NL8001464A - PLASMIDA DNA AND METHOD FOR OBTAINING IT. - Google Patents

Description

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Piasmid 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 micro-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 Streptomyees at high frequency", Nature 398-UOO.

Several Streptomycetes have been found to contain DNA plasmids. See Huber, M.L.B. and Godfrey, 0. 1978: "A general method for lysis of Streptomyees species", Can. J. Microbiol. 21, 631-632; Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W. 1975: "Isolation of covalently closed circular deoxyribonucleic acid from Streptomyees coelicolor A3 (2)", J. Bacteriol. 121, 15, 16-21; Umezawa, H., 1977 * · "Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)", Biomedicine 26_, 236-2U9 · Nevertheless, only three Streptomycete plasmids have so far been isolated and described by Schrempf supra . That 20 more plasmids exist in the genus Streptomyees can be deduced from results of genetic studies described by: (1) Akagawa, H., Okanishi, M..en Umezawa, H. 1975.

"A plasmid involved in chloramphenicol production in Streptomyees venecuelae: Evidence 25 from genetic mapping", J. Gen. Microbiol. 90 336-3½.

(2) Freeman, R.F. and Hopwood, D.A. 1978.

"Unstable naturally occurring resistance to 800 1 4 64 - 2 - 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 controlling 5 fertility in an industrial strain of Strepto myces rimosus". J. Gen. Microbiol. 106, 201-206.

[h) Hopwood, D.A. and Wright, H.M. 1973.

"A plasmid of Streptomyces coelicolor carrying a chromosomal locus and its inter-specific 10 transfer". J. Gen. Microbiol. J9, 331-3 ^ 2, (5) Hotta, K., Okami, Y. and Umezawa, H. 1977 · "Elimination of the ability of a kanamycin producing strain to biosynthesize deoxy-streptamine moiety by acriflavine". J. Anti-15 biotics 30, IIU6-HU9.

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

20 (7) Kirby, R and Hopwood, D.A. 1977 * "Genetic determination of methylenomycin synthesis by 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 by episomic factors". J. Antibiotics 33, ^ 5- ^ 7. ____ 30 (9) Okanishi, M. 1977 .::.

Involvement of plasmids in the production of secondary metabolites, Amino Acid-Nucleic Acid 35, 15-30.

(10) Schrempf, H. and Goebel, W. 1977 '35 Characterization of a plasmid from Streptomyces 80 0 1 4 64 * * τ * - 3 - eoelicolor Ag, J. Bacteriol. 131, 251-258.

(11) Shaw, PiD »and Piwowarski, J. 1977:

Effect of ethidium bromide and acriflavine on streptomycin production by Streptomyces 5 bikiniensis, J. Antibiotics 30, HoH-J + 08.

(12) Yagisawa, M., Huang Rossana T-S., And Davies, J.E. 1978:

Possible involvement of plasmids in biosynthesis of neomycin, J. Antibiotics 3J_, 809-81310 In Journal of Antibiotics, Vol. XXX No. 10, 897-899 is through

Dr. Vedpal S. Malik described a method for isolating piasmi from the DNA.

The microorganism Strentomyces sp.3022a NRRL 11 ^ i ^ 1 has been found to contain a plasmid not previously described.

This plasmid, which was coded p UC3, has a molecular weight of about 20 x 10 ^ daltons and is degraded by restriction endonuleases according to the fragmentation pattern shown in the diagram, which is based on plasmid p UC3 with a molecular weight of about 20 x 10 ^ dalton and a molecule length of 20 about 30 kilobase pairs. The restriction sites are indicated as percentages with the total plasmid length set at 100% and one of the EcoRI restriction sites at 0%. The abbreviations used for the restriction endonuleases have the following meaning: (1) EcoRI, an enzyme from Escherichia coli; (2) HindIII, an enzyme: from Hemophilus influenzae; (3) Xhol, an enzyme, from Xanthomonas holcicola; (if) PstI, an enzyme from Providencia stuartii; and (5) BglII, an enzyme from Bacillus globigii.

PUC3 is obtained from Streptomyces sp. 3022a NRRL 1lUhl. This biologically pure culture is deposited with the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, America, under the aforementioned number, and is described by 35 (1) Smith, C.G. 1958: Effect of halogens on the 800 1 4 64 - k -

Chloramphenicol fermentation, J. or Bacteriol. J2 .: 577-583 .; (2) Vining, L.C. and D.W.S. Westlake (196U): Biosynthesis of the phenylpropanoid moiety of chloramphenicol, Can. J. or Microbiol. K): 705-716 .; and 5 (3) Malik, U.S. and L.C. Vining (1970): Metabolism of Chloramphenicol by the producing organism, Can. J. or Microbiol.

l £: 173-179.

