CA1335964C - Use of psg5 as a temperature-sensitive plasmid - Google Patents
Use of psg5 as a temperature-sensitive plasmidInfo
- Publication number
- CA1335964C CA1335964C CA000594516A CA594516A CA1335964C CA 1335964 C CA1335964 C CA 1335964C CA 000594516 A CA000594516 A CA 000594516A CA 594516 A CA594516 A CA 594516A CA 1335964 C CA1335964 C CA 1335964C
- Authority
- CA
- Canada
- Prior art keywords
- plasmid
- psg5
- temperature
- marker
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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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
-
- 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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- General Health & Medical Sciences (AREA)
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- Saccharide Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Pyrrole Compounds (AREA)
Abstract
The Streptomycetes plasmid pSG5 is temperature-sensitive.
It is thus possible with the aid of the pSG5 replicon to prepare temperature-sensitive hybrid plasmids. The latter are suitable for screening for DNA sections which are homologous with DNA segments of the hybrid plasmid.
It is thus possible with the aid of the pSG5 replicon to prepare temperature-sensitive hybrid plasmids. The latter are suitable for screening for DNA sections which are homologous with DNA segments of the hybrid plasmid.
Description
Description The use of pSG5 as a temperature-sen~itive plasmid European Patent (EP-B) No. 0,158,872 (published 23 Oct 85) discloses the Streptomycetes plasmid pSG5 which can be isolated from a culture of Streptomyces ghanaensis DSM 2932. This plasmid is suitable for the preparation of hybrid vectors, for example for what are called shuttle vectors, which can, because of an incorporated E. coli replicon, be multiplied in E. coli strains.
Vectors of this type are disclosed, for example, in the European Patent Application with the publication No.
(EP-A) 158,201 (published 16 Oct 85).
It has now been found that pSG5 i8 temperature-sensitive.
The property of temperature-sensitivity for pSG5 is ~urprising because as yet no natural Streptomycetes plasmids having this property have been disclosed. Since pSG5 and the hybrid plasmids prepared with its replicon exhibit a wide host range, the new use of this plasmid according to the invention provides those skilled in the art with a large number of possibilities:
- When a plasmid which has the replicon of pSG5, contAi~;ng a marker, is co~structed and used to transform a com-patible host ~train, and then the temperature is raised above the threshold, the only transformants obt~i ne~
after selection for the marker are ~hose wh;ch have the plasmid integrated, in whole or in part, into the genome.
Since such integrations essentially take place only in homologous regions of the genome, this method i8 suitable for finding such homologous regions in the genome of the host strain.
EP-A 0,243,856 (published 04 Nov 87) discloses a method for the preparation of mutants, which comprises isolating from the starting A`' ~
. ,~,;.-~ - 2 - 1 3 3 5 9 ~ ~
strain the complete DNA, converting it into short frag-ments, integrating these into a plasmid which contains a marker,~is temperature-sensitive and replicates in the starting strain, snd transforming the resulting hybrid population into the starting strain, selecting the transformants by selection for the marker, eliminating the hybrid plasmids by increasing the temperature above the threshold of the temperature-sensitive plasmid, and selecting the mutants by renewed selection for the marker.
The term "short" fragments denotes DNA sections which are obt~ineA with restriction enzymes which cut many times, such as Sau3A or TaqI, but also with mechanical methods (ultrasound, shearing) and which contain neither the promoter region nor the translation stop signals.
Because the only cells surviving after elimination of the plasmids in a ~election for the marker are those which have taken up the plasmid DNA into their chromosome (which preferably takes place via the homologous DNA
integrated in the plasmid), the mutants are obt~ineA
directly.
The plasmid pSG5 is mentioned in EP-A O, 243,856 as a suitable starting plasmid. However, it is now possible according to the invention to dispense with mutation to give temperature-sensitive replication mutants when this plasmid is used. This means that not only are the muta-tion and the particularly elaborate selection dispensed with, but also the risk of generating undesired multiple mutations is avoided. The plasmid pSG5 is thus particu-larly well suited for this method.
