NL8001379A - PLASMID DNA AND METHOD FOR OBTAINING IT. - Google Patents

Description

ψ

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 Streotomvces has been described by Bibb, MJ, Ward, JM, and Hopwood, DA, 1978: "Transformation of plasmid DNA into Streptomvces at high freouency" , Nature 274, 389-400.

Several Strepiomycetes have been found to contain DNA nsmids. See Huber, M.L.B. and Godfrey, O., 1978: "A general method for lysis of Strentomyces 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 Strentomyces coelicolor A3 (2)", J, Bacteriol.

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, U.S.

1977: "Preparative method for the isolation of supercoiled DNA from a chloramphenicol producing Streptomycete, J. Antiobiotics 3, 20 897-899. Nevertheless, only one Streptomycete plasmid has so far been isolated and described by Schrempf supra. That in the genus Streptomyces Other plasmids may be derived from the results of genetic studies described by: 1) Akagawa, H., Okanishi, M. and Umezawa, H., 1975: 25 "A plasmid involved in chloramphenicol production in Strepbmvces venezuelae: Evidence from genetic mapping ", J. Gen. Microbiol. 90, 336-346.

2) Freeman, R.F. and Hopwood, D.A., 1978: "Unstable. 800 1 3 79 * * 2 naturally occurring resistance to antibiotics in Streptonryces," 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 rltnosus, J. Gen. Microbiol. 106, 201-206.

4) Hopwood, D.A. and Wright, H.M, 1973: "A plasmid 10 of Streptomyces coelicolor carrying a chromoso mal 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 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 antiobiotic synthesis and resistance in Streptomyces coelicolor", Nature 254, 265-267.

7) Kirby, R. and Hopwood, D.A, 1977: "Genetic determination of methylenomycin synthesis by the SCPI plasmid of Streptomyces coelicolor A3 (2), 25 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 11444 has been found to contain a plasmid not previously described. This plasmid, which was coded p UC2, has a molecular weight of about 29.8 megadalton, is degraded by restriction endonuleases according to the fragmentation pattern shown in the 80 0 1 3 79 + 3 diagram and comes in 1-2 copier , per cell £. fradiae NRRL 11444 for.

The diagram is based on plasmid p UC2 with a molecular weight of about 29.8 + 0.7 megadalton or a molecular length of about 45.0 kilobases. The restriction sites are indicated as kilo-5 base coordinates to the Xba I restriction site at 0.0 / 45.0 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 (3) Xba I, an enzyme from Xanthomonas badrii.

p UC2 is isolated from Streptomvces fradiae NRRL 11444 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.

Since p UC2 is degraded or "cut" by various restriction endonucleases, it can be easily recombined and adapted for a number of host vectors. p UC2 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 bacterium and / or veAtor. Characteristics of p UC2 Molecular weight: approximately 29.8 megadalton.

25 Number of copies per cell: 1-2.

Place restriction endonuclease (see diagram):

Number of restriction places Number of restriction places

Enzyme pUC2 Enzyme pUC2

BamHI 10 Hind III 2 30 EcoRI 0 Κρη I 10

Pst I> 11 Xho I 9

Xba I 2 Sma I £ 12

Bgl II 4 Call I 5

These values were obtained by fragmenting p OC2 DNA in the presence of excess restriction endonucleases and determining the number of soluble fragments in 0.7 and 1.0% agarose gels.

püC2 can be used to obtain recombinant plasmids that can be introduced into host bacteria. The DNA of the circular vectorial plasmid 5 with restriction endonuclease, for example Hind III or Xba I, is cut open at specific locations, whereby linear DNA is created. Between the ends thereof, a piece of foreign, non-vector 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 micro-organisms. For example, frog DNA encoding ribosomal RNA can be incorporated into pUC2. The circular recombinant plasmid then consists of püC2 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.

PöC2 derives its significance from its ability to function as a plasmid vector in microorganisms of importance for industrial applications such as streptomvces. Cloning of DNA from Streptomyces into püC2 offers the possibility to increase the formation of economically important products from these organisms, such as antibiotics. This proposal can be compared with the cloning of genes for antibiotics in Escherichia coll K- 12. This Grara- negative bacteria, however, have the drawback that genes from certain ram-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 Otam positive hosts. This underlines the advantage of a Gram-positive host vector system and thus the utility of pUC2 in such a system.

