IL89687A - Method for the selection of large dna sections - Google Patents

Abstract

DNA segments from mutants can be obtained very straightforwardly by isolating from the strain to be mutated the total DNA, cleaving the latter into small fragments and integrating into a temperature- sensitive plasmid with another selection marker which is selectable in E. coli, transforming the hybrid plasmid population obtained in this way into the starting strain, selecting the transformants by selection for the marker, eliminating the hybrid plasmids by raising the temperature above the threshold of the temperature-sensitive plasmid, and selecting the mutants by renewed selection for the marker. Construction of a cosmid gene bank with the DNA of these mutants results, by selection for the marker which has been introduced and is selectable in E. coli and for the cosmid-intrinsic marker, directly in cosmids which contain the mutated gene.

Description

A METHOD FOR THE SELECTION OF LARGE DNA SECTIONS . .3.T bv ο*ί?πλ t yu n bw n^^ipbob hoiy HOECHST AKTIENGESELLSCHAFT HOE 88/F 070 Dr. KL/ml Description A method for the selection of large DNA sections The European Patent Application with the publication number (EP-A) 0 , 243 , 856 discloses a method for the preparation of mutants, in which the complete DNA is isolated from the initial strain and converted into short fragments, the latter are integrated into a plasmid which contains a marker, is temperature-sensitive and repli-cates in the initial strain, the resulting hybrid plasmid population is transformed into the initial strain, the transformants are selected by selection for the marker, the hybrid plasmids are eliminated by increasing the temperature above the threshold of the temperature-sensitive plasmid, and the mutants are selected by renewed selection for the marker. Thus, the only cells obtained in this last selection step are those which have taken up the plasmid DNA with the marker into the chromosome. Since it is preferentially via the DNA cloned into the plasmid that the plasmid DNA is integrated into a corresponding homologous DNA region of the host cell, this process leads to inactivation of one gene in this homologous region if the DNA fragment cloned in the plasmid contains neither the promoter region nor the translation stop signals of this gene. If the genome of the mutants obtained in this way is now converted into a gene bank, the clones which contain the mutated gene are obtained directly by selection for the marker.

In contrast to the method, which has been known for some time, using the phage J0C31 , in which only short DNA fragments can be isolated, the method described in EP-A 0 , 243 , 856 makes it possible to isolate somewhat longer DNA sections which, for example, may embrace adjacent unmutated genes besides the mutated gene. However, the precondition for this is that suitable cleavage sites for - - restriction enzymes are available and that the desired genes are arranged in the hoped-for manner on the cloned DNA sections.

It has now been found that the stated limitations on the length and the arrangement of restriction cleavage sites in the mutated DNA fragment which is to be isolated, and in its adjacent regions, can be overcome if a marker which is selectable in E. coli is chosen in this method. This is because it is then possible to select the mutated gene directly from a cosmid gene bank in E. coli. A cosmid gene bank of the mutants in E. coli is constructed for this purpose. Immediately after selection for the integrated marker, expediently at the same time as the selection for the marker intrinsic to the cosmid, the cosmid of interest is selected. This makes the very elaborate screening by hybridization, which is otherwise necessary, superfluous.

The term "cosmid" in this connection is intended to embrace not only the actual cosmids but also E. coli plasmids which act in the same way and which, like cosmids, are capable of stable integration and of replication of DNA fragments of about 40-50 kb. Particularly suitable plasmids are those which are present in small copy number in the cell.

The method according to the invention thus allows the isolation of large DNA regions (of the order of 40 kb in size) as can be integrated in cosmids in a manner known per se. This method is therefore particularly suitable for the isolation of gene clusters and facilitates the elucidation of entire biosynthetic pathways, whose genes are often arranged in the form of clusters.

The marker which is selectable in E. coli need not be identical to the marker used for the first selection steps in the known method. On the contrary, it is pos-sible to use for the method according to the invention - plasmids which are temperature-sensitive or have been made temperature-sensitive and which, on the one hand, have markers which are selectable in E. coli and, on the other hand, have DNA regions which can replicate in the strain to be mutated and can be selected. The construction of such vectors is known.

Examples of resistance markers which are expressed both in E. coli and, for example, in Streptomycetes from their own promoter are the kanamycin- and gentamicin-resistance genes (EP-A 0,248,207) .

Reference may be made to EP-A 0,243,856 for further details .

A preferred embodiment of the invention relates to the preparation and isolation of Streptomycetes mutants. It is possible to use for this particularly advantageously the plasmid pS65, which is disclosed in European Patent (EP-B) 0,158,872 (and the published Australian Application 40599/85). This is because, surprisingly, it has emerged that this plasmid is temperature-sensitive and thus can be used directly after insertion of a marker for the method according to the invention. The Patent Application with the title "The use of pSG5 as a temperature-sensitive plasmid" filed on the same date (HOE 88/F 069, corresponding to German Patent Application P 38 09 692.7 of March 23, 1988) relates to the use of the plasmid pSG5 as a temperature-sensitive plasmid.

The figure shows the plasmid pGM8, a derivative of pSG5 with two markers.

The invention is explained in detail in the examples which follow.

