FI95394B - Use of pSG5 as a temperature sensitive plasmid - 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

Use of pSG5 as a temperature sensitive plasmid

The invention relates to the use of the pSG5 replicon for the preparation of a temperature-sensitive hybrid plasmid and for the detection of IS elements 5 and transposons. The invention further relates to a method for producing Streptomyces mutants and to the use of these mutants to isolate mutated genes and corresponding wild-type genes.

EP 0 158 872 B discloses the Strep-10 tomyces plasmid pSG5, which can be isolated from a culture of Streptomyces ghanaensis, DSM 2932. This plasmid is suitable for the production of hybrid vectors, for example for the production of so-called pendulum vectors, which can be propagated in E. coli species due to the built-in E. coli rep-15 lycon. Such vectors are known, for example, from EP patent application 158 201 A.

It was now found that pSG5 is temperature sensitive.

The temperature sensitivity property of pSG5 is surprising, as natural Streptomyces plasmids with this property are not yet known. Because pSG5 and its replicon hybrid plasmids have a wide variety of hosts, the novel use of this plasmid in accordance with the invention opens up many possibilities for the person skilled in the art: When constructing a plasmid comprising a pSG5 replicon containing a tag, it is transformed compatible host species and then raising the temperature above the threshold, only transformed individuals that have fully or partially integrated the plasmid into

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systems. Since such integrations occur mainly only in homologous regions of the genome, this method is suitable for searching for such homologous regions in the genome of a host species.

EP 953 856 A discloses a method for preparing mutants in which all DNA is isolated from a starting strain, converted into short fragments, integrated into a plasmid containing a tag, heat-sensitive and replicated in the starting strain, and the resulting hybrid population is transformed. to the starting strain, transformants are screened for label, hybrid plasmids are eliminated by raising the temperature above the temperature-sensitive plasmid threshold, and mutants are screened using repeated screening for 10 characters.

The term "short" fragments refers to pieces of DNA obtained by densely cleaving restriction enzymes such as Sau3A or Taal. but also by mechanical methods (ultrasound, cleavage), and which do not contain 15 promoter regions or translation termination signals.

Since, after plasmid elimination, only cells that have taken up plasmid DNA into their chromosome (which is preferably through homologous DNA integrated into the plasmid) are retained for character screening, mutants are obtained immediately.

Plasmid pSG5 is described in EP 0 243 856 A as a suitable starting plasmid. However, according to the invention, the mutation into temperature-sensitive mutants can now be omitted when using this plasmid. This not only saves on mutation and particularly laborious screening, but also avoids the risk of producing unwanted multiple mutations. Plasmid pSG5 is therefore particularly well suited to this method.

The temperature sensitivity of pSG5 is such that this plasmid is unstable from 36 ° C and ♦♦ no longer replicates. The upper limit of the useful temperature range depends on the host cell: for example, in S. venezuela it is 38 ° C, in S. lividans 45 ° C and in S. ghanaensis 45 ° C. When incubating cultures containing pSG5 or a derivative of this 35 plasmid at or above 36 ° C, "the plasmid is diluted" and is no longer detectable after a few generations.

Thus, the use of the pSG5 replicon of the invention can be exploited to search for homologous genes for an existing gene. Thus, it is a useful alternative for gene bank preparation and screening with labeled DNA probes. This option is valuable especially when hybridization has not yielded a convincing result. Another advantage of this method is that it is possible to avoid working with radioactive substances.

Similarly, insertion elements (IS elements) and transposons that have entered the genome of the host species studied can be searched for: in fact, when only a labeled plasmid without other in-15 inserted DNA is used, homologous regions are only available when the IS element or the transposon has migrated from the chromosome to the plasmid before raising the temperature. Thus, screening by tag allows the search for such DNA elements.

The use of the pSG5 replicon according to the invention for the detection of IS elements and transposons is characterized by the addition of a marker to the plasmid comprising the pSG5 replicon, the transformation of Streptomyces organisms with the hybrid plasmid thus obtained, the incubation of the transformants. Above 36 ° C, screen for the marker and analyze the region of DNA where recombination occurred.

Said studies can be carried out in a manner analogous to the method for isolating mutants described in EP 0 243 856 A.

The method of the invention for preparing Streptomyces mutants is thus characterized by isolating the entire DNA from the Streptomyces starting strain, converting it into short fragments, integrating these into a hybrid plasmid comprising the pSG5 replicon containing marker 35, transforming the resulting hybrid plasmid population into a transformant population obtained. based screening, hybrid plasmids are eliminated by raising the temperature above the threshold and mutants are screened using repeated marker-based screening. As noted, mutants can be used to isolate both mutated genes and corresponding wild-type genes.

The invention thus brings a number of advantages: 1. Based on the pSG5 plasmid, due to the wide range of hosts, there is already a versatile family of vectors comprising various screening markers, such as resistance neomycin, thiostrepton, kanamycin (EP-A 0 158 201) and gentamicin. (EP-A 0 248 207) and 15 color markers such as melanin and other dyes or pigments (EP-A 0 257 416 and 0 257 417).

2. When a plasmid containing a marker that can be screened in E. coli from said family of vectors is used, it can be found that EP 0 243 856 A

The known method extends to the generation of a cosmid gene bank and thus to the isolation of large DNA fragments of about 40 kb in size: by integrating a screenable marker in E. coli into the host chromosome, the genome of the mutant thus formed can be transferred to the cosmid gene bank.

