NO178157B - Use of pSG5 as a Temperature Sensitive Plasmid and Method for Using Hybrid Plasmid with the pSG5 Replicon for Studies of Streptomyces - 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

The present invention relates to the use of natural temperature-sensitive pSG5 replicons and a method for using a hybrid plasmid with the pSG5 replicon for studies of Streptomyces.

From the European Patent (EP-B) No. 0 158 872 streptomycete plasmid pSG5 is known, and this plasmid can be isolated from a culture of streptomyces ghanaensis DSM 2932. This plasmid is suitable for the production of hybrid vectors, for example for so-called shuttle vectors, which due to an inserted E. coli replicon can be propagated in E. coli strains. Such vectors are known, for example, from the European patent application with Veroffentlichungs no. (EP-A) 158,201.

It has now been discovered that pSG5 is temperature sensitive.

It is surprising that pSG5 has the property of being temperature sensitive, due to the fact that natural streptomycete plasmids have not previously been discovered to have this property. Because pSG5 and those with a replicon-produced hybrid plasmid have a wide host range, the new use according to the invention of this plasmid opens up many possibilities for the person skilled in the art: When one constructs a plasmid with the replicon of pSG5 containing a marker, and transforms a compatible host strain with this and then increases the temperature above the threshold value, then after selection with regard to the marker only such transformants are provided which have integrated the plasmid in its entirety or in part into its genome. Due to the fact that such integrations are achieved essentially only in homologous regions of the genome, this method is suitable for finding such homologous regions in the genome of the host strain.

From EP-A 0 243 856, a method for the production of mutants is known and characterized by isolating the entire DNA from the starting strain and transferring them to short fragments, and integrating these into a plasmid containing a marker, which is temperature-sensitive and which is replicated in the starting strain, transforms the obtained hybrid population in the starting strain, selects the transformants by selection on the marker, by increasing the temperature above the threshold value of the temperature-sensitive plasmids, eliminates the hybrid plasmids and selects the mutants by renewed selection on the marker.

The term "short" fragments means DNA fragments which are obtained with frequent cutting restriction enzymes such as Sau3A or Taql, or with mechanical methods (ultrasound, shearing), and those which, due to the promoter region, still contain the signal for translational stop.

After elimination of plasmids by selection on the marker for surviving cells that have plasmid DNA in their chromosome (which preferably occurs via the homologous DNA integrated in the plasmid), the mutants are immediately obtained.

The plasmid pSG5 is listed in EP-A 0 243 856 as a suitable starting plasmid. According to the invention, by inserting this plasmid one can now disregard mutation to temperature-sensitive replication mutants. In this way, one is spared not only the mutation and the specially applied selection, but one also avoids the danger of producing unwanted multiple mutations. Plasmid pSG5 is also particularly suitable for this method.

The temperature sensitivity of pSG5 deteriorates in that this plasmid becomes unstable at temperatures from 36° C and no longer replicates. The upper limit of the applicable temperature range depends on the host cell: In S. venezuelae, it is e.g. at 38°C, in S. lividans at 39°C and in S. ghananensis at 45°C. If cultures containing pSG5 or a progeny of this plasmid are incubated at a temperature of 36°C or above, the plasmid is "thinned out" and after a few generations is no longer detectable.

The present invention therefore relates to the use of natural temperature-sensitive pSG5 replicons for the production of temperature-sensitive hybrid plasmids and corresponding use for the detection of IS elements and transposons.

The invention also relates to a method for using a hybrid plasmid with the pSG5 replicon for studies of streptomyces, characterized in that for the production of streptomycete mutants for isolation of the mutated gene, and for isolation of the corresponding wild-type gene, the entire DNA is isolated from streptomycete starting strains, transfer them to short fragments, integrate these into a hybrid plasmid with the pSG5 replicon, which contains a marker, transform the obtained hybrid plasmid population in the starting strain, select by selection on the marker of the transformant, eliminate the hybrid plasmids by raising the temperature above the threshold value and select by new selection on the marker of the mutant.

Use according to the invention of the pSG5 replicon can also be used to find homologous genes to an existing gene. One thus has a usable alternative for creating a gene bank and "screening" with labeled DNA probes. These options are particularly valuable if a correct hybridization result is not achieved. Another advantage of this method is that you can avoid working with radioactive compounds.

Correspondingly, insertion elements (IS elements) and transposons, which have been taken up in the genome of the host strains under investigation, can also be found. Namely, if you only use the plasmid with the marker without further inserted DNA, then homologous regions are only available when an IS element or transposon from the chromosome has been transferred to the plasmid before the temperature increase. Thus, selection on the marker enables such DNA elements to be found.

The aforementioned investigations can be carried out in the same way as the corresponding method for isolating mutants in EP-A 0 243 856.

The invention thus provides a number of advantages:

1. On the basis that pSG5 plasmids exist, due to the large host range, diverse useful vector families with different 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 dyes, respectively pigments (EP-A 0 257 416 and 0 257 417). 2. If you use a plasmid from the aforementioned vector family with a marker that is selectable in E. coli, you can, based on the method known in EP-A 0 243 856 by inserting cosmid banks, isolate large DNA fragments with an extent -ning of approximately 40 kb. If the marker which is selectable in E. coli is integrated into the chromosome of the host, then the genome of the resulting mutant can be transferred to a cosmid gene bank. Selection on the marker (it is appropriate that it is at the same time as the cosmid's own marker) then immediately leads to the cosmid clones of interest. One is thus spared the labour-intensive, widely used "screening" through hybridisation. Compared to the known method in EP-A 0 243 856, large gene regions in the vicinity of the mutated gene can thus be obtained in one step. In this way, gene "clusters" can be isolated, including, for example, the gene for an entire biosynthesis pathway. One is also no longer dependent on the existence of suitable cleavage sites for restriction enzymes in the vicinity of the mutated gene.

