DK175475B1 - 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

DK 175475 B1 I

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Strep-I is known from European Patent No. 158,872

the tomycet plasmid pSG5, which can be isolated from a culture of I

Streptomyces ghanaensis DSM 2932. This plasmid is suitable for I

producing hybrid vectors, especially for so-called pendulums

5 vectors that due to a built-in E. coli replicon I

can be propagated in E. coli strains. Such vectors are e.g. IN

You know from the European patent application with publication I

In SE number 158.201. IN

It has now been found according to the invention that pSG5 is I

In 10 temperature sensitive. IN

In pSG5's temperature sensitivity property is surprising, I

In that so far no natural Strep-I has been known

tomycet plasmids that have this property. Since pSG5 and the I

Hybrid plasmids prepared with its replicon present an I

15 wide host range, opens the application of the invention of I

In this plasmid, there are a number of possibilities for the skilled person:

When constructing a plasmid with the replicon from I

In pSG5 containing a marker transforms a compatible I

host strain thereby, and then the temperature rises above 1

At the 20 threshold, only after selection after the cursor is I obtained

In such transformants that have integrated the plasmid completely I

or partially in the genome. Since such integrations essentially only occur in homologous regions of the genome, this method is suitable for finding such homologous regions in the host strain genome.

From European Publication No. 243,856, a method for producing mutants is known, in which, from the starting strain, isolating the total DNA, converting it into small fragments, integrating them into a plasmid containing a marker is temperature sensitive and replicates in the starting strain, transforms the resulting hybrid H plasmid population into the starting strain, selects the transformants by selection after the marker, eliminates the hybrid I plasmids by raising the temperature above the temperature sensitive plasmid thresholds, and selects You re-select after the cursor.

DK 175475 B1

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The term "short" fragments means DNA sections that

H is available with frequent cutting restriction enzymes such as Sau3A

H or Taql, but also by mechanical methods (ultrasound, clipping H), and containing neither the promoter region nor the translation stop signals.

H Since, after elimination of the plasmids by selection after the marker, only cells that have incorporated H plasmid DNA into their chromosome (which preferably H occurs over the homologous DNA integrated into the plasmid) survive, the mutants are obtained immediately.

Plasmid pSG5 is listed as a suitable starting plasmid in European Publication No. 243,856. However, according to the invention, the mutation to temperature-sensitive replication mutants can now be eliminated using this plasmid. This not only saves the mutation and the particularly demanding selection, but also avoids the risk of creating unwanted multiple duplications. Thus, plasmid pSG5 is particularly well suited for this method.

The pSG5's temperature sensitivity indicates that this plasmid becomes unstable at temperatures of 36 ° C and no longer replicates. The upper limit of the applicable temperature range depends on the host cell. In S. venezuelae it lies, for example. at 38 ° C, in S. lividans at 39 ° C and at S. ghanaensis I at 45 ° C. Cultures containing pSG5 or a derivative of this plasmid are incubated at a temperature of 36 ° C or higher, the plasmid "diluted" and can no longer be detected after a few generations.

Thus, the use of the invention of the pSG5 replicon I can be utilized to find homologous genes for a present gene. Thus, there is a viable alternative I for constructing a gene bank and "screening" with labeled I DNA probes. This alternative is particularly valuable when you do not get an enlightening result in the hybridization. A further advantage of this method is that work with radioactive substances can be avoided.

Similarly, insert elements can also be found

3 I

DK 175475 B1 I

(IS elements) and transposons recorded in the sub-I

searched the host strain genome. For it is only used with mar- I

the plasmid bearing no further inserted DNA, they stand

homologous areas only available when before temperature I

5 the elevation transmits an IS element or transposon from I

the chromosome on the plasmid. This makes the selection according to I

the marker makes it possible to find such DNA elements. IN

The said investigations may be carried out in accordance with I

the method for isolating mutants according to European I

10,243,856. IN

The invention thus has a number of advantages:

1. Based on the pSG5 plasmid, I already exist

one because of the wide host range versatile usable I

vector family with various selection markers such as I

15 resistance to neomycin, thiostrepton, kanamycin (European I

Publication No. 158,201) and gentamycin (European I

Publication No. 248,207), as well as color markers, I

such as melanin and other dyes or pigments (euro-I

Peak Publication Publication No. 257,416 and No. 257,417). IN

2. From the said vector family a plasmid I is used

mid with a marker selectable in E. coli, it can I

from European Publication No. 243,856 known I

method is extended to the use of cosmid banks and I

thus the isolation of large DNA sections of approx. 40 kb: If the E. coli selectable marker is integrated into the chromosome of the host, the genome of the resulting mutants can be transferred into a cosmid gene bank. Selection according to the marker (conveniently together with the cosmid's own marker) then immediately leads I to the cosmid clone of interest. Thus, you save 30 hitherto extremely necessary "screening" by hybridization. Contrary to the method known from European Publication No. 243,856, one can thus obtain large genomes in the neighboring region of the mutant gene in one step. In this way, one can isolate "clusters", i.e. eg. the genes for a whole biosynthetic pathway. Also, you are no longer referred to the fact that, in the vicinity of the gene mutated in DK 175475 B1H, suitable cut sites are present for restriction H enzymes.

Η In the following examples, the invention is further explained.

Percentages hereby refer to the weight if no other information is available.