Characteristics of pUC3 molecular weight: approximately 20 x 10 6 daltons.

10 restriction endonuclease sites (see diagram): number of sites number of sites enzyme pUC3 enzyme pUC3

Bgll> 10 BglII U

BamHI h Hpal 0 15 HindlII 1

EcoRI 3 Kpnl> 5

PstI 5 PvuII 9

MboII> 10 Aval> 10

Xbal no Xhol 2 20 Sail> 10 Hphl> 10

Haell> 7 Hinfl> 10

Narrow> 10 Hindi> 10

These values were obtained by fragmentation of DNA in the presence of excess restriction endonuclease and determination of the number of fragments soluble in 0.7 or 1.0 µg agarose gels.

pUC3 can be used to obtain recombinant plasmids that can be introduced into host bacteria. In addition, the DNA of the circular vectorial plasmid with a restriction endonuclease, for example HindlII, is cut open at 30 specific places, whereby linear DNA is created.

Between the ends thereof, a piece of foreign, non-vector DNA is pasted (by mixing the tween linear plasmids) under cyclization by mixing the two linear plasmids in the presence of a polynucleotide ligase. The foreign obtained with the same enzyme DNA can come from higher organisms.

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For example, frog DNA encoding ribosomal RNA can be incorporated into pUC3. The circular recombinant piasmid then consists of pUC3 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.

pUC3 derives its significance from being able to function as a plasmid vector in microorganisms of interest for industrial applications, such as Strepto myces. Cloning DNA from Streptmyces into pUC3 offers the opportunity to increase 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 Bac-illus, are not properly expressed therein. Likewise, genes from Gram negative bacteria are poorly expressed or not expressed at all in Gram positive hosts. This underscores the benefit of a Gram positive host vector system and thereby the utility of pUC3 in such a system.

In addition to the gene expression difficulties outlined above, difficulties may also arise in the cultivation of the microorganism and in the extraction and purity of the product. These difficulties could be addressed by introducing a plasmid vector, for example pUC3, into a host that forms the product and cloning the genes for the product in question into that 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 all the genes for biosynthesis, but only the regulatory genes or genes encoding the enzymes that control production rate. Since pUC3 is a streptomycete plasmid, it lends itself to 35 Streptomyces for these purposes. Furthermore, since pUC3 is a plasmid from a Gram-positive 800 1 4 64-6 micro-organism, it can serve as a vector in a number of other micro-organisms such as Bacillus and Arthrobacter.

Streptomyces sp.3022a NRRL 11UU1 can be grown in water reef nutrient medium under sugar aerobic conditions. The nutrient medium can 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 brewer's yeast with milk solids, soybean meal, cottonseed meal, corn meal, milk solids, pancreatically digested casein, yeast meal, distiller solids, animal peptone liquids and meat and bone offal . Combinations of the aforementioned carbon and nitrogen sources are also eligible. Since tap water and raw materials are further 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 18-30 ° C and preferably 20-28 ° C. After about 2-5 days, the growth of the microorganism is optimal.

During cultivation, the medium becomes basic and chloro-amphenicol forms.

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 grafting in order to avoid excessive growth in the microorganism. avoiding improper 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 and active vegetative graft is then placed under aseptic conditions in a culture vessel or culture ank. The medium in which the vegetative graft is formed can be the same as used for the growth of the microorganism.

Example - Isolation of plasmid pUC3 from a biologically pure culture of Stre-ptorayces sp. 3022a NRRL 11 ^ kl

Traces of Strentomyces sp. 3022a NRRL 11 Uii-1 were inoculated into 100 ml of a medium of the following composition bacto tryptone 0.05 $ brer rabbit molasses 1.0 $ glycerol 1.0 $

Difco yeast extract 0.25 $ 10 pH with 1 N NaOH at 7.2 adjusted tap water 1000 ml

The medium was previously sterilized in a 500 ml Erlenmeyer flask. After seeding, the flask contents were incubated at 25 ° C for about 36-1 * 8 hours in a Gump rotational shake direction at 250 rpm.

