DE3008646A1 - PLASMID PUC8 AND METHOD FOR INSULATING PLASMID PUC8 FROM STREPTOMYCES FRADIAE - Google Patents

Description

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Flasmid pUC8 and method for isolating Piasaid pUC8

from Streptomyces fradiae

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description

The development of plasmid vectors for recombinant DNA genetic engineering in microorganisms is known. A detailed summary of DNA research can be found in Science, Volume 196, April 1977.

Similar DNA work is currently in progress with industrially important microorganisms of the genus Streptomyces (cf. MJ Bibb, JM Vard and DA Hopwood in “Nature”, Volume 274, pages 398 to 400 (1978) “Transformation of plasmid DNA into Streptomyces at high frequency "). Although plasmid DNAs were detected in various Streptomycetes (cf. MLB Huber and O. Godfrey in "Can", J. Microbiol. ", Volume 24, pages 631 and 632 (1978) ¹¹ A general method for lysis of Streptomyces species "; H. Schrempf, H. Bujard, DA Hopwood and W. Goebel in" J. Bacteriol. ", Volume 121, pages 416 to 421 (1975)" Isolation of covalently closed circular deoxy-ribonucleic acid from Streptomyces coelicolor A3 (2 ) "; H. Ttoezawa in" Biomedicine ", Volume 26, pages 236 to 249 (1977)" Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds) "and VS Malik in" J. Antibiotics ", Volume 30, pages 897 to 899 (1977) "Preparative Method for the isolation of super-coiled DNA from a chloramphenicol producing Streptomycete"), only a Streptomycete plasmid was physically isolated and described in detail in the literature (cf. Schrempf loc. Cit.). The presence of other plasmids in the genus Streptomyces was confirmed by the genetic data published in the following publications closed:

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(1) H. Akagawa, H. Okanishi, and H. Umezawa in "J. Gen. 'Microbiol. ", Vol. 90, pp. 336-346 (1975)" A plasmid involved in chloramphenicol production in Streptomyces venezuelae: Evidence from genetic mapping ^

(2) RF Freeman and DA Hopwood in "J. Gen. Microbiol. ^N , Volume 106, pages 377 to 381 (1978)" Unstable naturally occurring resistance to antibiotics in Streptomyces ^M ;

(3) E.J. Friend, M. Warren and D.A. Hopwood in "J. Gen. Microbiol. ", Volume 106, pages 201-206 (1978)" Genetic evidence for a plasmid controlling fertility in an industrial strain of Streptomyces rimosus ";

(4) DA Hopwood and HM Wright in "J. Gen. Microbiol.", Volume 79, pages 331 to 342 (1973), ^M A plasmid of Streptomyces coelicolor carrying a chromosomal locus and its inter-specific transfer ¹¹ ;

(5) K. Hotta, Y. Okami and H. Umezawa in "J. Antibiotics", Volume 30, pages 1146 to 1149 (1977) "Elimination of the ability of a kanamycin-producing strain to biosynthesize deoxystreptamine moiety by acriflavine ";

(6) R. Kirby, L.F. Wright and D.A. Hopwood in "Nature", Volume 254, pages 265 to 267 (1975) "Plasmiddetermined antibiotic synthesis and resistance in Streptomyces coelicolor ";

(7) R. Kirby and D.A. Hopwood in "J. Gen. Microbiol."

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Volume 98, Selten 239-252 (1977) "Genetic determination of methylenomycin synthesis by the SCPI plasmld of Streptomyces coelicolor A3 (2) "tmd

(8) H. Okanishi, T. Ghta and H. Umezawa in "J. Antibiotics", Volume 33, pages 45 to 47 (1969) "Possible control of formation of aerial Mycelium and antibiotic production in Streptomyces by episomic factors "

Plasmld pUC8 is obtained from the microorganism Streptomyces fradiae (DSM). This plasmid is obtained from the microorganism Streptomyces fradiae (DSM) by growing the culture on a suitable medium, fragmenting the mycelium, incubating the fragmented mycelium, harvesting the culture after a suitable time and then lysing the mycelium. Leaves from the lysate obtained isolate essentially pure plasmid pUC8. Its sensitivity to a wide variety of restriction endonucleases should be easily modified accessible and adaptable to a number of host vector systems.

The plasma pUC8 is identified by standard labeling tests, e.g., its molecular weight of about 45.0 megadaltons and the presence of one copy per Streptomyces fradiae (DSM) cell.

The plasmid pUC8 is suitable as a cloning vector in DNA work in which the plasmid contains the desired genes are incorporated and then the plasmid is transformed into a suitable host.