25 Screen-based screening (expediently at the same time as a cosmid-specific label) then immediately leads to a cosmid clone of interest. Thus, hitherto, the necessary, extremely laborious screening using hybridization is avoided. Thus, in contrast to the method V known from EP 0 243 856 A, large gene regions can be examined in one step around the environment of the modified gene. In this way, gene clusters can be isolated ", i.e. for example genes for the entire biosynthetic pathway. It is also no longer dependent on the availability of suitable cleavage sites for restriction enzymes in the vicinity of the mutated gene.

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The following examples further illustrate the invention. Percentages are based on weight unless otherwise indicated.

Example 1 Isolation of total DNA 0.1 g of the mycelium of a three-day-old, homogenized S. qha-naensis strain (ATCC 14672, US 3,674,866) of a Streptomyces culture that does not produce melanin (mel) is pelletized 1, In a 5 ml Eppendorf reaction vessel in an Eppendorf-10 centrifuge for 1 min and then wash 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 of 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. 0.2 ml of 5% SDS solution is added to the mixture, mixed, incubated for 10 min at 65 ° C and then cooled again to room temperature. Then add 100 μΐ phenol / chloroform (5 g. * Phenol, 5 ml chloroform, 5 mg 8-hydroxyquinoline, 1 ml 20 0.1 M Tris, pH 8) and mix gently on a shaker (^ 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 to a new reaction vessel. To the DNA-containing solution is added 70 μΐ of 3 M buffer V 25 unattended Na acetate and 700 μΐ of isopropanol. After mixing and incubation for 15 minutes at room temperature, the DNA is pelleted by centrifugation (5 min in an Eppen-Dorf centrifuge) and the supernatant is removed quantitatively. The DNA is resuspended in 300 μl of 30 TE and then incubated for 45 min at 37 ° C with 10 μl of RNase solution (50 g RNase / ml H 2 O). The RNase is inactivated with 100 μl: 1ΐ3 phenol / chloroform and the denatured proteins are pelleted (5 min in an Eppendorf centrifuge). The DNA-containing solution is re-treated with isopropanol (add 30 μΐ of 3 M Na acetate and 400 μΐ of isopropanol, 15 6 95394 min incubation at room temperature). The DNA pellet obtained after centrifugation is washed twice with 70% ethanol and repelleted. After drying, the DNA is taken up in 300 .mu.l of TE and used for further steps.

Example 2

Cleavage of total DNA with Sau3A 1 μς DNA is incubated in cleavage buffer (50 mM Tris-HCl, pH 8, 10 mM MgCl, 50 mM NaCl) in the presence of 1 unit of 10 Sau3A (manufactured by BRC-Gibco, Karlsruhe) 1 h at 37 ° C. The reaction is stopped by the addition of phenol and the DNA is purified by ethanol precipitation.

Example 3

Cloning of total DNA fragments into plasmid pGM4 pGM4 (EP-A 0 257 416 and 0 257 417) is linearized analogously to Example 2 with BamHI. Both DNA samples are mixed in digestion buffer, heated to 70 ° C, and the addition of mercaptoethanol (final concentration: 10 mM) and ATP (0.1 mM) are adjusted to ligase reaction conditions. In the presence of 1 unit of T4 ligase (Boehringer Mannheim), the mixture is incubated for 12 h at 14 ° C. The mixture is then transformed into protoplasts of the starting strain and applied to regeneration plates. These are coated after about 20 h with soft agar containing so much

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25 thiostrepton to a final concentration of 50 βg / ml in the dish.

Example 4

Production and screening of mutants

Transformants are incubated in shake culture in 30 S medium (Hopwood et al., "Genetic Manipulation of '! Streptomyces, a Laboratory Manual", The John Innes Foundation, Norwich 1985) for about 36 h at 28 ° C. The temperature is then raised to 39 ° C and incubated for another 36 h at this temperature. The culture is collected sterile, washed with TE-f in 10% sucrose and incubated for a further 48 h at 39 7 95394 ° C in complete medium (Volume) with thiostrepton (20 mg / l). The mutants produced are then plated and characterized (incubation at 39 ° C).

Example 5 Isolation of mutated DNA

DNA from the mutants is isolated according to Example 1 with a restriction enzyme that has no cleavage sites in the integrated plasmid, digested and separated by T4 DNA ligase. Thus, an integrated plasmid containing the mutated gene is formed as the only replicable cyclic DNA. After transformation into a suitable strain such as S. lividans. screened for thio-strepton resistance and a plasmid containing the mutated gene is isolated from the transformants.

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Claims (6)

Use of the pSG5 replicon to produce a temperature sensitive hybrid plasmid. Use of the pSG5 replicon for detection of IS elements and transposons, characterized in that a marker is inserted into a plasmid comprising the pSG5 replicon, Streptomyces organisms are transformed with the so-called hybrid plasmid, the transformants are incubated at a temperature above 36 ° C, is screened on the basis of the marker and the DNA region in which recombination has taken place is analyzed. A method for producing Streptomyces mutants, characterized in that the entire DNA is isolated from a Streptomyces lineage, it is converted to short fragments, these are integrated into a hybrid plasmid comprising the marker and the pSG5 replicon, the resulting hybrid plasmid population is transformed into the starting strain, the transformants are screened using marker-based screening, the hybrid plasmids are eliminated by raising the temperature above the threshold, and the mutants are screened using repeated marker-based screening. Use of Streptomyces mutants prepared by the method of claim 3 for isolating mutant genes, characterized in that mutated genes are isolated from the screened mutants. Use of Streptomyces mutants prepared by the method of claim 3 for isolating corresponding wild-type genes, characterized in that the corresponding wild-type genes are isolated by *; the mutated genes of claim 4. Use according to claim 5, characterized in that the hybrid plasmid comprising the pSG5 replicon contains a marker that can be screened in E. 3. coli and that a cosmid bank is used in isolation of the mutated genes.