GB2045254A - A plasmid and its microbiological preparation - Google Patents
A plasmid and its microbiological preparation Download PDFInfo
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- GB2045254A GB2045254A GB8007081A GB8007081A GB2045254A GB 2045254 A GB2045254 A GB 2045254A GB 8007081 A GB8007081 A GB 8007081A GB 8007081 A GB8007081 A GB 8007081A GB 2045254 A GB2045254 A GB 2045254A
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- plasmid
- puc9
- nrrl
- dna
- fradiae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
- C12R2001/54—Streptomyces fradiae
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Abstract
The plasmid pUC 9 can be obtained from the microorganism Streptomyces fradiae, NRRL 11446. The plasmid can be used 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.A. 1978. "Transformation of plasmid DNA intoStreptomyces 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, W. 1975. "Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolorA3(2)".J. Bacteriol, 121,416421.] [Umezawa, H, 1977. "Microbial secondary metabolities 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 su percoiled 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 chloram
phenicol production in Streptomyces ven
ezuelae: 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. Mic
robiol. 106,377-381.
(3) Friend, E.J., Warren, M. and Hopwood, D.A.
1978. "Genetic evidence for a plasmid con
trolling fertility in an industrial strain of Strep
tomyces 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-specific
transfer". 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 deoxystrep
tamine moiety by acriflavine". J. Antibiotics
30,1146-1149.
(6) Kirby, R., Wright, L.F. and Hopwood, D.A.
1975. "Plasmid-determined antibiotic synth
esis and resistance in Streptomyces
coelicolor". Nature254, 265-267.
(7) Kirby, R. and Hopwood, D.A. 1977. "Genetic
determination of methylenomycin synthesis by the SCPI plasm it ofStreptomyces 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 Strep
tomyces by episomic factors". J. Antibiotics
33,45-47.
Plasmid pUC9 is obtainable from the microorgan
ism Streptomyces fradiae, NRRL 11446. This plasma can be obtained from NRRL 11446 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 pUC9. Plasmid pUC9 sensitivities to a variety of restriction endonucleases should allow its ready modification and adaptation to a number of host vector systems.
pUC9 is characterized by standard characterization tests which includes its molecular weight, approximately 6.0 megadaltons, and presence at two copies per S. fradiae NRRL 11446 cell.
pUC9 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
pUC9 is obtainable from Streptomyces fradiae,
NRRL 11446. 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 pUC9
Molecular Weight: ca. 42.4 megadaltons.
Copies Per Cell: two.
Restriction Endonuclease Sensitivities:
pUC9 has the following sensitivities to restriction endonucleases.
Plasmid Sensitivities To Restriction Endonucleases
#Cleavage Sites #Cleavage Sites
Enzyme pUC9 Enzyme pUC9
BamHI > 10 Bglll 5 Pst > 15 Hind lil 2
Xbal 2 Xhol > 14
These results were obtained by digestion of pUC9
DNA in the presence of an excess of restriction endonuclease. The number of restriction sites were determined from the number of resolvable fragments in either 0.7 or 1.0% agarose gels.
pUC9 can be used to create recombinant plasmids which can be introduced into host bacteria by transformation. The process of creating recombinant plasmids is well known in the art. Such a process comprises cleaving the isolated vector plasmid, e.g., pUC9, 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 pUC9. For example, the DNA which codes for ribosomal RNA in the frog can be mixed with pUC9 DNA that has been cleaved. The resulting circular DNA molecules con sistofplasmid pUC9with an inserted length of frog rDNA.
The recombinant plasmids containing a desired genetic element, prepared by using pUC9, can be introduced into a host organism for expression.
Examples of valuable genes which can be inserted into host organisms by the above described processes are genes coding for somatostatin, rat proinsulin, and proteases.
The usefulness of plasmid pUC9 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 pUC9 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. The E. coli 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 coll 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 pUC9 in such a system.
