GB2045251A - A plasmid and its microbiological preparation - Google Patents

Abstract

The novel plasmid pUC8 can be obtained from the microorganism Streptomyces fradiae, NRRL 11445. The plasmid can be uses as a cloning vehicle in recombinant DNA work. By way of example, the insulin gene can be inserted into the plasmid and then a suitable host containing the resulting plasmid can be used to produce insulin.

Description

SPECIFICATION A novel plasmid and its microbiological preparation The present invention relates to a novel plasmid which can have utility as a cloning vehicle in recombinant DNA work.

The development of plasmid vectors useful for recombinant DNA genetics among microorganisms is well known. The editorial in Science, Vol. 196, April, 1977, gives a good summary of DNA research.

This editorial is accompanied by a number of supporting papers in the same issue of Science.

Similar DNA work is currently being done on industrially important microorganisms of the genus Streptomyces. [Bibb, M. J., Ward, J. M., and Hopwood, D. 1978. "Transformation of plasmid DNA into Streptomyces at high frequency". Nature274, 398400]. Though plasmid DNA's have been detected in several Streptomycetes[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, 1975. "Isolation ofcovalentlyclosed circular deoxyribonucleic acid from Streptomyces coelicolor A3(2)". J. Bacteriol. 121, 416-421.] [Umezawa, H. 1977. "Microbial secondary metabolites with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)".

Biomedicine 26,236-249.], [Malik, V. S. 1977. Preparative Method for the isolation of super-coiled DNA from a chloramphenicol producing Streptomycete. J. Antibiotics 30,897-899.], only one Streptomycete plasmid has been physically isolated and extensively characterized in the literature [Schrempf, supra]. The existence of other plasmids in the genus Streptomyces has been inferred from reported genetic data as follows: (1) Akagawa, H., Okanishi, M. and Umezawa. H.

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.

"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 controlling 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-specifictransfer". 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 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 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)". 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.

Plasmid pUC8 is obtainable from the microorganism Streptomyces fradiae, NRRL 11445. This plasmid can be obtained from NRRL 11445 by growing the culture on a suitable medium, fragmenting the mycelia, incubating the fragmented mycelia, harvesting the culture after a suitable time, and then lysing the mycelia. From this lysate it is possible to isolate essentially pure pUC8. Plasmid pUC8 sensitivities to a variety of restriction endonucleases should allow its ready modification and adaptation to a number of host vector systems.

pUC8 is characterized by standard characterization tests which includes its molecular weight, approximately 45.0 megadaltons, and presence at one copy per S. fradiae NRRL 11445 cell.

pUC8 is useful as a cloning vector in DNA work wherein desired genes are incorporated into the plasmid, and the plasmid then transformed into a suitable host.

The Microorganism pUC8 is obtainable from Streptomyces fradiae, NRRL 11445. This biologically pure culture is available from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A.

Characteristics Of pUC8 Molecular Weight: ca. 45.0 megadaltons.

Copies Per Cell: one.

Restriction Endonuclease Sensitivities: pUC8 has the following sensitivities to restriction endonucleases.

Plasmid Sensitivities to Restriction Endonucleases #Cleavage Sites #Cleavage Sites Enzyme pUCB Enzyme pUC8 BamHI > 10 Bgl II 5 Post 1 910 Hind lit 2 Xbal 1 Who 1 > 13 These results were obtained by digestion of pUC8 DNA in the presence of an excess of restriction endonuclease. The number of restriction sites were determined from the number of resolvable frag ments in either 0.7 or 1.0% agarose gels.

pUC8 can be used to create recombinant plasmids which can be introduced into host bacteria by trans formation. The process of creating recombinant plasmids is well known in the art. Such a process comprises cleaving the isolated vector plasmid, e.g., pUC8, at a specific site(s) by means of a restriction endonuclease, for example, Hind IlI,Xba I, and the like. The plasmid, which is a circular DNA molecule, is thus converted into a linear DNA molecule by the enzyme which cuts the two DNA strands at a specific site. Other non-vector DNA is similarly cleaved with the same enzyme. Upon mixing the linear vector or portions thereof and non-vector DNAs, their single stranded or blunt ends can pair with each other and in the presence of a second enzyme known as polynucleotide ligase can be covalently joined to form a single circle of DNA.

The above procedure also can be used to insert a length of DNA from a higher animal into pUC8. For example, the DNA which codes for ribosomal RNA in the frog can be mixed with pUC8 DNA that has been cleaved. The resulting circular DNA molecules consist of plasmid pUC8 with an inserted length of frog rDNA.