The temperature sensitivity of pSG5 is manifested in sucha way that this plasmid becomes unstable and is no longer replicated at temperatures at or above 36C. The upper limit of the utilizable temperature range depends on the host cell: it is, for example, 38DC in S. venezuelae, 39C
` _ 3 _ ~335~
in S. lividans and 45C with S. ghanaensis. When cultures which contain pSG5 or a derivative of this plasmid are incubated a~ a temperature of 36C or above, the "plasmid becomes diluted out" and is no longer detectable after a few generations.
The use, according to the invention, of the pSG5 replicon can thus be utilized to find genes which are homologous to an existent gene. Thus a utilizable alternative to setting up a gene bank and screening with labeled DNA
probes is available. This alternative i8 especially valuable when the result obtsined from hybridization has not been conclusive. Another advsntage of this method is that it is possible to avoid working with radioactive 6ubstances.
It i8 also possible correspondingly to find insertion elements (IS elements) and transposons which have been taken up into the genome of the host strain investigated:
If, ~pecifically, the plasmid which ha~ been provided only with the marker and contains no other inserted DNA
i~ u~ed, then homologous regions are available only if an IS element or transposon has been tran~ferred from the chromosome to the plasmid before raising the temperature.
Hence selection for the marker makes it possible to find such DNA elements.
The said investigations can be carried out in accordance with the method for isolating mutants described in EP-A
0,243,856 (published 04 Nov 87).
Thus the invention is associated with a number of advantages:
1. There already exists a family of vectors which i8 based on the pSG5 plasmid and, because of the wide host range, has a large variety of possible uses and which has various selection markers such as resistance to neomycin, thiostrepton, kanamycin (EP-A 0,158,201, published 16 Oct 85) and gentamicin (EP-A 0,248,207, published 09 ec 87) as well as color markers such as melanin ~ i . .
4 1 3359~
and other coloring substances or pigments (EP-A
0,257,416, published 02 Mar 88 and 0,257,417, published 02 Mar 88).
Vectors of this type are disclosed, for example, in the European Patent Application with the publication No.
(EP-A) 158,201 (published 16 Oct 85).
It has now been found that pSG5 i8 temperature-sensitive.
The property of temperature-sensitivity for pSG5 is ~urprising because as yet no natural Streptomycetes plasmids having this property have been disclosed. Since pSG5 and the hybrid plasmids prepared with its replicon exhibit a wide host range, the new use of this plasmid according to the invention provides those skilled in the art with a large number of possibilities:
- When a plasmid which has the replicon of pSG5, contAi~;ng a marker, is co~structed and used to transform a com-patible host ~train, and then the temperature is raised above the threshold, the only transformants obt~i ne~
after selection for the marker are ~hose wh;ch have the plasmid integrated, in whole or in part, into the genome.
Since such integrations essentially take place only in homologous regions of the genome, this method i8 suitable for finding such homologous regions in the genome of the host strain.
EP-A 0,243,856 (published 04 Nov 87) discloses a method for the preparation of mutants, which comprises isolating from the starting A`' ~
. ,~,;.-~ - 2 - 1 3 3 5 9 ~ ~
strain the complete DNA, converting it into short frag-ments, integrating these into a plasmid which contains a marker,~is temperature-sensitive and replicates in the starting strain, snd transforming the resulting hybrid population into the starting strain, selecting the transformants by selection for the marker, eliminating the hybrid plasmids by increasing the temperature above the threshold of the temperature-sensitive plasmid, and selecting the mutants by renewed selection for the marker.
The term "short" fragments denotes DNA sections which are obt~ineA with restriction enzymes which cut many times, such as Sau3A or TaqI, but also with mechanical methods (ultrasound, shearing) and which contain neither the promoter region nor the translation stop signals.
Because the only cells surviving after elimination of the plasmids in a ~election for the marker are those which have taken up the plasmid DNA into their chromosome (which preferably takes place via the homologous DNA
integrated in the plasmid), the mutants are obt~ineA
directly.
The plasmid pSG5 is mentioned in EP-A O, 243,856 as a suitable starting plasmid. However, it is now possible according to the invention to dispense with mutation to give temperature-sensitive replication mutants when this plasmid is used. This means that not only are the muta-tion and the particularly elaborate selection dispensed with, but also the risk of generating undesired multiple mutations is avoided. The plasmid pSG5 is thus particu-larly well suited for this method.