In addition to the difficulties with gene expression 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 pUC2, 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 pUC2 is a streptomycete plasmid, it lends itself pre-eminently to Streotomvces for these purposes. Furthermore, because pUC2 is a plasmid from a ram-positive microorganism, it can also serve as a vector in a number of other microorganisms such as Bacillus and Arthrobacter . Streotomyces fradiae NRRL 11444 can be grown in a watery 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, brewers' yeast with autolysed milk solids, soy bean meal, cottonseed meal, corn meal, milk solids, casein digested digestions, yeast meal, solid distillers, animal peptone liquids and meat and bone waste. Combinations of the aforementioned carbon and nitrogen sources are also eligible. Furthermore, because 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 and preferably 20-37 ° C. The growth of the micro-organism is optimal after about 3-15 days. The medium 35 remains acidic during the gree cycle. The final pH is partly 80013 79 6 depending 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-5 form in order to inoculate an excessive layer in the growth of the micro-organism. -organism and therefore 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, 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 pUC2 from a pure culture of Streptomyces fradlae NRRL 11444

Traces of a pure culture of Streptomyces fradiae NRRL 11444 were inoculated into 10 ml medium containing 1 wt% glucose, 0.4 wt% peptone, 0.4 wt% yeast extract, 0.05 wt% MgS0 4. 7H 2 O, 0.2 wt% kh 2 PO 4 and 0.4 wt% I ^ 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 was placed in a 50 ml

Erlenmeyer flask mixed with 10 ml of the above medium to which 0.5-2.0 w / v glycine was added. The presence of gly-. cine 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 800 1 3 79 7 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 wt / vol. sucrose was added. After adding 0.3 ml of a 5 mg / ml solution of Ivsozyme and 0.15 ml of a 15 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 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 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 isonropanol 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, pH 7.4) yielding practically pure pUC2.

Characterization of ptjc2.

The size of pUC2 was determined by sedimentation in neutral and basic sucrose gradients using an internal marker plasmid DNA with an irolecular 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 pUC2 was determined at 64S. The molecular weight 800 1 3 79 Λ 8 was calculated from the equations of Hudson et al (Hudson, B., Clayron, DA and Vinograd, J. 1968): "Complex mitochondrial DNA", Cold Spring Harbor Symp. Quant, Biol. 33, 435-442) and was in good agreement with the basic sucrose assay 5 gradients.

The molecular weight of pUC2 was also determined by electron microscopy of individual DNA molecules (Kleinschmidt, AK (1968): "Monolayer techniques in electron microscopy of nucleic acid molecules": "Method in Emzymology" 10 (SP Colowick and NO Kaplan, eds) Vol. 12B, biz. 361-377, Academic

Press, New York).

For the mean contour length, a value of 15.2 +0.6 µm was found and a corresponding molecular weight of 29.8 +0.7 megadaltons.

The percentage of plasmid DNA in Streptomyces fradiae NRRL 11444 was determined by staining the culture with (methyl-3H) thymidine, preparing the crude lysates, and centrifuging samples of the lysates in cesium chloride ethidiurabromide density gradients. The gradients were fractionated and the radio-activity 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.

25 Nat. Acad. Sci. USA 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 a 2-fold excess of endonuclease. In some assays, the plasmid DNA was fragmented with more than one endonuclease. Assays were performed by two methods, first the plasmid DNA was first fragmented with the enzyme requiring a lower ionic strength and then with the enzyme requiring a higher ionic strength after adding an equal volume of 800 1 3 79 a 9 2X buffer of the second enzyme.

According to the second method, restriction fragments of one enzyme were isolated from a nrenarative agarose gel as described by Tanaka and Weisblum (Tanaka, T., and Weisblum, B. 1975 "Constnuction 5 of a colicin El-R factor composite plasmid in vitro: Means for amplification or deoxyribonucleic acid, J, Bacteriol. 121, 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 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 parainfluenzae using analvti-20 cal agarose-ethidium bromide electrophoresis, Biochemistry 12, 3055-3063). The molecular weights of the fragments were determined relative to the standard migration patterns of bacteriophage}. DNA fragmented with enzyme Ecori (Helling, RB, Goodman, H, M. And Boyer, HW 1974: Analysis of endonuclease R. EcoRl fragments of DNA 25 from lambdiod bacteriophages and other viruses by agarose gel electrophoresis, J. Virology 14, 1235 -1244).

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

30 80 0 1 3 79

Claims (6)

1. Piasmid DNA, characterized in that piasmid DNA with the code pUC2 in a pure or practically pure state has a molecular weight of about 29.8 megadaltons and a restriction endonuclease fragmentation pattern as shown in the diagram. A method of isolating plasmid DNA from a microorganism, characterized in that pure or substantially pure pUC2 as defined in claim 1 is isolated from Streotomyces fradiae NRRL 11444 wherein 10 (a) S.fradiae NRRL 11444 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 lysate pure or practically pure puc2 is isolated, 3. A method according to claim 2, characterized in that the microorganism is grown for about 24-36 hours at a temperature of about 32 ° C. The method according to claim 3, characterized in that the fragmented mycelium is incubated in a growth medium containing glycine. 5. Plasmid pUC2 available by the method as described in the description and the example. 6. Products and methods as described in the description and the example. 30 80 0 1 3 79