The recombinant plasmids containing a desired genetic element, prepared by using pUC8, can be introduced into a host organism for expression.

Examples of valuable genes which can be inserted into host organisms by the above described process are genes coding forsomatostatin, rat proinsulin, and proteases.

The usefulness of plasmid pUC8 is derived from its capacity to function as a plasmid vector in industrially important microorganisms, e.g. Streptomyces.

Hence, cloning of genetic information from Streptomyces into pUC8 provides a means of increasing the production of commercially important products from these organisms, e.g. antibiotics.

This approach is compared to the concept of cloning genes for antibiotic production into the well characterized Escherichia coli K-12 host-vector system. TheE. coll system has the disadvantage that it has been found that genes from some Gram-positive organisms, e.g. Bacillus, do not express well in the Gram-negativeE. coli host. Likewise, plasmids from Gram-negative organisms are not maintained in Gram-positive hosts, and Gram-negative genetic information is either expressed poorly or not at all in Gram-positive hosts. This clearly argues for the advantage of a Gram-positive host-vector system and argues the usefulness of plasmid pUC8 in such a system.

In general, the use of a host-vector system to produce a product foreign to that host requires the introduction of the genes for the entire biosynthetic pathway of the product to the new host. As discussed above, this may lead to problems of genetic expression, but may also generate new and/or increased problems in the fermentation of the microorganisms and in the extraction and purification of the product. A perhaps more useful approach is to introduce a plasmid vector, e.g. pUC8, into a host which normally produces the product and clone onto that plasmid the genes for biosynthesis of the product. At the very least, problems of fermentation and product extraction and purification should be minimized.Additionally, in this cloning system it may not be necessary to clone and amplify all the genes of the biosynthetic pathway, but rather it may be necessary only to clone regulatory genes or genes coding forthe enzymes that are rate limiting in product biosynthesis. Since pUC8 is a streptomycete plasmid, it is ideally suited for these purposes in the genusStreptomyces. Furthermore, since pUC8 is also a plasmid from a Gram-positive organism, it may serve as a vector in a number of other microorganisms, e.g. Bacillus, Arthrobacter, etc.

Streptomyces fradiae, NRRL 11445, can be grown in an aqueous nutrient medium under submerged aerobic conditions. The organism can be grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously.Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

The inoculated medium can be incubated at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 50"C., and preferably between about 20 and 37"C.

Ordinarily, optimum growth of the microorganism is obtained in about 3 to 15 days. The medium normally remains acidic during the growth cycle. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the growth of the microorganism and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquotfrom a soil liquid N2 agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred asepticallyto large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the growth of the microorganism so long as a good growth of the microorganism is obtained.

The following examples are illustrative of the process and products of the subject invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example i-Isolation OfPlasmidpUC8 From A Biologically Pure Culture Of Streptomyces fradiae, NRRL 11445 The spores from a biologically pure culture of Streptomyces fradiae, NRRL 11445 are inoculated into 10 ml of the following medium which contains 1% glucose; 0.4% peptone; 0.4% yeast extract; 0.05% MgSO4 7H2O; 0.2% KH2PO4; and 0.4% K2HPO4.

The medium has previously been sterilized in a 50 ml Erlenmeyer flask. After inoculation, the flask is incubated at 32"C. for about 24 to 36 hours on a Gump or New Brunswick rotary shaker operating at 100-250 rpm. Upon completion of the incubation, 0.5 ml of the culture is transferred into 10 ml of the above medium containing 0.5 to 2.0% (w/v) glycine in a 50 ml Erlenmeyer flask. The addition of glycine facilitates the subsequent lysing of the cells. The amount of glycine in the medium can be varied by routine adjustments with the goal being to facilitate the subsequent lysing of the cells. The flask is then incubated further for another 24 to 36 hours at 32"C.

on a Gump rotary shaker, as above. After this incubation, the mycelia are separated from the broth by low speed centrifugation, for example, at 6000 x g for 15 minutes at4"C. and decantation of the supernatant from the mycelial pellet.

The supernatant is discarded and the pellet is resuspended in 1.5 ml of an isotonic buffer, e.g. TES buffer [0.03 M tris(hydroxymethyl)aminomethane (Tris), 0.005M EDTA and 0.05M NaCI; pH = 8.0] containing 20% (w/v) sucrose. Next, 0.3 ml of a 5 mg/ml lysozyme and 0.15 ml of a 1 mg/ml RNase in the same buffer are added and the mixture is incubated at 37"C. for 30 minutes with occasional mixing.