In general, the use of a host-vector system to produce a product foreign to that host requires the introduction if 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. pUC9, 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 for the enzymes that are rate limiting in product biosynthesis. Since the pUC9 is a streptomycete plasmid, it is ideally suited for these purposes in the genusStreptomyces. Furthermore, since pUC9 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 11446, 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 aliquot from a soil liquid N2 agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to 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 pro- - cess 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 1 - Isolation OfPlasmidpUC9 From A
Biologically Pure Culture Of Strep
tomyces fradiae, NRRL 1146
The spores from a biologically pure culture of
Streptomyces fradiae, NRRL 1146 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 Erlenmeyerflask. 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 thins incu
bation, the mycelia are separated from the broth by
low speed centrifugation, for example, at 6000 x g
for 15 minutes at 40 C. and decantation of the super
natant 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[0.03M tris(hydroxymethyl)aminomethane
(Tris), 0.005M EDTA and 0.05M NaCI; pH = 8.0]
containing 20% (w/v) sucrose. Next, 0.3 ml of a5 mg/ml lysozyme and 0.15 ml of a 1 mg/ml RNase in
the same buffer are added and the mixture is incu
bated at 37"C. for 30 minutes with occasional mixing.
Then, 0.6 ml of 0.25 M 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 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 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 cov
alently 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 pre
pared 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 NaCI,0.0015M Na3C6H,O7 . 2H2O; pH
= 7.4) to yield essentially pure pUC9.
Procedures For Characterizing pUC9
The size of pUC9 was determined by sedimenta
tion 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 approxi
mately 58S. From the neutral sucrose gradients the
sedimentation value of pUC9 was determined to be
78S. The molecular weight for pUC9 was calculated
from the equations by Hudson et al. [Hudson, B.,
Clayton, D.A. and Vinograd, J. 1968. "Complex
mitochondrial DNA". Cold Spring Harbor Symp.
Quaint. Biol33,435442]. This molecular weight is in
good agreement with that estimated from the alkaline sucrose gradients.
The percent plasmid DNA in Streptomyces fradiae
NRRL 11446 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 Endonuclease Digestion AndAgarose
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 electrophorsies. J. Virology 14, 1235-1244].
The work described herein was all done in conformity with physical and biological containment requirements specificed in the NIH Guidelines.
Claims (5)
1. Essentially pure plasmid pUC9 which is characterized by a molecular weight of approximately 42.4 megadaltons, and sensitivity to the following restriction endonucleases:BamHI > 10;post I > 15;Xba I Bgl it 5; Hind 1112; andXho I > 14.
2. A process for isolating essentially pure pUC9 from Streptomyces fradiae, NRRL 11446, which comprises:
(a) growing S. fradiae, NRRL 11446 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 pUC9 from the
lysate.
3. A process, according to claim 2, which com
prises cultivating Streptomyces fradiae, NRRL
11446, 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.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US2489079A | 1979-03-29 | 1979-03-29 |
Publications (2)
Publication Number | Publication Date |
---|---|
GB2045254A true GB2045254A (en) | 1980-10-29 |
GB2045254B GB2045254B (en) | 1982-11-10 |
Family
ID=21822894
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB8007081A Expired GB2045254B (en) | 1979-03-29 | 1980-03-03 | Plasmid and its microbiological preparation |
Country Status (6)
Country | Link |
---|---|
JP (1) | JPS55133399A (en) |
DE (1) | DE3008648A1 (en) |
FR (1) | FR2452519A1 (en) |
GB (1) | GB2045254B (en) |
IT (1) | IT1130966B (en) |
NL (1) | NL8001381A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4703009A (en) * | 1983-03-08 | 1987-10-27 | Merck & Co., Inc. | RDNA cloning vector pVE1, deletion and hybrid mutants and recombinant derivatives thereof products and processes |
-
1980
- 1980-03-03 GB GB8007081A patent/GB2045254B/en not_active Expired
- 1980-03-06 DE DE19803008648 patent/DE3008648A1/en not_active Withdrawn
- 1980-03-07 NL NL8001381A patent/NL8001381A/en not_active Application Discontinuation
- 1980-03-14 IT IT20656/80A patent/IT1130966B/en active
- 1980-03-21 JP JP3484780A patent/JPS55133399A/en active Pending
- 1980-03-28 FR FR8006948A patent/FR2452519A1/en active Granted
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4703009A (en) * | 1983-03-08 | 1987-10-27 | Merck & Co., Inc. | RDNA cloning vector pVE1, deletion and hybrid mutants and recombinant derivatives thereof products and processes |
Also Published As
Publication number | Publication date |
---|---|
GB2045254B (en) | 1982-11-10 |
DE3008648A1 (en) | 1980-11-20 |
JPS55133399A (en) | 1980-10-17 |
IT8020656A0 (en) | 1980-03-14 |
NL8001381A (en) | 1980-10-01 |
FR2452519B1 (en) | 1984-03-16 |
FR2452519A1 (en) | 1980-10-24 |
IT1130966B (en) | 1986-06-18 |
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