 sites and Xho "* / fragmented in more than 13 sites. A method of isolating plasmid DNA 10 from a microorganism, characterized in that pure or substantially pure pUC8 as defined in claim 1 is isolated from Streptomvces fradiae NRRL 11445 wherein (a) S.fradiae NRRL 11445 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 20 (f) from the lysate pure or practically pure pUC8 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. The method according to claim 3, characterized in that the fragmented mycelium is incubated in a growth medium containing glycine. 5. Plasmid pUC8 available by the method as described in the description and the example. 6. Products and methods as described in the description and the example. . 35 SO0 1 3 80