The temperature sensitivity of pSG5 is manifested in sucha way that this plasmid becomes unstable and is no longer replicated at temperatures at or above 36C. The upper limit of the utilizable temperature range depends on the host cell: it is, for example, 38DC in S. venezuelae, 39C
` _ 3 _ ~335~
in S. lividans and 45C with S. ghanaensis. When cultures which contain pSG5 or a derivative of this plasmid are incubated a~ a temperature of 36C or above, the "plasmid becomes diluted out" and is no longer detectable after a few generations.
The use, according to the invention, of the pSG5 replicon can thus be utilized to find genes which are homologous to an existent gene. Thus a utilizable alternative to setting up a gene bank and screening with labeled DNA
probes is available. This alternative i8 especially valuable when the result obtsined from hybridization has not been conclusive. Another advsntage of this method is that it is possible to avoid working with radioactive 6ubstances.
It i8 also possible correspondingly to find insertion elements (IS elements) and transposons which have been taken up into the genome of the host strain investigated:
If, ~pecifically, the plasmid which ha~ been provided only with the marker and contains no other inserted DNA
i~ u~ed, then homologous regions are available only if an IS element or transposon has been tran~ferred from the chromosome to the plasmid before raising the temperature.
Hence selection for the marker makes it possible to find such DNA elements.
The said investigations can be carried out in accordance with the method for isolating mutants described in EP-A
0,243,856 (published 04 Nov 87).
Thus the invention is associated with a number of advantages:
1. There already exists a family of vectors which i8 based on the pSG5 plasmid and, because of the wide host range, has a large variety of possible uses and which has various selection markers such as resistance to neomycin, thiostrepton, kanamycin (EP-A 0,158,201, published 16 Oct 85) and gentamicin (EP-A 0,248,207, published 09 ec 87) as well as color markers such as melanin ~ i . .
4 1 3359~
and other coloring substances or pigments (EP-A
0,257,416, published 02 Mar 88 and 0,257,417, published 02 Mar 88).
2. It is possible, by use of a plasmid which belongs to the said family of vectors and contains a marker which can be selected in E. coli, to extend the method disclosed in EP-A 0,243,856 (published 04 Nov 87) to the use of cosmid banks and thus to the isolation of large DNA sections of about 40 kb: if the marker which can be selected in E. coli is integrated into the host chromosome, the genome of the mutants produced in this way can be transferred into a cosmid gene bank.
Selection for the marker (expediently at the same time as the marker intrinsic to the cosmid) then immediately leads to the cosmid clone of interest. In this way the extremely elaborate screening by hybridization which was hitherto necessary is dispensed with. In contrast to the method disclosed in EP-A 0,243,856 (published 04 Nov 87), it is thus possible to detect in one step large gene regions in the neighborhood of the mutated gene.
Gene clusters can be isolated in this way, this is to say, for example, the genes for an entire biosynthetic pathway. Nor is there any longer a need for cleavage sites suitable for restriction enzymes to be present in the vicinity of the mutated gene.
The invention is explained in detail in the examples which follow. Unless indicated otherwise, percentage data herein relate to weight.
Example 1: Complete DNA isolation 0.1 g of mycelium from a 3-day old homogenized Strepto-mycetes culture of the strain S. ghanaensis (ATCC 14672;
US Patent 3,674,866), which produces no melanin (mel~), is pelleted in a 1.5 ml Eppendorf*reaction tube in an Eppendorf*centrifuge for 1 min and then washed once with 0.5 ml of TE tlO mM tris-HCl, 1 mM EDTA (pH 8) containing 10% sucrose). The pellet is then resuspended in 0.5 ml of lysozyme solution (0.3 M sucrose, 25 mM tris-HCl (pH 8), 25 mM EDTA, lO mg/ml lysozyme) and incubated at 37C for * denotes trade m~rk ,-~
~ _ 5 _ ~ 3~
60 min. 0.2 ml of 5% strength SDS solution is added and then the solution is thoroughly mixed and incubated at 65C for 10 min and subsequently cooled again to room temperature. Then 100 ~1 of phenol/chloroform (5 g of phenol, 5 ml of chloroform, 5 mg of 8-hydroxyquinoline, 1 ml of 0.1 M tris (pH 8)) are added, and the suspension is mixed cautiously on a shaker ((R)Vortex) until it is homogeneous. The mixture is then centrifuged in an Eppendorf centrifuge for 5 min, and the upper aqueous phase is transferred into a new reaction tube. 70 ~1 of 3 M unbuffered sodium acetate and 700 ~1 of isopropanol are added to the DNA-contAining solution. After mixing and incubation at room temperature for 15 minutes, the DNA is pelleted by centrifugation (5 min in an Eppe~orf centrifuge), and the supernatant is removed quantitative-ly. The DNA is resuspended in 300 ~1 of TE and then incubated with 10 ~1 of RNase solùtion (50 ~g of RNase/ml of H20) at 37C for 45 min. The RNase is inactivated by 100 ~1 of phenol/chloroform, and the denatured proteins are pelleted (5 min in an Eppendorf centrifuge). The DNA-contAining solution is again treated with isopropanol (addition of 30 ~1 of 3 M sodium acetate and 400 ~1 of isopropanol, incubation at room temperature for 15 min).