Example 1 Construction of the Streptomycetes vector pGM8 The temperature-sensitive vector pGM8 is a derivative of the plasmid pSG5 which is disclosed in EP-B 0,158,872 (and is deposited under the provisions of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German microorganism and cell-culture collection) under No. DSM 2932). This EP-B has already described two vectors which are derived from pSG5 and harbor antibiotic-resistance genes. pGM8 is constructed in an anologous manner: Initially a 1.1 kb fragment on which is located the thiostrepton-resistance gene tsr is isolated with Bell from the commercially available plasmid pIJ6 - as descri-bed in EP-B 0,158,872. This fragment is ligated into the commercially available vector pUC19 which has been opened with BamHI. The plasmid pSLE60 is obtained in this way. pSLE60 is initially opened with Hindlll, after which the protruding ends are filled in with Klenow polymerase. Cutting with EcoRI is then carried out, and the 1.1 kb fragment on which is located the tsr gene is isolated.

The plasmid pSG5 is now totally digested with EcoRI and PvuII, and the 3.2 kb fragment which carries the origin of replication is isolated. This fragment is now ligated to the fragment which carries the tsr gene. The plasmid pGM5 which is 4.3 kb in size is obtained in this way.

A gentamicin-resistance gene which is active in Streptomycetes and in E. coli is now inserted as second resistance marker into pGM5. For this purpose, the 2.3 kb fragment which carries the Gmr gene is isolated with Sail from the plasmid pSLE80 (cf. EP-A 0,248,207; deposited under the provisions of the Budapest Treaty under No. DSM 3710). pGM5 is now opened with Xhol and ligated to the 2.3 kb fragment, resulting in the vector pGM8. Cleavage sites for EcoRI, Sstl, Hindlll, Xbal, Clal ( inactivation of tsr) and Bglll ( inactivation of Gmr) are available in the latter.

The figure shows the ^restriction map of pGM8.

Example 2 The starting material is the strain S. hygroscopicus ATCC 21705, which is specified in EP-A 0,173,372, 0,242,236 and 0,242,246 and is generally available. This strain contains a gene for resistance to the antibiotic phosphinothricyl-alanyl-alanine (PTT) . Thompson et al., EMBO J. 6 (9), 2519-2523, 1987, disclose that double digestion with Sail and Kpnl produces a DMA fragment which embraces parts of the resistance gene without promoter and without translation stop.

For this purpose, the complete DNA is isolated from the said strain, the DNA is cleaved with Sail and Kpnl, and a DNA fragment about 250 bp in size is isolated. This DNA is amplified in the commercially available vector pUC18 which has been opened with Sail and Kpnl. After the amplification, the DNA fragment with the resistance gene is cut out with HindiII and EcoRI and, after attachment of an appropriate linker, inserted either into the EcoRI or into the Hindlll cleavage site of the Streptomycetes vector pGM8.

The ligation mixture is transformed into S. hygroscopicus. Plasmid-harboring cells are selected for resistance to thiostrepton and/or gentamicin. The transformants are incubated on non-selective medium at a temperature above 36°C in order to eliminate all autonomous plasmids.

Renewed selection of replica-plated spores on selection medium yields only those colonies which have integrated plasmid DNA into the chromosome. Because recombination via the cloned homologous regions is preferred, the thiostrepton-resistant cells are essentially mutants, from which the PTT-sensitive cells can now be selected.

Example 3 Isolation of the mutated PTT-resistance gene and adjacent DNA regions A gene bank is now constructed, with the aid of the commercially available cosmid pHC79 (Boehrxnger Mannheim; cf. E.-L. Winnacker, Gene und lone (Genes and Clones), VCH Verlagsgesellschaft, Weinheim, 1985, page 150), from the mutants obtained as in Example 2. The clones which harbor the mutated gene and the circumadjacent DNA regions are obtained directly by simultaneous selection for resistance to gentamicin and tetracycline or ampicil-lin. Since it is known from Murakami et al., Mol. Gen. Genet. 205, 42-50, 1986, that the PTT-resistance gene is present as part of the 16 kb biosynthesis cluster, there are obtained in this direct way cosmids which harbor these biosynthesis genes.

Claims (3)

- 7 - HOE 88/F 070 Patent claims large

1. A method for the selection of/DNA sections from mutants, entailing the complete DNA being isolated from an initial strain which is to be mutated and being converted into short fragments, the latter being integrated into a plasmid which is temperature-sensitive, contains a selection marker and replicates in the initial strain, the hybrid plasmid population obtained in this way being transformed into the initial strain, the transfor- mants being selected by selection for the marker, the hybrid plasmids being eliminated by increasing the temperature above the threshold of the temperature-sensitive plasmid, the mutants having the plasmid DNA integrated into the genome being isolated by renewed selection for the marker, a gene bank being constructed from the DNA of the mutants, and clones having the mutated gene being selected by selection for the marker, wherein the temperature- sensitive plasmid contains a selection marker expressing in E. coli, and wherein the gene bank constructed with the DNA of the mutants is a cosmid bank.

2. The method as claimed in claim 1, wherein the selection marker expressible in E. coli is identical to that used for the selection of the transformants in the initial strain.

3. The method as claimed in claim 1 or 2, wherein DNA sections which harbor gene clusters are isolated.