DE3824670A1 - DNA SEGMENT, PLASMIDES CONTAINING THIS SEGMENT, TRANSFORMED BACTERIA, AND TRANSFORMED PLANT MATERIAL - Google Patents

Abstract

This invention concerns a DNA segment whose gene product is suitable for making an oriV sequence capable of replication in cis or trans form. The gene product is a fusion peptide of trfB and trfA. Recombinant plasmids containing this DNA sequence, transformed bacteria and transformed plant material, as well as seeds of this plant material are also disclosed.

Description

The invention relates to a DNA segment whose gene product is suitable for making an oriV sequence capable of replication in cis or in trans, as well as plasmids containing this segment, bacteria transformed by these plasmids, as well as transformed plant material and seeds from transformed plant material.

The naturally occurring plasmid RK2, which is identical or almost identical to the plasmids RP1, RP4, R18 and R86 of the incompatibility group P1, is a broad host range plasmid that contains resistance genes to the antibiotics tetracycline, ampicillin and kanamycin. The sequence of the vegetative origin (oriV) and the gene product of the transacting factor A gene (trfA) is sufficient for replication ( Fig. 1). The base sequence of the trfA gene was elucidated in Christopher A. Smith and Christopher M. Thomas, J. Mol. Biol. (1984), 175, pp 251-262. It enables the replication of oriV in many species of gram-negative bacteria. However, the plasmid has a complicated replication system. For routine use, its size of 56 kilobase pairs (Kb) is also a major disadvantage. Attempts have been made to overcome these disadvantages by constructing smaller plasmids based on the RK2 plasmid. For example, a tetracycline-resistant plasmid component of the RK2 plasmid, called pRK 290, is known from Ditta et al., Proc. Natl. Acad-Sci. Vol 77 (12), pages 7347-7351, USA 1980 ( Fig. 2). pRK 290 can be transferred using an auxiliary plasmid, but does not have the ability to transfer itself or other plasmids. The plasmid is 20 Kb in size, making it a relatively large plasmid that is also difficult to isolate and is only present in low copy numbers (5-8 copies per chromosome equivalent in Eschericha coli).

It has now been found that the trfA gene contained in the plasmids RK2 and pRK 290 can be fused with the trfB gene, the base sequence of which is described in Nucleic Acid Res. 14 (11), 4453, with deletion of intervening sequences, so that a DNA sequence is formed whose gene product enables the replication of oriV.

The present invention therefore relates to a DNA segment whose gene product is suitable for making an oriV sequence capable of replication in cis or in trans, which is characterized in that the segment contains a sequence selected from a group containing the following nucleotide sequence

and subfragments and mutants thereof which have essentially the same ability to replicate oriV.

The gene product of this sequence and derived sequences enables the replication of plasmids containing oriV in a number of microorganisms, particularly in Gram-negative bacteria.

The invention further relates to a process for producing the DNA segment according to the invention, which is characterized in that the plasmid pRK 290 is cut with the restriction endonuclease SpH I, the DNA piece between 17.8 kb and 8.3 kb is ligated with the aid of the T4 DNA ligase, again with the Restriction endonuclease SpH I is cut, the plasmid pUC 18 is inserted into the cleavage site, ligated using T4 DNA ligase, this plasmid is linearized using restriction endonuclease Kpn I and shortened from both ends using exonuclease Bal 31, the extent of shortening being controlled by gel electrophoresis, after which the ends are ligated using T4 DNA ligase.

The DNA segment according to the invention is produced from plasmid pRK 290 using various DNA-modifying enzymes. These are initially restriction endonucleases that cut double-stranded DNA in the area of a defined recognition sequence. This creates fragments at the ends of which either single-stranded overhangs (cohesive ends, sticky ends) remain or both strands are cut at the same base pair, creating so-called blunt ends or blunt ends. These fragments are then reassembled (ligated) in the desired manner using a DNA ligase. It is possible to freely combine blunt end fragments, whereas with sticky end fragments ligation is only possible if the single-stranded overhangs are correctly paired with the bases.

To delete larger areas of the plasmid DNA, the exonuclease Bal 31 is used, which continuously breaks down single- or double-stranded linearized DNA from the ends in a roughly symmetrical manner, leaving behind blunt ends. The extent of the shortening is checked using gel electrophoresis and the plasmid DNA is then ligated again using a DNA ligase. The DNA segment according to the invention is a fusion gene of transacting factor B and transacting factor A, although the complete base sequences of the structural genes are not retained. In the sequence given as an example in claim 1, for example, in the gene product of this sequence the first 25 amino acids of the trf-A protein would be replaced by the first 117 amino acids of a protein IncC encoded on the trfB gene or by the first 12 amino acids of another peptide (P 259) (C. M. Thomas, C. A. Smith, Nucleic Acid Res. 14 (11), 4453 (1986)). The nucleotide sequence GTG, which encodes the amino acid valine, is formed at the fusion site. However, the DNA segment according to the invention is not restricted to this nucleotide sequence, but also includes subfragments and mutants thereof whose gene products have essentially the same ability to make an oriV sequence capable of replication in cis or trans.