The DNA pellet obtained after centrifugation is washed twice with 70% strength ethanol and again pelleted. After the DNA has been dried it is taken up in 300 ~1 of TE and used for the further steps.
Example 2: Cleavage of the complete DNA with Sau3A
1 ~g of DNA is incubated in cleavage buffer (50 mM tris-HCl (pH 8), 10 ~M MgCl2, 50 mM NaCl) in the presence of 1 unit of Sau3A (manufactured by BRL-Gibco, Karlsruhe) at 37C for 1 h. The reaction is stopped by phenol treatment, and the DNA is purified by ethanol precipitation.
6 l 335964 Example 3: Cloning of fragments of complete DNA into the J plasmid pGM4 pGM4 (EP-A 0,257,416 and 0,257,417) is completely linear-ized with BamHI in analogy to Example 2. The two DNA
samples are mixed in cleavage buffer, heated to 70OC and adjusted to the ligase reaction conditions by addition of mercaptoethanol (final conc. 10 mM) and ATP (0.1 mM). The mixture is incubated in the presence of 1 unit of T4 DNA
ligase (Boehringer Mannheim) at 14C for 12 h. The mixture is then transformed into protoplasts of the starting strain and plated out on regeneration plates. After about 20 h, a top layer of soft agar which contains sufficient thiostrepton for the final concentration in the plate to be 50 ~g/ml is placed on the latter.
Example 4: Generation and selection of mutants The transformants are incubated in S medium (Hopwood et al., "Genetic Manipulation of Streptomyces, a Laboratory Manual", The John Innes Foundation, Norwich 1985) in a shake culture at 28C for about 36 h. The temperature is then raised to 39C and incubation is continued at this temperature for about 36 h. The culture is harvested sterile, washed in TE + 10% sucrose and incubated in complete medium containing thiostrepton (20 mg/l) at 39C
for a further 48 h. The mutants generated in this way are then plated out and characterized (incubation always at 39C) Example 5: Isolation of the mutated DNA
The DNA is isolated as in Example 1 from the mutants, is cut with a restriction enzyme which has no cleavage sites in the integrated plasmid, and is religated with T4 DNA
ligase. This results in the in~egrated plasmid, which now contains the mutated gene, being formed as the only cyclic DNA capable of replication. Retransformation into a suitable strain such as S. lividans is followed by 7 1 3359~
..
selection for thiostrepton resistance and isolation of the plasmid which harbors the mutated gene from the transformants.
The following published Australian patent applications correspond to the above-mentioned European specifications:
AU-A 40,599/85 = EP-B 0,158,872 (published 23 Oct 85);
AU-A 40,600/85 = EP-A 0,158,201 (published 16 Oct 85);
AU-A 76,803/87 = EP-A 0,257,416 (published 02 Mar 88);
AU-A 76,802/87 = EP-A 0,257,417 (published 02 Mar 88).
Selection for the marker (expediently at the same time as the marker intrinsic to the cosmid) then immediately leads to the cosmid clone of interest. In this way the extremely elaborate screening by hybridization which was hitherto necessary is dispensed with. In contrast to the method disclosed in EP-A 0,243,856 (published 04 Nov 87), it is thus possible to detect in one step large gene regions in the neighborhood of the mutated gene.
