DE3008647A1 - PLASMID PUC1 AND METHOD FOR INSULATING PLASMID PUC1 FROM STREPTOMYCES FRADIAE - Google Patents

Description

3Q08647

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Plasmid pUC1 and method for isolating plasmid pUC1

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 refer to "Science", Volume 196, April 1977.

Similar DNA work is currently in progress on industrially important microorganisms of the genus Streptomyces (cf. M.J. Bibb, J.M. Ward and D.A. Hopwood in "Nature", volume 274, Pages 398-400 (1978) "Transformation of plasmid DNA into Streptomyces at high frequency"). Although in different Streptomycete plasmid DNAs were detected (cf. M.L.B. Huber and 0. Godfrey in "Can. J. Microbiol.", Volume 24, pages 631 and 632 (1978) "A general method for lysis of Streptomyces species"; H. Schrempf, H. Bujard, D.A. 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. Umezawa 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 V.S. 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 has been physically isolated and extensively in the literature described (see Schrempf loc. cit.). The presence of other plasmids in the genus Streptomyces was determined from the in genetic data published in the following publications:

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(1) H. Akagawa, M. 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) R.F. Freeman and D.A. Plopwood in "J. Gen. Microbiol." Volume 106, pages 377-381 (1978) "Unstable naturally occurring resistance to antibiotics in Streptomyces ";

(3) EJ Friend, M. Warren and DA Hopwood in "J. Gen. Microbiol. ¹¹ , Volume 106, pages 201-206 (1978)" Genetic evidence for a plasmid controlling fertility in an industrial strain of Streptomyces rimosus ";

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

(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, pages 239 to 252 (1977) "Genetic determination of methylenomycin synthesis by the SCPI plasmid of Streptomyces coelicolor A3 (2) "and

(8) M. Okanishi, T. Ohta 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 episomid factors ".

The plasmid pUC1 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 it of the mycelium, incubating the fragmented mycelium, harvesting the culture after a suitable time and then Lysing the mycelium. Essentially pure plasmid pUC1 can be isolated from the lysate obtained. Its sensitivity in relation to the most diverse restriction endonucleases, modifications should be made in a simple manner accessible and adaptable to a number of host vector systems.

The plasmid pUC1 is identified by standard labeling tests, e.g., its molecular weight of about 47.1 megadaltons Restriction image or a restriction map according to the drawing and the presence of one copy per Streptomyces fradiae (DSM) cell.

The plasmid pUC1 is suitable as a cloning vector in DNA work in which the plasmid contains the desired genes and then a transformation of the plasmid takes place in a suitable host.

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In the drawing, this is restriction endonuclease cleavage MId for the plasmid pUC1. The image is based on the pUC1 plasmid of molecular weight about 47.1 - 1.2 megadaltons or a molecule length of about 7 ^ 1 Constructed kilobases. The image positions of the various restriction sites are relative as kilobase coordinates indicated to the Xba-I restriction site at 0.0 / 71.1 kilobases. The following abbreviations are used for the restriction endonucleases:

(1) BgI II is an enzyme from Bacillus globigii;

(2) Hind III is an enzyme from Haemophilus influenzae; and

(3) Xba I is an enzyme from Xanthomonas badrii.

As already mentioned, the plasmid pUC1 is obtained from Streptomyces fradiae (NRRL 11443). The biologically pure culture is in the permanent collection of the Worthern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA, under the accession number NRRL 11443.

Characterization of the plasmid pUC1:

Molecular weight: about 47.1 - 1.2 megadaltons Number of copies per cell: one

Restriction endonuclease sensitivity:

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Cleavage points Plasmid pUC1 Cleavage points Plasmid pUC1 enzyme > 15 enzyme 2 BamHI 0 Hind III * 15 EcoRI t 18 Κρη Ι 11 Pst I 2 Xho I * 15 Xba I. 5 Sma I. 6th BgI II At I

These results were obtained by digesting pUC1 DNA in the presence an excess of restriction endonuclease. The number of restriction sites results from the number of the above agarose gels that can be resolved in 0.7 or 1 Fragments.

pUC1 can be used to create recombinant plasmids that can be introduced into host bacteria by transformation can be used. It is known how recombinant plasmids can be created. In such a procedure there is a cleavage of the isolated vector plasmid, for example pUC1, at specific (specific) site (s) with the aid of a restriction endonuclease, for example Hind III, Xba I and the like. The plasmid, which is a circular DNA molecule, is thereby used the enzyme that cuts the two strands of DNA at a special point is 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 polynucleotide ligase known second enzyme to covalently combine to form a single DNA ring.