Another object of the invention is a recombinant plasmid containing a DNA segment according to claim 1.

From the plasmid produced in the manner described above, which contains a DNA segment according to the invention, derivatives can be produced by methods known to the person skilled in the art, which contain, for example, other resistance genes such as kanamycin or ampicillin resistance genes or the border sequences of Ti (tumor inducing) or Ri (root inducing) plasmids. Agrobacterium tumefaciens and Agrobacterium rhizogenes contain natural plasmids, Ti plasmids and Ri plasmids, respectively, which, due to certain functional components, have the ability to transfer DNA segments into plants. In particular, these segments contain sequences that contain information on the synthesis of growth hormones. For transfer into the plant cell, the flanking base pairs of these sequences are required, which contain around 30 base pairs and are almost identical on both sides. These border sequences are the actual requirement for the transfer of the intervening DNA pieces into the plant cell. Any DNA fragments located between the border sequences can be transferred into the plant cell.

The invention further relates to microorganisms, in particular gram-negative bacteria, which have been transformed with a plasmid containing one of the DNA sequences according to the invention.

For transformation, a suitable bacterial strain, for example E. coli HB 101, E. coli JM 103, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Pseudomonas species, etc., is treated in a conventional manner with a suitable transformation buffer and plasmid DNA is added. The transformants are then selected, for example by plating.

Methods for transforming microorganisms, particularly bacteria, are known to the person skilled in the art and are described, for example, in Meth. in Enzymology Vol. 101, p. 507 ff (1983), Mol, Gen. Gen. Vol 163, 181 (1978), J. Mol. Biol. 53, 159-162 (1970), Proc. Natl. Acad. Sci., USA 69, 2110-2114 (1972). Since transformed bacteria have a more or less pronounced tendency to degenerate, the bacteria must be kept under favorable living conditions in order to retain their artificially created abilities. The person skilled in the art will easily be able to determine these conditions depending on the bacterial strain used.

The invention further relates to plant material which has been transformed by agrobacteria which contain a plasmid according to the invention, as well as seeds from this plant material. The transformation of plant material using agrobacteria is described, for example, in Chilton M. D., Drummond M. J., Merlo D. J., Sciaky D., Montoja, A. L., Gordon M. P., Nester E. W., Cell 11, (1977) pp 263-271; Chilton M. D., Tepfer D. A., Davin C., Casse-Dellart F., Tempe J., Nature 295 (1982) pp 432-434 and Barton K. A., Chilton M. D., Methods in Enzym. 101 (1983) pp 527-539.

As already described, it is possible to transfer any sequence located between the border sequences of the Ti or Ri plasmids to the plant and thus influence its properties.

Examples 1. Construction of a plasmid containing a DNA segment according to the invention ( Fig. 2). (pPS 99)

The plasmid pRK 290 was modified with the restriction endonucleosis Sph I (from Steptomycees phaeochromogenes) at the cleavage sites Sp ( Fig. 2) with the recognition sequence

The reaction is carried out with plasmid DNA in an incubation buffer of 10 mmol/l TrisHCl, 50 mmol/l NaCl, 10 mmol/l MgCl₂ and 1 mmol/l dithiothreitol (pH 7.5) at 37°C. The large fragment is then closed again using T4 DNA ligase in 40 µl 50 mM Tris/HCl pH 7.8, 10 mM MgCl₂, 10 mM dithiothreitol and 1 mM adenosine triphosphate to form a plasmid which contains the region between 17.8 Kb and 8.3 Kb of the original plasmid pRK 290 (plasmid I in Fig. 2).

In a further step, the 2.7 Kb plasmid pUC18 (described in Vieira J., Messing J., 1982, Gene 19 p. 259) was inserted into plasmid I at the Sp cleavage site. To do this, plasmid I and pUC18 are linearized with Sph I under the conditions described above and then ligated again with T4 DNA ligase. (Plasmid II in Fig. 2.) The plasmid thus produced contains a single KpnI restriction site (K) from the pUC18 fragment with the recognition sequence

which is the starting point for a subsequent deletion with the exonuclease Bal 31. The sequences of trfB and trfA are approximately equidistant from this KpnI restriction site due to the insertion of pUC18.

Plasmid II was first linearized with the restriction endonuclease Kpn I (from in an incubation buffer consisting of 10 mmol/l Tris HCl (pH 7.5), 10 mmol/l MgCl₂ and 1 mmol/l dithiothreitol) and then shortened from both ends with the exonuclease Bal 31 (from Alteromonas espeijiani).

The extent of the truncation was checked by gel electrophoresis on 1% agarose gel. Finally, a 6.6 Kb plasmid containing a DNA segment of claim 1 was ligated using T4 DNA ligase.

2. Transformation of E. coli HB 101

The transformation of the E. coli strain can be carried out in a manner known per se, for example in the procedure given by Mandel M., and Higa A. J. Mol. Biol. 53 159-162 (1970), and Cohen, S. N., Chang, A. C. Y., and Hsu, L. Proc. Natl. Acad. Sci., USA 69, 2110-2114 (1972).