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As already mentioned, the plasmid pUC8 is obtained from Strep tomyces fradiae (NRRL 11445). The biologically pure culture is in the permanent collection of Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA, on deposit under No. NRRL 11445.

Characterization of the plasmid pUC8i Molecular weight: about 45.0 megadaltons Number of copies per cell: one Restriction Endonuclease Sensitivity:

Plasmid sensitivity properties to restriction endonucleases:

Cleavage points Cleavage points 5 Enzyme plasmid pUCB 2 BamHI> 10 Enzyme - plasmid pUC8 > 13 Pst I> 10 BgI II Xba I 1 Hind III Xho I

These results were obtained by digesting pUC8 DNA in the presence of excess restriction endonuclease obtain. The number of restriction sites results from the number of fragments that can be resolved in 0.7 or 1.0 # agarose gels.

pUC8 can be used to create recombinant plasmids that can be introduced into host bacteria by transformation can be used. It is known how recombinant piasaids can be created. In such a process, the isolated vector plasmid is cleaved,

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for example pUC8, at specific (specific) site (s) with the aid of a restriction endonuclease, for example Hind III, Xba I and the like. The plasmid in which it is is a circular DNA molecule, is thereby activated by the enzyme that attaches the two DNA strands to a special Site cut, converted into a linear DNA molecule. Another non-vector DNA is made with the same enzyme in similarly split. When mixing the linear vector or parts thereof with non-vector DNAs, can have their single-stranded or blunt ends pair with each other and in the presence of one as a folynucleotide ligase known second enzyme to covalently combine to form a single DNA ring.

The measures outlined can also be used to incorporate a certain length of DNA from a higher animal into pUC8 will. For example, the DNA that is responsible for coding ribosome RNA in frogs is to be mixed with the cleaved pUC8 DNA. The circular DNA molecules obtained consist of plasmid pUC8 with an inserted length of frog rDNA.

The one obtained using pUC8, a desired one Recombined plasmids containing genetic element can be introduced into a host organism for printing purposes will. Examples of valuable genes that are introduced into host organisms in the manner described are genes with somatostatin, rat proinsulin and protease coding.

The utility of plasmid pUC8 is based on its ability to act as a plasmid vector in industrially important

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Microorganisms, e.g. Streptomyces. So you get through Cloning of genetic information from Streptomyces into pUC8 a way of increasing industrially valuable Products from these organisms, e.g. from antibiotics.

This approach is based on the concept of cloning genes for antibiotic production into the well characterized Escherichia coli K-12 host / vector system comparable. The E. coli system has the disadvantage that genes from some gram-positive Organisms such as Bacillus in the gram negative E. coli host are not easy to clasp (do not express well). In the same way, plasmids from gram-negative remain Organisms do not exist in gram-positive hosts, so gram-negative genetic information in gram-positive hosts either is poorly or not at all expressed. This clearly illustrates the benefit of a gram-positive host / vector system and the utility of plasmid pUCB in such a system.

Usually requires the use of a host / vector system to produce a product foreign to the host the introduction of genes for the entire biosynthetic pathway of the product into the new host. As already stated, this can lead to problems with genetic expression. Furthermore, new and / or increased problems arise in the cultivation of the microorganisms and in the extraction and purification of the product. Perhaps a more useful attempt is to convert a plasmid vector, e.g., pUC8, into a normally smuggle in the host supplying the product and clone the genes for the biosynthesis of the product on the plasmid. At least this should alleviate the problems with the breeding

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and product extraction and purification to a minimum let lower. In addition, it should be with this cloning system it should not be necessary to clone and amplify all genes of the biosynthetic pathway, rather it should all that is required is regulatory genes or genes coding for the speed of the Cloning enzymes that limit product biosynthesis. Oa the plasmid pUC8 is a streptomycete plasmid, it is in in this respect ideally suited for the genus Streptomyces. Oa also the plasmid pUC8 a plasmid from a gram-positive Organism ^ it can be found in a number of other microorganisms, e.g. Bacillus, Arthrobacter and the like, serve as a vector.

Streptomyces fradiae (DSM) can be grown in an aqueous nutrient medium under submerged aerobic conditions. The cultivation can, for example, in a nutrient medium with an assimilable carbohydrate as a carbon source, an assimilable nitrogen compound or a protein-like or proteinaceous material as a nitrogen source be allowed to grow. Preferred carbon suppliers are glucose, brown sugar, sucrose, glycerine, starch, Corn starch, lactose, dextrin, molasses and the like. Preferred sources of nitrogen are corn steep liquor, Yeast, autolyzed brewer's yeast with milk solids, soybean meal, Cottonseed Flour, Corn Flour, Milk Solids, Pancreatic Digestion Products of Casein, Fish Meal, Distillation Solids, Animal peptone liquids, meat and bone scraps, and the like. Appropriately be Combinations of these carbon and nitrogen sources are used. Trace metals such as zinc, magnesium, manganese, cobalt, Iron and the like do not need to be added as they are part of the medium before it is sterilized.