Gene clusters can be isolated in this way, this is to say, for example, the genes for an entire biosynthetic pathway. Nor is there any longer a need for cleavage sites suitable for restriction enzymes to be present in the vicinity of the mutated gene.
The invention is explained in detail in the examples which follow. Unless indicated otherwise, percentage data herein relate to weight.
Example 1: Complete DNA isolation 0.1 g of mycelium from a 3-day old homogenized Strepto-mycetes culture of the strain S. ghanaensis (ATCC 14672;
US Patent 3,674,866), which produces no melanin (mel~), is pelleted in a 1.5 ml Eppendorf*reaction tube in an Eppendorf*centrifuge for 1 min and then washed once with 0.5 ml of TE tlO mM tris-HCl, 1 mM EDTA (pH 8) containing 10% sucrose). The pellet is then resuspended in 0.5 ml of lysozyme solution (0.3 M sucrose, 25 mM tris-HCl (pH 8), 25 mM EDTA, lO mg/ml lysozyme) and incubated at 37C for * denotes trade m~rk ,-~
~ _ 5 _ ~ 3~
60 min. 0.2 ml of 5% strength SDS solution is added and then the solution is thoroughly mixed and incubated at 65C for 10 min and subsequently cooled again to room temperature. Then 100 ~1 of phenol/chloroform (5 g of phenol, 5 ml of chloroform, 5 mg of 8-hydroxyquinoline, 1 ml of 0.1 M tris (pH 8)) are added, and the suspension is mixed cautiously on a shaker ((R)Vortex) until it is homogeneous. The mixture is then centrifuged in an Eppendorf centrifuge for 5 min, and the upper aqueous phase is transferred into a new reaction tube. 70 ~1 of 3 M unbuffered sodium acetate and 700 ~1 of isopropanol are added to the DNA-contAining solution. After mixing and incubation at room temperature for 15 minutes, the DNA is pelleted by centrifugation (5 min in an Eppe~orf centrifuge), and the supernatant is removed quantitative-ly. The DNA is resuspended in 300 ~1 of TE and then incubated with 10 ~1 of RNase solùtion (50 ~g of RNase/ml of H20) at 37C for 45 min. The RNase is inactivated by 100 ~1 of phenol/chloroform, and the denatured proteins are pelleted (5 min in an Eppendorf centrifuge). The DNA-contAining solution is again treated with isopropanol (addition of 30 ~1 of 3 M sodium acetate and 400 ~1 of isopropanol, incubation at room temperature for 15 min).
The DNA pellet obtained after centrifugation is washed twice with 70% strength ethanol and again pelleted. After the DNA has been dried it is taken up in 300 ~1 of TE and used for the further steps.
Example 2: Cleavage of the complete DNA with Sau3A
1 ~g of DNA is incubated in cleavage buffer (50 mM tris-HCl (pH 8), 10 ~M MgCl2, 50 mM NaCl) in the presence of 1 unit of Sau3A (manufactured by BRL-Gibco, Karlsruhe) at 37C for 1 h. The reaction is stopped by phenol treatment, and the DNA is purified by ethanol precipitation.
6 l 335964 Example 3: Cloning of fragments of complete DNA into the J plasmid pGM4 pGM4 (EP-A 0,257,416 and 0,257,417) is completely linear-ized with BamHI in analogy to Example 2. The two DNA
samples are mixed in cleavage buffer, heated to 70OC and adjusted to the ligase reaction conditions by addition of mercaptoethanol (final conc. 10 mM) and ATP (0.1 mM). The mixture is incubated in the presence of 1 unit of T4 DNA
ligase (Boehringer Mannheim) at 14C for 12 h. The mixture is then transformed into protoplasts of the starting strain and plated out on regeneration plates. After about 20 h, a top layer of soft agar which contains sufficient thiostrepton for the final concentration in the plate to be 50 ~g/ml is placed on the latter.