The cell suspension was used to inoculate flasks with 1% glycerol-serine-lactate medium. The glycerol serine lactate medium was composed as follows: glycerol 2 $ 20 60 $ sodium lactate 2.8 $ dl-serine 0.3 $ sodium chloride 0.6 $

Difco yeast extract 0.025 $ KH2P0 ^ 0.15% 25 Κ2ΗΡ0ΐ * 0.2 $

MgSO4 -THgO 0.05 $

MnSO ^ - ^ 0 0.0008 $

CuS0jj * 5H20 0.0006 $

ZnSOlj. 0.0012 $ 30 pH with 1 NaOH adjusted to 7.0

After incubation at 28 ° C for 3 days, the cells were separated by low speed centrifugation such as 10000 x g for 10 min at U ° C followed by decanting the supernatant. The mycelium was then suspended in 200 ml of 25% sucrose with 50 ml of Tris buffer adjusted to pH 8.0.

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After adding UO ml of a 2 mg / ml solution of lysozyme in TES buffer (0.03 M tris (hydroxymethyl) aminomethane (Tris), 0.005 M EDTA and 0.05 M NaCl, pH 8.0), the mixture became 30 min. incubated at 25 ° C with stirring from time to time. After adding 100 ml of 0.25 M EDTA (pH 8.0), incubation at 25 ° C was continued for an additional 30 min. Afterwards, the cell suspension was lysed by adding 320 ml of a lytic mixture of 1.0 w / v. Brij-58 (a detergent from Pierce Chem. Co., Rockford, Illinois), Q, w / v deoxycholic acid, 0.05 M Tris (pH 8.0) and 0.06 M EDTAi 10, then incubated at 25 ° C for 30 min. The lysate was incubated at 50 ° C for 30-60 min, then 60 min. was centrifuged in a Gum Rotation Shaker at 10000 rpm. using a Sorvall centrifuge.

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 1 × 5,000 x g (isopynic density graph). 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 it twice with 1/3 volume of isopropanol, after which the aqueous phase was dialyzed against a mixture of 10 ml Tris and 10 ml EDTA, pH 8.0, practically pure pUC3 was obtained. Characterization of pUC3

The size of pUC3 was determined by electron microscopy.

Restriction endonuclease fragmentation and agarose gel electrophoresis

The restriction enzymes used were from New England Biolabs.

Fragmentation of plasmid DNA was performed in a restriction buffer consisting of 10 mM Tris «HCl, pH 7» ^; 5mM

80 0 1 4 64 9

MgC ^ j 1 mM DTT for Ava I, Hphl, Mbo II, Pvu II, Hae II, Κρη I, Hinc II, Hpa I and Bgl II. The same buffer to which added 50 mM NaCl was used for Bam H-I, Hinf I, Sal I, Xba I, Bgl I,

EcoRI, Xho, Hind III and Pst I. Sma I fragmentation mixtures contains -5 to 15 mM Tris.HCl, pH 9.0; 15 mM KC1; 6 mM mgC ^ (Post, L.E., Arfsten, A.E., Reusser, F., and Nomura, M, 1978; DNA sequences of promoter regions for the str and spc ribomosal protein operons in E. coli, Cell J5, 215-229).

The fragmentation mixtures were analyzed in 10 1% agarose gels prepared according to Shinnick et al. (Shinnick, TM, Lund, E., Smithies, 0. and Blattner, FR, 1975; Hybridization of labeled RNA to DNA in agarose gels, Nucl Acids Res. 2, 1911-1229).

As reference for molecular weight, Hind 15 III fragmented λ DNA was used (Murray, K. and Murray, N.E.

1975: Phage Lambda Receptor Chromosomes for DNA Fragments made with Restriction Endonuclease III of Haemophilus influenzae and Restriction Endonuclease I of Escherichia coli, J. Mol. Biol. 98, 551-56A).

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

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Claims (5)

1. Plasmid DNA, characterized in that plasmid DNA with the code pUC3 in a pure or practically pure state has a molecular weight of about 20 x 10 6 daltons and a restriction endonuclease fragmentation pattern as shown in the diagram. 2. A method of isolating plasmid DNA from a microorganism, characterized in that pure or practically pure pUC3 as defined in claim 1 is isolated from Streptomyces sp. 3022a NRRL 11441, wherein (a) the microorganism is grown until the mycelium has grown sufficiently; (b) the culture is harvested; (C) the harvested mycelium is lysed; and (d) isolated from the lysate pure or substantially pure pUC3. 3. A method according to claim 2, characterized in that the cultivation is carried out for about 36-48 hours at a temperature of about 28 ° C. 4. Plasmid pUC3 available by the method as described in the description and the example. 5. Products and methods as described in the description and the example. 800 1 4 64