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tion tap water and unpurified components may be used.

The inoculated medium can be incubated, preferably between about 20 ° and 37 ⁰ C, at any temperature at which an acceptable growth of the microorganism is possible, for example, at a temperature between about 18 ° and 50 °. Usually, optimal growth of the microorganism is achieved in about 3 to 15 days. The medium usually remains acidic during the growth cycle. The final pH depends partly on the buffers that may be present and partly on the initial pH of the culture medium.

If the growth or cultivation is carried out in large vessels and tanks, it is preferable to use the vegetative form and not the spore form of the microorganism for inoculation in order to significantly delay the growth of the microorganism and to avoid inefficient use of the device due to this.

Consequently, it is expedient to create a vegetative Ihokulate in a nutrient broth by inoculating it with an aliquot from a soil / liquid N ₂ agar plug or an inclined culture. When a young, active, vegetative inoculate is formed, it is aseptically transferred into large vessels or tanks. The medium in which the vegetative inoculate is produced can consist of the same medium or a different medium (from that used for culturing the microorganisms) as long as good microorganism growth is ensured.

The following example is intended to illustrate the invention in more detail. Unless otherwise stated, all

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before percentages "wt .-% ^M and all ratios for solvent mixtures" volume information ".

example

Isolation of the plasmid pUC8 from a biologically pure culture of Streptomyces fradiae (DSM):

10 ml of a medium with 1% glucose, 0.4% peptone, 0.4% yeast extract, 0.05% MgSO ^ .7H ₂ O, 0.2% KH ₂ PO ₄ and 0.4% K ₂ HPO ₄ inoculated with the spores of a biologically pure culture of Streptomyces fradiae (DSM).

The medium had previously been sterilized in a 50 ml Erlenmeyer flask. After the inoculation, the flask is incubated for about 24 to 36 hours on a rotary shaker rotating at 100 to 250 rpm at a temperature of 32 ° C. After the incubation has ended, 0.5 ml of the culture is transferred to 10 ml of the medium of the stated composition, which is in a 50 ml Erlenmeyer flask and which contains 0.5 to 2.0% (w / v) glycine. The subsequent lysing of the cells is facilitated by the addition of glycin. The amount of glycine in the medium can be routinely adjusted with a view to facilitating the subsequent lysing of the cells. The flask is then incubated again for 24 to 36 hours at a temperature of 32 ° C. on the rotary shaker. After completion of the incubation, the mycelium is removed by centrifugation at low speed (for example 15 minutes at a temperature of 4 ⁰ C with 6OOO χ g) from the broth. The supernatant liquid is then poured off from the mycelial pellet.

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The supernatant liquid is discarded while the pellet is in 1.5 ml of an isotonic buffer, e.g. TES buffer (0.03m-tris- (hydroxymethyl) -aminomethane, 0.05m-ethylenediaminetetraacetic acid and 0.05m-NaCl; pH value : 8.0) with 20% (W / V) sucrose, is resuspended. Then 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 for 30 minutes at a temperature of 37 ° C. with occasional mixing. After addition of 0.6 ml of O, 25m-ethylenediaminetetraacetic acid (pH: 8.0) the mixture is incubated for 15 minutes at a temperature of 37 ⁰ C. Thereafter, 0.3 ml of a 5 added mg / ml Pronase and incubated for 10 minutes at a temperature of 37 ⁰ C. Finally, the cell suspension is lysed by adding 3.0 ml of a 2% Sarkosyl solution in TES buffer and incubating the mixture at a temperature of 37 ° C. for 20 to 30 minutes. The lysate is then sheared by passing 5 to 10 times through a 50 ml, needle-free, disposable syringe.

The crude lysate obtained is then treated with a salt such as cesium chloride (preferred) or cesium sulfate, and the intercalating dye ethidium bromide mixed, a solution having a density of 1.550 is obtained. This solution will contribute to equilibrium 145000 χ g (isopycnic density gradient centrifugation) centrifuged. The covalently closed circular plasmid DNA is then exposed to light in the centrifuge tube long-wave ultraviolet (320 nm) as a weak fluorescent stripe under the intensely fluorescent one Strips of the linear chromosome and plasmid DNAs visible.