In the following examples, the invention is explained in more detail. The percentages are based on weight, unless otherwise stated.

EXAMPLE 1

Isolation of whole DNA

. an Eppendorf centrifuge for 1 min. and then washed once with 0.5 ml TE (10 mM Tris-HCl, 1 mM EDTA (pH 8) with 10% sucrose). The pellet is then resuspended in 0.5 ml lysozyme solution (0.3 M sucrose, 25 mM tris-HCl (pH 8), 25 mM EDTA, 10 mg/ml lysozyme) and incubated for 60 min at 37°C. After adding 0.2 ml of 5 µg SDS solution and mixing the solution, this is incubated in

10 minutes at 65 "C and then cooled at room temperature. Then 100 µl phenol/chloroform (5 g phenol, 5 ml chloroform, 5 mg 8-hydroxyquinoline, 1 ml 0.1 M Tris (pH 8)) is added and mixed gently on a stirrer ((<R>)vortex), until the suspension is homogeneous. The mixture is then centrifuged for 5 min in an Eppendorf centrifuge and the upper, aqueous phase is transferred into a new test tube. To the DNA-containing solution is added 70 µl of 3 M unbuffered Na acetate and 700 µl isopropanol. After mixing and incubation for 15 min at room temperature, the DNA is pelleted by centrifugation (5 min in an Eppendorf centrifuge) and the supernatant quantitatively removed. The DNA is resuspended in 300 µl TE and incubated in 45 min at 37°C with 10 µl RNase solution (50 pg RNase/ml). The RNase is inactivated with 100 µl phenol/chloroform and the denatured proteins pelleted (5 min in Eppendorf centrifuge). The DNA-containing solution is re-treated with isopropanol (addition of 30 µl 3 M Na-acetate and 400 µl isopropanol l, 15 min. incubation at room temperature). The DNA pellet obtained after centrifugation is washed twice with 70% ethanol and pelleted again. After drying, the DNA is taken up in 300 µl TE and used for the further steps.

EXAMPLE 2

Cleavage of whole DNA with Sau3A

1 pg of DNA is incubated in cleavage buffer (50 mM Tris-HCl (pH 8), 10 mM MgCl 2 , 50 mM NaCl) in the presence of 1 unit of Sau3A (manufacturer: BRL-Gibco, Karlsruhe) for 1 h at 37°C. The reaction is stopped by phenol treatment and the DNA is purified by ethanol precipitation.

EXAMPLE 3

Cloning of fragments of whole DNA in plasmid pGM4

pGM4 (EP-A 0 257 416 and 0 257 417) is, as in example 2, completely linearized with BamHI. Both DNA probes were mixed in the cleavage buffer and heated to 70°C by addition of mercaptoethanol (final conc. 10 mM) and ATP (0.1 mM) adjusted to the ligase reaction conditions. In the presence of 1 unit of T4 DNA ligase (Boehringer Mannheim), the mixture is incubated for 12 h at 14°C. Finally, the mixture is transformed into the protoplasts of the starting strains and sown on regeneration dishes. After 20 hours, these are coated with soft agar that contains so much thiostrepton that the final concentration in the dish contains 50 pg/ml.

EXAMPLE 4

Generation and selection of mutants

The transformants are incubated in a shuttle culture in an S medium (Hopwood et al., "Genetic Manipulation of Streptomyces, a Laboratory Manual", The John Innes Foundation, Norwich 1985) for approx. 36 hours at 28°C. The temperature is then raised to 39° C and incubated again for approx. 36 h. at this temperature. The culture is harvested sterile, washed in TE + 10% sucrose and incubated in complete medium with thiostrepton (20 mg/l) for a further 48 h at 39°C. Finally, the mutants obtained in this way are sown and characterized (all by incubation at 39°C).

EXAMPLE 5

Isolation of mutated DNA

DNA is isolated from the mutants according to example 1 and cleaved with a restriction enzyme which has no cleavage site in the integrated plasmid, and ligated with T4 DNA ligase. Hereby, the integrated plasmid, which now contains the mutated gene, is formed as the only cyclic DNA capable of replication. After transformation into a suitable strain such as S. lividans, one selects for thiostrepton resistance and isolates from the transformants the plasmid containing the mutated gene.

Claims (6)

1. Use of natural temperature-sensitive pSG5 replicons for the production of temperature-sensitive hybrid plasmids. 2. Application of naturally temperature-sensitive pSG5 replicons for detection of IS elements and transposons. 3. Method for using hybrid plasmid with the pSG5 replicon for studies of streptomycetes, characterized in that for the production of streptomycete mutants for isolation of the mutated gene, and for isolation of the corresponding wild-type gene, the entire DNA is isolated from streptomycete starting strains , transfers them into short fragments, integrates these into a hybrid plasmid with the pSG5 replicon, which contains a marker, transforms the obtained hybrid plasmid population into the starting strain, selects by selection on the marker of the transformant, eliminates the hybrid plasmids by raising the temperature above the threshold value and selects by new selection on the marker of the mutant. 4. Method according to claim 3, characterized in that the mutated gene is isolated from the selected mutant. 5 . Method according to claim 4, characterized in that the corresponding wild-type gene is isolated using the mutated gene. 6. Method according to claim 4, characterized in that the hybrid plasmid with the pSG5 replicon contains an E. coli selectable marker and that a cosmid bank is inserted upon isolation of the mutated gene.