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The measures outlined can also be used to incorporate a certain length of DNA from a higher animal into pUC1 will. For example, the DNA that is responsible for coding ribosome RNA in frogs is to be mixed with the cleaved pUC1 DNA. The circular DNA molecules obtained consist of plasmid pUC1 with an inserted length of frog rDNA.

The recombined plasmids containing a desired genetic element obtained using pUC1 can be introduced into a host organism for printing purposes. Examples of valuable genes found in the can be smuggled into host organisms as described, genes with somatostatin, rat proinsulin and protease coding.

The utility of plasmid pUC1 is based on its ability to act as a plasmid vector in industrially important Microorganisms, e.g. Streptomyces. By cloning genetic information from Streptomyces in pUC1 a possibility to increase industrially valuable products from these organisms, e.g. from antibiotics.

This experiment is based on the concept of gene cloning for Antibiotic production in 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 not in gram-positive hosts, so that gram-negative genetic information is produced in gram-positive hosts.

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is neither badly or not at all expressed. This clearly illustrates the benefit of a gram positive host / vector system and the utility of plasmid pUC1 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., pUC1, into a normally smuggle in the host supplying the product and clone the genes for the biosynthesis of the product on the plasmid. At the very least, this should minimize the problems associated with breeding and product extraction and purification let lower. In addition, this cloning system should not require all genes of the To clone and amplify the biosynthetic pathway, rather it should only be necessary to identify control genes (regulatory genes) or to clone genes encoding enzymes that limit the rate of product biosynthesis. Since that Plasmid pUC1 represents a streptomycete plasmid, it is ideally suited in this regard for the genus Streptomyces. Furthermore, since the plasmid pUC1 is 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.

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Streptomyces frauiae (DSM) leaves grow in an aqueous nutrient medium under submerged aerobic conditions. Breeding can take place, for example, in a nutrient medium with an assimilable carbohydrate as a carbon source, an assimilable nitrogen compound or a proteinaceous or proteinaceous material can be grown as a nitrogen source. Preferred carbon suppliers are glucose, brown sugar, sucrose, glycerine, starch, corn starch, lactose, dextrin, Molasses and the like. Preferred nitrogen sources are Corn steep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, Cornmeal, Milk Solids, Pancreatic Digestion Products of Casein, Fishmeal, Distillation Solids, Animal Peptone Liquids, Meat and bone scraps and the like. Appropriately, combinations of these carbon and nitrogen sources are used. Trace metals such as zinc, magnesium, manganese, cobalt, iron and the like must be are not added, since tap water and unpurified components are part of the medium before it is sterilized be used.

The inoculated medium can be at any temperature at which an acceptable growth of the microorganism is possible, for example, at a temperature between about 18 ° and 50 °, are preferably incubated between about 20 ° and 37 ⁰ C, typically to reach an optimal growth of the Microorganism 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.

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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 Mkr ο organism for inoculation, a significant delay in microorganism growth and an inefficient one due to it Avoid exploitation of the device.

Accordingly, it is useful to create a vegetative inoculate 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 specified, all mean Percentages “weight percent” and all ratios for solvent mixtures “volume”.

example

Isolation of the plasmid pUC1 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 ₄ .7H ₂ O, 0.2% KH ₂ PO ₄ and 0.4% K ₂ HPO ₄ inoculated with the spores of an officially pure culture of Streptomyces fradiae (DSM).