Example 4: Generation and selection of mutants The transformants are incubated in S medium (Hopwood et al., "Genetic Manipulation of Streptomyces, a Laboratory Manual", The John Innes Foundation, Norwich 1985) in a shake culture at 28C for about 36 h. The temperature is then raised to 39C and incubation is continued at this temperature for about 36 h. The culture is harvested sterile, washed in TE + 10% sucrose and incubated in complete medium containing thiostrepton (20 mg/l) at 39C
for a further 48 h. The mutants generated in this way are then plated out and characterized (incubation always at 39C) Example 5: Isolation of the mutated DNA
The DNA is isolated as in Example 1 from the mutants, is cut with a restriction enzyme which has no cleavage sites in the integrated plasmid, and is religated with T4 DNA
ligase. This results in the in~egrated plasmid, which now contains the mutated gene, being formed as the only cyclic DNA capable of replication. Retransformation into a suitable strain such as S. lividans is followed by 7 1 3359~
..
selection for thiostrepton resistance and isolation of the plasmid which harbors the mutated gene from the transformants.
The following published Australian patent applications correspond to the above-mentioned European specifications:
AU-A 40,599/85 = EP-B 0,158,872 (published 23 Oct 85);
AU-A 40,600/85 = EP-A 0,158,201 (published 16 Oct 85);
AU-A 76,803/87 = EP-A 0,257,416 (published 02 Mar 88);
AU-A 76,802/87 = EP-A 0,257,417 (published 02 Mar 88).
Claims (7)
1. The use of pSG5 as a temperature-sensitive plasmid.
2. The use of the pSG5 replicon for the preparation of temperature-sensitive hybrid plasmids.
3. The use of the pSG5 replicon for finding IS elements and transposons.
4. A method for the preparation of Streptomycetes mutants, which comprises isolating from a starting Streptomycetes strain the complete DNA, converting it into short fragments, integrating these into a hybrid plasmid which has the pSG5 replicon, containing a marker, and transforming the resulting hybrid plasmid population into the starting strain, selecting the transformants by selection for the marker, eliminating the hybrid plasmids by increasing the temperature above the threshold, and selecting the mutants by renewed selection for the marker.
5. A method of obtaining a mutated gene, comprising obtaining a mutant according to claim 4; and isolating a mutated gene from the selected mutant.
6. A method of isolating wild-type genes, comprising obtaining a mutated gene according to claim 5; and hybridizing at least a portion of the mutated gene to a wild-type gene.
7. The method as claimed in claim 5, wherein the hybrid plasmid having the pSG5 replicon contains a marker which can be selected in E. coli, and wherein a cosmid bank is used in the isolation of the mutated genes.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3809692A DE3809692A1 (en) | 1988-03-23 | 1988-03-23 | USE OF PSG5 AS A TEMPERATURE-SENSITIVE PLASMIDE |
| DEP3809692.7 | 1988-03-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1335964C true CA1335964C (en) | 1995-06-20 |
Family
ID=6350421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000594516A Expired - Lifetime CA1335964C (en) | 1988-03-23 | 1989-03-22 | Use of psg5 as a temperature-sensitive plasmid |
Country Status (16)
| Country | Link |
|---|---|
| EP (1) | EP0334282B1 (en) |
| JP (1) | JP2770975B2 (en) |
| KR (1) | KR970003961B1 (en) |
| AT (1) | ATE108485T1 (en) |
| AU (1) | AU615524B2 (en) |
| CA (1) | CA1335964C (en) |
| DE (2) | DE3809692A1 (en) |
| DK (1) | DK175475B1 (en) |
| ES (1) | ES2056987T3 (en) |