For this purpose, 5 ml of Lurea Broth - a nutrient medium consisting of meat extract, yeast extract and salt - is inoculated with 1 ml of preculture of E. coli HB 101 and shaken at 37°C until a quarter of the maximum cell density (extinction in the photometer approx. 0.4-0.6) is reached.

The cells are harvested by centrifugation at 5000 rpm and suspended in ice in 25 ml of transformation buffer consisting of 150 mmol KCl, 50 mmol CaCl₂, 5 mmol MgCl₂ and 1 mmol Tris HCl (pH 7.5). After centrifugation again, the cells are resuspended in 2 ml of transformation buffer. 0.1 ml of this suspension is mixed with plasmid DNA (pPS 99) under ice cooling, warmed to 42°C after 1 hour and 1.2 ml of Lurea Broth at 37°C is added after 90 seconds. After 5-15 minutes, the cells are plated on selection agar (Lurea Broth with 1.5% agar and a suitable antibiotic). The transformants are visible after 15-24 hours.

A transformed E. coli HB 101 strain obtained in this way was deposited on 16 June 1988 with the German Collection of Microorganisms and Cell Cultures GmbH under the number DSM 4673.

3. Transformation of agrobacteria

Among other things, the constructions shown in Fig. 3 were tested for their replication ability in Agrobacteria.

100 ml of YEP (1% yeast extract, 1% peptone, 0.5% NaCl) was inoculated with 2 ml of preculture at 30°C for 4-6 hours. The cells were grown at 30°C for 4-6 hours and harvested by centrifugation at 5000 rpm for 10 minutes. The cells were then suspended in 10 mM NaCl and centrifuged again. They were then resuspended in 1 ml of YEP.

0.2 ml of this suspension was mixed with 1 µg plasmid DNA and immediately frozen at -80°C in an EtOH bath. After 5 min, the cells were thawed and kept at 37°C for 25 minutes. They were then diluted with 0.5 ml YEP and incubated for 1-2 h at 30°C.

The cells were then plated on selection agar nutrient broth (0.8%, 1.5% agar) or YEP (with 1.5% agar) with the desired antibiotic.

In this way, Agrobacteria were transformed with the plasmids shown in Fig. 3 and in each case transformants were obtained that had the desired phenotype (antibiotic resistance(s)). The plasmids used could be recovered from these transformants, which could be demonstrated by transforming Escherichia coli with this DNA and subsequent restriction analysis.

The abbreviations shown in Fig. 3 mean:

Amp ^r ampicillin resistance gene, Tet ^r tetracycline resistance gene, NPT: neomycin phosphotransferase gene, encodes for kanamycin resistance, Pnos: promoter of nopaline synthetase, SNPT: structural gene of NPT, Tnos: terminator of the nopaline synthetase gene, ori (pUC) origin of replication of the plasmid pUC18, oriV: origin of replication of the plasmid RK2, trfBA the described fragment - essential for the replication of oriV, F: fusion site of the trfA structural gene with the trfB region, recl: piece of the T-DNA of the Ti plasmid as left recombination fragment. EcoRI, BglIII, PvuII, BamHI, AvaI, HindIII, KpnI: restriction enzymes. The unit PnosSNPTTnos can cause neomycin and kanamycin resistance in plants.

Claims (10)

1. DNA segment whose gene product is suitable for making an oriV sequence capable of replication in cis or in trans, characterized in that the segment contains a sequence selected from a group containing the following nucleotide sequence

and subfragments and mutants thereof which have essentially the same ability to replicate oriV are omitted. 2. Process for the preparation of a DNA segment according to claim 1, characterized in that the plasmid pRK 290 is cut with the restriction endonuclease SpH I, the DNA piece between 17.8 kb and 8.3 kb is ligated with the aid of the T4 DNA ligase, cut again with the restriction endonuclease SpH I, the plasmid pUC 18 is inserted into the cleavage site, ligated with the aid of the T4 DNA ligase, this plasmid is treated with the Restriction endonuclease Kpn I is linearized and shortened from both ends with exonuclease Bal 31, the extent of shortening being controlled by gel electrophoresis, after which the ends are ligated with T4 ligase. 3. A recombinant plasmid containing a DNA segment according to claim 1. 4. Recombinant plasmid according to claim 3, characterized in that it contains a resistance gene against at least one antibiotic. 5. Recombinant plasmid according to one of claims 3 or 4, characterized in that it has the border sequences of Ti or Ri plasmids. 6. Transformed bacteria containing a plasmid according to any one of claims 3 to 5. 7. Transformed bacteria according to claim 6, characterized in that the bacteria are selected from the group of gram-negative bacteria. 8. Transformed bacteria according to claim 7, characterized in that they are selected from the group of agrobacteria. 9. Plant material transformed by bacteria according to claim 8. 10. Seed from plant material according to claim 9.