IL89686A - Use of pSG5 replicon for the preparation of temperature-sensitive plasmid and uses thereof - Google Patents

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 segments which are homologous with DNA segments of the hybrid plasmid.

Description

' 89686/2 THE USE OF pSG5 REPLICON FOR THE PREPARATION OF TEMPERATURE-SENSITIVE PLASMID AND USES THEREOF European Patent (EP-B) No. 0,158,872 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.

It has now been found that pSG5 is temperature-sensitive.

The invention relates to a method for the preparation of temperature sensitive hybrid plasmids, which comprises ligating DNA which contains the replicon of pSG5 with foreign DNA, wherein the property of temperature sensitivity is imparted by the replicon of pSG5 that has not been mutated.

Passages which are not in the ambit of the claims do not belong to the invention. The scope of protection is as defined in the claims, and as stipulated in the Patent Law (1968).

The property of temperature-sensitivity for pSG5 is surprising 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, containing a marker, is constructed and used to transform a compatible host strain, and then the temperature is raised above the threshold, the only transformants obtained after selection for the marker are those which 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 is suitable for finding such homologous regions in the genome of the host strain.

EP-A 0,243,856 discloses a method for the preparation of mutants, which comprises isolating from the starting strain the complete DNA, converting it into short fragments, integrating these into a plasmid which contains a marker, is temperature-sensitive and replicates in the starting strain, and 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 obtained with restriction enzymes which cut many times, such as Sau3A or Taql , 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 selection 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 obtained directly.

The plasmid pSG5 is mentioned in EP-A 0,243,856 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 mutation and the particularly elaborate selection dispensed with, but also the risk of generating undesired multiple mutations is avoided. The plasmid pSG5 is thus particularly well suited for this method.

The temperature-sensitivity of pSG5 is manifested in such a way that this plasmid becomes unstable and is no longer replicated at temperatures at or above 36°C. The upper limit of the utilizable temperature range depends on the host cell: it is, for example, 38°C in S. venezuelae, 39°C in S. lividans and 45°C with S. ghanaensis. When cultures which contain pSG5 or a derivative of this plasmid are incubated at a temperature of 36°C 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 is especially valuable when the result obtained from hybridization has not been conclusive. Another advantage of this method is that it is possible to avoid working with radioactive substances .

It is 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, specifically, the plasmid which has been provided only with the marker and contains no other inserted DNA is used, then homologous regions are available only if an IS element or transposon has been transferred 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.

Thus the invention is associated with a number of advantages : 1. There already exists a family of vectors which is 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) and gentamicin (EP-A 0,248,207) as well as color markers such as melanin and other coloring substances or pigments (EP-A 0,257,416 and 0,257,417) . 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 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, 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, that 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 (10 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, 10 mg/ml lysozyme) and incubated at 37°C for 60 min. 0.2 ml of 5% strength SDS solution is added and then the solution is thoroughly mixed and incubated at 65°C for 10 min and subsequently cooled again to room temperature. Then 100 μΐ 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 (lRVortex) 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 μΐ of 3 M unbuffered sodium acetate and 700 μΐ of isopropanol are added to the DNA-containing solution. After mixing and incubation at room temperature for 15 minutes, the DNA is pelleted by centrxfugation (5 min in an Eppendorf centrifuge), and the supernatant is removed quantitatively. The DNA is resuspended in 300 μΐ of TE and then incubated with 10 μΐ of RNase solution (50 μg of RNase/ml of H20) at 37°C for 45 min. The RNase is inactivated by 100 μΐ 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 μΐ of 3 M sodium acetate and 400 μΐ of isopropanol, incubation at room temperature for 15 min). The DNA pellet obtained after centrxfugation is washed twice with 70% strength ethanol and again pelleted. After the DNA has been dried it is taken up in 300 μΐ 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-HC1 (pH 8), 10 mM MgCl2, 50 mM NaCl) in the presence of 1 unit of Sau3A (manufactured by BRL-Gibco, Karlsruhe) at 37°C for 1 h. The reaction is stopped by phenol treatment, and the DNA is purified by ethanol precipitation.

Example 3: Cloning of fragments of complete DNA into the plasmid pGM4 pGM4 (EP-A 0,257,416 and 0,257,417) is completely linearized 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 cone. 10 mM) and ATP (0.1 m ) . The mixture is incubated in the presence of 1 unit of T4 DNA ligase (Boehringer Mannheim) at 14°C 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 μg/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 28°C for about 36 h. The temperature is then raised to 39 °C 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/1) at 39°C for a further 48 h. The mutants generated in this way are then plated out and characterized (incubation always at 39°C) .

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 integrated 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 selection for thiostrepton resistance and isolation of the plasmid which harbors the mutated gene from the transformants .

The following published Australian patent applications, which are hereby incorporated by reference, correspond to the above-mentioned European specifications: EP-B 0,158,872 = AU-A 40,599/85 EP-A 0,158,201 = AU-A 40,600/85 EP-A 0,257,416 = AU-A 76,803/87 EP-A 0,257,417 = AU-A 76,802/87

Claims (9)

CLAIMS :

1. A method for the preparation of temperature sensitive hybrid plasmids, which comprises ligating DNA which contains the replicon of pSG5 with foreign, DNA wherein the property of temperature sensitivity is imparted by the replicon of pSG5 that has not been mutated.

2. The method as claimed in claim 1, wherein the foreign DNA contains a marker.

3. The method as claimed in claim 1, wherein the foreign DNA is a linearized plasmid.

4. A method of using non-mutated pSG5 for preparing temperature sensitive plasmids which are capable of replicating in Streptomycetes, which comprises ligating DNA containing the replicon of pSG5 with foreign DNA, wherein the property of temperature sensitivity is imparted by the replicon of pSG5 that has not been mutated.

5. A method of using non-mutated pSG5 for finding IS elements and transposons, which comprises preparing a hybrid plasmid as claimed in claim 1, by providing pSG5 that has not been mutated with a selection marker, using said hybrid plasmid that does not contain a wild-type or mutated IS element or transposon, to transform Streptomycetes, incubating the transformants at a temperature above 36°C, selecting for marker, and isolating the IS element or transposon from the DNA region via which the recombination has taken place. 89686/3

6. A method of using non-mutated pSG5 for the preparation of Streptomycetes mutants, which comprises isolating from a starting Streptomycetes strain the complete DNA , converting it into short fragments, integrating at least one of said DNA fragments into a starting plasmid that ocntains both a selection marker and a non- mutated temperature sensitive pSG5 replicon of claim 1, and transforming the resulting hybrid plasmid population into the starting Streptomycetes strain, selecting the transformants by selection for the selection marker, eliminating the hybrid plasmids by heating the transformants to a temperature at which the plasmids are no longer replicated, and selecting the mutants, which contain mutated genes, by renewed selection for the selection marker.

7. A method of using non-mutated pSG5 for obtaining a mutated gene, which comprises obtaining a mutant according to claim 6; and isolating a mutated gene from the selected mutant.

8. A method of using non-mutated pSG5 for isolating wild-type genes, which comprises obtaining a mutated gene according to claim 7; and hybridizing at least a portion of the mutated gene to a wild-type gene.

9. A method according to claim 7, wherein the hydrid 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. OHEN ZEDEK * RAPAPORl