| FI (1) | FI95394C (en) |
| HU (1) | HU213350B (en) |
| IE (1) | IE63496B1 (en) |
| IL (1) | IL89686A (en) |
| NO (1) | NO178157C (en) |
| PT (1) | PT90090B (en) |
| ZA (1) | ZA892123B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3809691A1 (en) * | 1988-03-23 | 1989-10-12 | Hoechst Ag | METHOD FOR SELECTION OF LARGE DNA SECTIONS |
| DE4011863A1 (en) * | 1990-04-12 | 1991-10-17 | Hoechst Ag | REGULATED GENE EXPRESSION IN STREPTOMYCETES |
| FR2688515B1 (en) * | 1992-03-13 | 1995-03-31 | Institut Rech Agronomique | THERMOSENSITIVE PLASMID. |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0158872B1 (en) * | 1984-03-31 | 1989-01-18 | Hoechst Aktiengesellschaft | Streptomycetes plasmid psg5, process for its preparation and its use |
| DE3412093A1 (en) * | 1984-03-31 | 1985-10-10 | Hoechst Ag, 6230 Frankfurt | HYBRID PLASMIDE WITH A STREPTOMYCETE AND ESCHERICHIA COLI REPLICON |
| DE3614310A1 (en) * | 1986-04-28 | 1987-10-29 | Hoechst Ag | METHOD FOR ISOLATING MUTED GENES AND THE CORRESPONDING WILD-TYPE GENES |
| DE3627392A1 (en) * | 1986-08-13 | 1988-04-28 | Hoechst Ag | COLOR MARKER FOR CLONING IN STREPTOMYCES LIVIDANS |
-
1988
- 1988-03-23 DE DE3809692A patent/DE3809692A1/en not_active Withdrawn
-
1989
- 1989-03-21 FI FI891335A patent/FI95394C/en not_active IP Right Cessation
- 1989-03-21 DE DE58908022T patent/DE58908022D1/en not_active Expired - Lifetime
- 1989-03-21 AT AT89105012T patent/ATE108485T1/en not_active IP Right Cessation
- 1989-03-21 ES ES89105012T patent/ES2056987T3/en not_active Expired - Lifetime
- 1989-03-21 ZA ZA892123A patent/ZA892123B/en unknown
- 1989-03-21 IL IL8968689A patent/IL89686A/en not_active IP Right Cessation
- 1989-03-21 EP EP89105012A patent/EP0334282B1/en not_active Expired - Lifetime
- 1989-03-22 PT PT90090A patent/PT90090B/en not_active IP Right Cessation
- 1989-03-22 DK DK198901460A patent/DK175475B1/en not_active IP Right Cessation
- 1989-03-22 AU AU31582/89A patent/AU615524B2/en not_active Expired
- 1989-03-22 IE IE89889A patent/IE63496B1/en not_active IP Right Cessation
- 1989-03-22 JP JP1067773A patent/JP2770975B2/en not_active Expired - Lifetime
- 1989-03-22 KR KR1019890003551A patent/KR970003961B1/en not_active IP Right Cessation
- 1989-03-22 CA CA000594516A patent/CA1335964C/en not_active Expired - Lifetime
- 1989-03-22 HU HU891388A patent/HU213350B/en unknown
- 1989-03-22 NO NO891267A patent/NO178157C/en not_active IP Right Cessation
Also Published As
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|---|---|
| FI891335A (en) | 1989-09-24 |
| DE58908022D1 (en) | 1994-08-18 |
| KR970003961B1 (en) | 1997-03-24 |
| EP0334282A2 (en) | 1989-09-27 |
| EP0334282A3 (en) | 1989-11-29 |
| DK146089A (en) | 1989-09-24 |
| JPH029376A (en) | 1990-01-12 |
| IL89686A0 (en) | 1989-09-28 |
| EP0334282B1 (en) | 1994-07-13 |
| HU213350B (en) | 1997-05-28 |
| DK146089D0 (en) | 1989-03-22 |
| PT90090A (en) | 1989-11-10 |
| ATE108485T1 (en) | 1994-07-15 |
| AU3158289A (en) | 1989-09-28 |
| IL89686A (en) | 1994-12-29 |
| HUT50511A (en) | 1990-02-28 |
| DK175475B1 (en) | 2004-11-08 |
| NO891267L (en) | 1989-09-25 |
| IE890898L (en) | 1989-09-23 |
| NO178157C (en) | 1996-01-31 |
| KR890014741A (en) | 1989-10-25 |
| PT90090B (en) | 1994-06-30 |
| FI95394B (en) | 1995-10-13 |
| JP2770975B2 (en) | 1998-07-02 |
| NO891267D0 (en) | 1989-03-22 |
| FI95394C (en) | 1996-01-25 |
| FI891335A0 (en) | 1989-03-21 |
| DE3809692A1 (en) | 1989-10-12 |
| ES2056987T3 (en) | 1994-10-16 |
| IE63496B1 (en) | 1995-05-03 |
| NO178157B (en) | 1995-10-23 |
| AU615524B2 (en) | 1991-10-03 |
| ZA892123B (en) | 1989-11-29 |
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