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For identification purposes, covalently closed circular plasmid DNA is represented by removing it from the isopycnic gradient, extracting the ethidium bromide by two treatments with a third volume of isopropanol and finally the aqueous phase against a suitable buffer, for example 0.1X SSC buffer (0.015 m-NaCl, 0.0015111-Na ₅ C ₆ H ₅ O ₇ .2H ₂ O, pH value: 7.4), is dialyzed. This essentially gives pure plasmid pUC8.

Measures for labeling and isolating pUCS:

The size of the plasmid pUC8 is determined by sedimentation in neutral and alkaline sucrose gradients using an internal marker plasmid DNA of one molecular weight of about 28.0 megadaltons and a corresponding sedimentation value of about 58S. From the neutral Sucrose gradient results in a sedimentation value for pUC8 of 76S. The molecular weight of the plasmid pUCS is calculated from the equations according to Hudson and co-workers (cf. B. Hudson, D.A. Clayton and J. Vinograd in "Complex mitochondrial DNA," Cold Spring Harbor Symp. Quant. Biol. 33, pp. 435-442 (1968)). This Molecular weight agrees well with the molecular weight estimated from the alkaline sucrose gradients.

The percentage of plasmid DNA in Streptomyces fradiae (DSM

) is done by marking the culture with [methyl%] thymidine, Preparation of raw lysates and centrifugation of the lysates in cesium chloride / ethidium bromide density gradients determined. The gradients are fractionated, which is followed by an isotope count. The radioactivity percentage

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AS

vity in the plasmid strip is used to determine the amount the plasmid DNA and to calculate the plasmid copy number (R. Radioff, ¥. Bauer and J. Vinograd (1967) "A dye-buoyant density method for detection and isolation of closed circular duplex DNA: Tlie closed circular DNA in HeLa cells " in "Proc. Nat. Acad. Sci. USA", Volume 57, pages 1514 bis 1520).

Restriction endonuclease digestion and agarose gel electrophoresis:

Commercial products are used as restriction endonucleases. The enzyme digestive preparations are made accordingly Prepared according to the instructions of the respective manufacturer, with at least a two-fold excess of endonuclease is used.

In some experiments, plasmid DNA is digested with more than one endonuclease. Two methods are used in these experiments. In the first method, the plasmid DNA is digested first with the enzyme that requires a lower ionic strength and then with the enzyme that requires a higher ionic strength, after adding an equal volume of 2X buffer to the second enzyme. In the second method, restriction fragments of an enzyme digest are isolated from a preparative agarose gel according to the instructions of Tanaka and Weisblum (cf. T. Tanaka and B. Weisblum in "J. Bacteriol. ^W , Volume 121, pages 354 to 362 (1975)" Construction of a colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonuclab acid "). The isolated restriction fragments are concentrated by ethanol precipitation and then with other restriction

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digestion enzymes. Double digestion attempts are compared with single digestion attempts »to ensure that no abnormal restriction patterns are obtained, i.e. that no non-specific DNA cleavage by a Enzyme occurs after the ionic strength of the digestive preparation or mixture is changed.

The digested samples are placed on 0.7 to 1% agarose gels applied and subjected to electrophoresis for 2 hours at a constant voltage of 10 to 15 V / cm gel height (cf. P.A. Sharp, J. Sugden and J. Sambrook in "Biochemistry", Volume 12, pages 3055-3063 (1973) "Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis. "). The molecular weights of the restriction fragments are based on the standard migration patterns of bacteriophage lambda DNA digested with the enzyme EcoRI, determined (see R.B. Helling, H.M. Goodman and H.V. Boyer in "J. Virology", Volume 14, pages 1235-1244 (1974) "Analysis of endonuclease R. EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis ").

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Claims (4)

Claims 1. Essentially pure plasmid pUC8, characterized by a molecular weight of about 45.0 hegadaltons and the following sensitivity values to the following restriction endonucleases: BamHI > 10; Pst I > 10; Xba I 1; BgI II 5; Hind III 2; and Xho I > 13. 2. Method for isolating substantially pure plasmid pUC8 from Streptomyces fradiae (DSM), thereby marked that one (a) Streptomyces fradiae (DSM) on a culture medium suitable for Streptomyces fradiae up to grows sufficient mycelial growth; (b) the mycelium fragments; (c) the fragmented mycelium is incubated in a suitable culture medium (cf. step (a)); (d) harvest the culture after an appropriate time; (e) lyse the harvested mycelium and (f) essentially pure plasmid pUCd from the lysate isolated. 3. The method according to claim 2, characterized in that one Streptomyces fradiae (DSM) for about 24 to 36 h at a temperature of 32 ° C in a nutrient medium breeds. 030041/0588 $ 300,864 4. The method according spoke 2, characterized in that the fragmented mycelium is grown in a culture medium containing glycine. 030041/0588