Then, 0.6 ml of0.25M EDTA (pH = 8.0) is added and this mixture is incubated 15 minutes at 37"C. Then 0.3 ml of 5 mg/ml pronase is added and the material is incubated 10 minutes at 37"C. Subsequently, the cell suspension is lysed by the addition of 3.0 ml of a 2% sarkosyl in TES buffer and incubation of this mixture at 37"C. for 20-30 minutes. The lysate is then sheared by passing it 5-10 times through a 50 ml disposable syringe without a needle.

This crude lysate material is then mixed with a salt, for example, cesium chloride (preferred), and cesium sulfate, and the intercalating dye ethidium bromide to give a solution of density = 1.550. This solution is centrifuged to equilibrium at 145,000 x g (isopycnic density gradient centrifugation). The covalently closed circular plasmid DNA is then visible in the centrifuge tube under long wave ultraviolet (320 nm) illumination as a faint fluorescent band below the intensely fluorescent band of linear chromosomal and plasmid DNAs.

Covalently closed circular plasmid DNA is prepared for characterization by removing it from the isopycnic gradients, extracting the ethidium bromide by two treatments with one third volume of isopropyl alcohol and then dialyzing the aqueous phase against an appropriate buffer, e.g. 0.1 X SSC buffer (0.015 M NaCl,0.0015M Na3C,,H5O7 2H2O; pH = 7.4) to yield essentially pure pUC8.

Procedures For Characterizing pUC8 The size of pUC8 was determined by sedimentation in neutral and alkaline sucrose gradients using an internal marker plasmid DNA having a molecular weight of approximately 28.0 megadaltons and a corresponding sedimentation value of approximately 58 S. From the neutral sucrose gradients the sedimentation value of pUC8 was determined to be 76S. The molecular weight for pUC8 was calculated from the equations by Hudson et al. [Hudson, B., Clayton, D. A. and Vinograd, J. 1968. "Complex mitochondrial DNA". Cold Spring HarborSymp.

Quant. Biol. 33,435442]. This molecular weight is in good agreement with that estimated from the alkaline sucrose gradients.

The percent plasmid DNA in Streptomyces fradiae NRRL 11445 was determined by labeling the culture with [methyl-3H]thymidine, preparing crude lysates, and centrifuging samples of the lysates in cesium chloride ethidium bromide density gradients. The gradients are fractionated, the isotopic counting performed, and the percent radioactivity in the plasmid band used to quantitate the plasmid DNA and calculate the plasmid copy number[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. Nat. Acad. Sci. USA57, 1514-1520].

Restriction En don uclease Digestion And Agarose Gel Electrophoresis Restriction endonucleases were obtained as commercial preparations from Miles Laboratories and New England Biolabs. Enzyme digestions were prepared in accordance with the conditions specified by the suppliers using at least a two-fold excess of endonuclease.

The digested samples were applied to 0.7-1% agarose gels and were electrophoresed for 2 hours at a constant applied voltage of 10-15 v/cm of gel height. [Sharp, P.A., Sugden, J. and Sambrook, J.

1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12,3055-3063]. The molecular weights of restriction fragments were determined relative to the standard migration patterns of bacteriophage lambda DNA digested with enzymeEcoRl[Helling, R. B., Goodman, H. M. and Boyer, H. W. 1974. Analysis of endonuclease R EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virology 14, 1235-1244].

The work described herein was all done in con formitywith physical and biological containment requirements specified in the NIH Guidelines.

Claims (5)

1. Essentially pure plasmid pUC8 which is characterized by a molecular weight of approximately 45.0 megadaltons, and sensitivity to the following restriction endonucleases:BamHl > 10; Pst I > 10;Xba i 1;Bgl II 5; Hind 1112; andXho 1 > 13.

2. A process for isolating essentially pure pUC8 from Streptomyces fradiae, NRRL 11445, which comprises: (a) growing S. fradiae, NRRL 11445 on a suitable S. fradiae growth medium until sufficient mycelial growth is obtained; (b) fragmenting said mycelia; (c) incubating said fragmented mycelia in a suit able growth medium, as above; (d) harvesting the culture after a suitable time; (e) lysing the harvested mycelia; and (f) isolating essentially pure pUC8 from the lysate.

3. A process, according to claim 2, which comprises cultivating Streptomyces fradiae, NRRL 11445, in a nutrient medium at a temperature of about 32"C. for about 24 to 36 hours.

4. A process according to claim 2 or claim 3 in which the fragmented mycelia are incubated in a growth medium containing glycine.

5. A process according to claim 2 substantially as described in the Example.