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The medium had previously been sterilized in a 50 ml Erlenmeyer flask. After inoculation, the flask is incubated about 24 to 36 on a rotating with 100 to 250 rpm rotary shaker at a temperature of 32 ⁰ 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. Thereafter, the flask is incubated again 2A to 36 hours at a temperature of 32 ⁰ C on the rotary shaker. After completion of the incubation, the mycelium is (beispielsv ice / 15 minutes at a temperature of 4 ⁰ C with 6000 g χ) by low speed centrifugation, separated from the broth. The supernatant liquid is then poured off from the mycelial pellet.

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, resuspended. Thereafter, 0.3 ml of a 5 mg / ml lysozyme, and 0.15 ml of a 1 mg / ml RHase added, and the mixture is incubated with occasional mixing for 30 minutes at a temperature of 37 ⁰ C in the same buffer. 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. Then 0.3 ml of a 5 mg / ml pronase is added

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and the whole 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 mixed with a salt, for example cesium chloride (preferred) or cesium sulfate, and the intercalating dye ethidium bromide, a solution having a density of 1.550 being obtained. This solution is centrifuged to equilibrium at 145,000 χ g (isopycnic density gradient centrifugation). The covalently closed circular plasmid DNA is then visible in the centrifuge tube when exposed to long-wave ultraviolet (320 mn) ^f as a weak fluorescent stripes under the intense fluorescent strip of the linear chromosomal and plasmid DNA en.

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 ₃ C ₆ H ₅ O ₇ .2H ₂ O, pH value: 7.4), is dialyzed. This essentially gives pure plasmid pUC1.

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Measures for labeling and isolating pUC1:

The size of the plasmid pUC1 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 pUC1 of 76S. The molecular weight of the plasma mids pUC1 is calculated from the equations according to Hudson and co-workers (cf. B. Hudson, D.A. Clayton and J. Vino grad 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.

An estimate of the pUC1 molecular weight is also possible on the basis of electron microscopic investigations of individual DNA molecules (cf. AK Kleinschmidt (1968) "Monolayer techniques in electron microscopy of nucleic acid molecules", "Method in Enzymology", edited by SP _O Colowick and NO Kaplan, volume 12B, pages 361 to 377, Academic Press, New York). It has been shown that the plasmid PUC1 Umrißlänge an average of 24.0 i 0.6 microns and a corresponding molecular weight of 47.1 - having 1.2 megadaltons.

The percentage of plaemid DNA in Streptomyces fradiae (DSM

) is determined by marking the culture with [methyl- ³ H] -thymidine, producing crude lysates and centrifuging the lysates in cesium chloride / ethidium bromide density gradients. The gradients are fractionated, whereupon

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an isotope count takes place. The percentage of radioactivity in the plasmid strip is used to determine the amount the plasmid DNA and for calculating the plasmid copy number (R. Radioff, W. Bauer and J. Vinograd (1967) "A dye-buoyant density method for detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells " in "Proc. Nat. Acad. Sci. USA", Volume 57, pages 1514 to 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 manufacturer's instructions, 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.", Volume 121, pages 354 to 362 (1975) "Construction of a colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. "). The isolated restriction fragments are

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concentrated by ethanol precipitation and then digested with other restriction enzymes. Double digestion attempts are compared to single digest experiments to ensure that no abnormal restriction patterns are obtained i.e. that no non-specific DNA cleavage by an 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. PoA. Sharp, J. Sugden and J. Sambrook in "Biochemistry", Volume 12, pages 3055 to 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.W. Boyer in "J. Virology", Volume 14, pages 1235 to 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. A substantially pure plasmid pUCö, characterized by a molecular weight of from about 47.1 to 1.2 megadaltons and a restriction endonuclease cleavage image corresponding to the drawing. 2. Method for isolating substantially pure plasmid pUC1 from Streptomyces fradiae (DSM), thereby marked that one (a) Streptomyces fradiae (DSM) on a culture medium suitable for Streptomyces fradiae bis grows to 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 pUC1 from the lysate isolated. 3. The method according to claim 2, characterized in that Streptomyces fradiae (DSM) is cultured for about 24 to 48 hours at a temperature of about 32 ^° C. in a nutrient medium. 030041/0589 4. The method according to claim 2, characterized in that the fragmented mycelium is contained in a glycine Culture medium grows. 030041/0589