Example 1 H Total DNA Isolation 10 0.1 g of mycelium from a 3 day old homogenized H streptomycet culture of S. ghanaensis strain (ATCC 14672; H United States Patent No. 3,674,866) not producing melanin (flour ") , pellet for 1 minute in an Eppendorf centrifuge in a 1.5 ml Eppendorf reaction vessel, pellet for 1 minute and

15 is then washed with 0.5 ml TE (10 ml 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 mmol EDTA, 10 mg / ml lysozyme) and incubated for 60 minutes at 37 ° C. After adding 0.2 ml of 5% SDS solution H 2 O and thoroughly mixing the solution, it is incubated for 10 l minutes at 65 ° C and then cooled again to room temperature.

Then add 10 μΐ phenol / chloroform (5 g phenol, 5

ml of chloroform, 5 mg of 8-hydroxyquinoline, 1 ml of 0.1 M tris (pH

H 8)), and gently mix on a shaker (Vortex ^), I 25 until the suspension is homogeneous. The mixture is then centrifuged for 5 minutes in an Eppendorf centrifuge and the upper aqueous phase transferred to a new reaction vessel. To DNA-containing solution is added 70 μΐ 3 M non-buffered sodium I acetate and 700 μΐ isopropanol. After mixing and 15 minutes of incubation at room temperature, the DNA is pelleted by centrifugation (5 minutes in Eppendorf centrifuge) and the phase I removed quantitatively. The DNA is resuspended in 300 μΐ TE and then incubated for 45 minutes at 37 ° C with I 10 μΐ RNase solution (50 μg RNase / ml H2O). The RNase is inactivated by 100 μΐ phenol / chloroform and the denatured proteins are pelleted (5 minutes in Eppendorfen

DK 175475 B1 I

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centrifuge). The DNA-containing solution is again treated with iso-I

propanol (addition of 30 μΐ 3 M sodium acetate and 400 μΐ I

isopropanol, 15 minutes incubation at room temperature). IN

The DNA pellet obtained after centrifugation is washed

5 twice with 70% ethanol and pelleted again. After dry I

ring, the DNA is taken up into 300 μΐ TE and used for the I

the following steps. IN

Example 2 I

10 I

Cleavage of total DNA with Sau3A. IN

In 1 µg DNA is incubated in cleavage buffer (50 mM Tris-HCl I)

I (pH 8), 10 mM MgCl 2 (50 mM NaCl) in the presence of a unit I

In Sau3A (produces BRL-Gibco, Karlsruhe) for 1 hour at 37 ° C. IN

The reaction is stopped by phenolization and the DNA is purified by I

In ethanol precipitation. IN

In Example 3 I 20 Cloning of the total DNA fragments of plasmid pGM4 I pGM4 (European Publication Nos. 257,416 and 257,417) is completely linearized analogously to Example I 2 with BamHI. Both DNA samples are mixed in the cleavage buffer, heated to 70 ° C and adjusted to the ligase reaction conditions by the addition of mercaptoethanol (final concentration I 10 mM) and ATP (0.1 mM). In the presence of a unit of T4 DNA-I (ligase (Bohringer Mannheim), the mixture is incubated for 12 hours at 14 ° C. The protoblast I mixture is then transformed from the starting strain and plated onto regeneration plates. These I.30 are superimposed after about 20 hours). with soft agar containing so much thiostrepton that the final concentration in the plate amounts to 50 µg / ml.

I 35 I DK 175475 B1

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Example 4

Preparation and Selection of Mutants H The trans formants are incubated in a shaking culture in S medium H 5 (Hopwood et al., "Genetic Manipulation of Streptomyces, a

Laboratory Manual ", The John Innes Foundation, Norwich 1985) for about 36 hours at 28 ° C. This temperature is then raised to 39 ° C and incubated for approximately 36 hours at this temperature.

H The culture is harvested sterile, washed in TE + 10% sucrose and H10 incubated an additional 48 hours at 39 ° C in complete medium with thiostrepton (20 mg / l). Then the mutants thus produced are ironed out and characterized (always incubating at 39 ° C).

Example 5

Isolation of the mutated DNA

From the mutants, DNA according to Example 1 is isolated, cut with a restriction enzyme having no cut sites in the integrated plasmid, and relegated I with T4 DNA ligase. Hereby the integrated plasmid, which now contains the mutated gene, is formed as the only replication-capable cyclic DNA. After retransformation in a suitable I strain such as S. lividans, select for thiostrepton resistance and from the mutants, the plasmid carrying the mutated gene is isolated.

H

Claims (6)

Use of the pSG5 replicon for the production of temperature-sensitive hybrid plasmids. A method for detecting IS elements and 5 transposons, characterized by equipping a plasmid containing the pSG5 replicon with a marker, transforming streptomycetes with the hybrid plasmid thus formed, incubating the transformants at a temperature above 36 ° C, selects after the marker and analyzes the DNA region over which the recombination has occurred. A method for producing streptomycet mutants, for isolating the mutated genes and for isolating the corresponding wild-type gene, characterized by isolating from the streptomycet starting strain, integrating it into 15 total DNA, integrating it into short fragments, In these, in a hybrid plasmid with the pSG5 replicon containing I a marker, the resulting hybrid plasmid population I transforms into the parent strain, selects the transformants at selection I after the marker, eliminates the hybrid plasmids at elevation I 20 by the temperature above the threshold, and selects the mutants I by re-selecting the cursor. 4. A method according to claim 3, characterized in that the mutated genes are isolated from the selected I mutants. I 25 5. A method according to claim 4, characterized in that the corresponding wild-type genes are isolated by the mutated genes. 6. A method according to claim 4, characterized in that the hybrid plasmid containing the pSG5 replication icon contains a marker selectable in E. coli and that in isolation I of the mutated genes a cosmid bank is used. I 35