FR2718152A1 - Process for selecting transformed eukaryotic cells and cells obtained - Google Patents

Abstract

Method for producing animal or plant eucaryotic cells or cell clusters, resistant to streptothricin or analogues thereof, including the steps of transforming said eucaryotic cells with a plasmid containing an exogenous DNA sequence coding for a streptothricin acetyl transferase (SAT), which comprises the DNA sequence coding for said streptothricin acetyl transferase (DNA-SAT) in an appropriate position, and one or more DNA sequences coding for elements controlling the expression of said DNA-SAT sequence in said cells, and selecting the cells resistant to streptothricin or its analogues after their transformation.

Description

 The present invention relates, in particular, to a new method for selecting eukaryotic, particularly animal or transformed plant cells.

 This invention relates to the use of genes encoding streptothricin inactivating proteins in the selection of transformed plant tissues, particularly streptothricin-acetyl transferases.

Streptothricins isolated from Streptomyces lavendulae are antimicrobial agents. They consist of three regions represented by gulosamine, streptolidine and a peptide chain of β-lysines. The length of this chain varies from 1 to 7 residues and characterizes the type of streptothricin A, B, C, etc. (Khoklov and
Shutova, 1972; Carter et al., 1952). This class of antibiotic has both a bacteriostatic and bactericidal action, both gram-positive and gram-negative bacteria, in particular enterobacteria. This type of antibiotic binds to the 30s subunit of ribosomes resulting in inhibition of protein synthesis.

 Following the use of streptothricin in livestock feed, Escherichia coli strains resistant to this antibiotic were isolated. It has been shown that in most cases this streptothricin resistance is carried by plasmids of different compatibility groups which code for a specific acetyl transferase called streptothricin acetyl transferase (SAT). Streptothricin acetylated on the amine group of the lysine residues is inactive.

 Two sat1 and sat2 genes have been characterized (Tietze et al., 1988).

They come respectively from transposons Tn1825 and Tn1826 related to transposon Tn7. The nucleotide sequence of these two genes has been determined (Heim et al., 1989, Tietze and Brevet, 1990). A very high degree of homology is found between these two sequences.

 In the context of the present invention, it has been possible to demonstrate the existence of a sat3 gene which also codes for a streptothricin acetyl transferase but does not show substantially homology with the sat1 or sat2 genes.

More particularly, the present invention relates to a process for obtaining eukaryotic cells, isolated or not, resistant to streptothricin or its analogs comprising the following steps - transformation of said eukaryotic cells using a vector
of Expression of a DNA Sequence Encoding a Streptothricin
acetyl transferase (SAT) having the DNA sequence coding for
said streptothricin acetyl transferase (DNA-SAT) located in
suitable orientation and one or more coding DNA sequences
for the elements controlling the expression of said DNA sequence
SAT in said cells, and - selection of cells resistant to streptothricin or its
analogs after transformation.

 By "expression vector" is meant any system allowing the incorporation and expression of the DNA sequence encoding a SAT, it may be self-replicating system, plasmid type, or integrative type virus or Naked DNA.

 Among the transformation techniques that can be used for the implementation of the present invention, it will be advantageous to use, for plant cells, the so-called "Agro-infection" technique using an Agrobacterium rhizogenes bacterium possessing a binary vector or a coding vector. integration, or the so-called DNA bombardment technique "or" biolistic ", and for animal cells, it will be advantageous to use sequences of a retrovirus.

It is important to specify that among the sequences coding for streptothricin acetyltransferase, the sequences chosen from among a) the sat1, sat2 and sat3 genes, b) the DNA sequences which hybridize with said genes and which encode
for a protein with SAT activity, and c) the DNA sequences that are obtained by degeneration from
sequences a) and b) and which encode a protein having a
SAT activity.

The sequence of sat3 is represented in FIG.
The satl sequence is found in particular in Heim et al, 1989,
The sequence of sat2 is shown in particular in Tietze and Brevet, 1990.

The DNA-SAT sequences are placed under the control of at least one functional promoter in the transformed cell, the use of such promoters will be the subject of examples, these are nevertheless elements which are known to the patient. skilled in the art for eukaryotic cells, whether they be animal cells or plant cells
It is also possible to provide processes implementing transposons.

 The present invention is, in particular, intended to be applied to plant cells, which is why the vectors used are more particularly vectors derived from Agrobactenum, in particular derived from A. tumefaciens or A. rhizogenes, also being a technology known to those skilled in the art.

 The present invention also relates to the use of the genes coding for these streptothricin acetyl transferases as selective markers of transformed eukaryotic cells, isolated or not, differentiated or otherwise, for example, in the form of a cell suspension, of cell aggregates. , embryos, tissues or plants.

However, it is understood that the expression of streptothricin acetyltransferase is only one element of selection, that in most cases the genetic construct used for the transformation will furthermore comprise a set of DNA sequences. allowing the transformed cell and the transformed tissues, organs or transformed plants to express a character of interest, particularly in the case of plant cells, a character of agronomic interest.

 It is, of course, not possible to make an exhaustive list of the characters of interest that can be carried by the vectors, one will simply quote the resistance to certain insects in the fight against the rodents of culture, the resistance to certain herbicides to facilitate for example the weeding of certain crops, also the resistance to certain types of diseases such as fungal diseases, or climatic conditions, one can also cite characters such as the increase of the content in certain active principles or products of the secondary metabolism, or the possibility for certain plants to be able to dispense with certain inputs such as, for example, nitrogen inputs, or the improvement of the technological or nutritional properties of certain plants.

 The present invention also relates to the cells obtained by the method according to the invention and the tissue cultures or the plants regenerated from these cells.

The present invention also relates to a DNA sequence encoding streptothricin acetyl transferase (SAT-DNA) selected from: a) the sat3 sequence shown in Figure 1, b) the DNA sequences that hybridize with said gene and code for
a protein with SAT activity, c) DNA sequences that are obtained by degeneration from
sequences a) and b) and which encode a protein having a
SAT activity.

 It should be noted that the comparison of this sat3 sequence with that of sat1 and its t2 shows no homology. This confirms the result of previously performed tests showing no hybridization between the sat3 sequence on the one hand and the sat1 and sat2 sequences on the other hand. The sat3 gene encodes a protein of 180 amino acids with a molecular weight of 335 daltons. The acetyl transferase activity of these three genes is streptothricin specific and is inactive on other antibiotics such as chloramphenicol, gentamycin, kanamycin and neomycin (Tschape et al., 1984).

 The present invention also relates to cloning vectors and expression of DNA-SAT sequences as well as plasmid vectors, integration vectors, transformed eukaryotic cells, isolated or not, differentiated or not, and plants, obtained by carrying out the method according to the present invention.

 Other features and advantages of the present invention will appear on reading the examples below.

 One of the interests of streptothricins is that a large number of prokaryotic and eukaryotic cells are susceptible.

 Before testing the effectiveness of the construction described above, the sensitivity of different organisms to streptothricin was sought. For example, the optimal inhibitory concentrations found for different cells are given in Table 1.

The optimal inhibitory concentration is the minimum concentration from which cell division is completely abolished.

The experienced experimenter will know that this optimal concentration varies according to the cells concerned and the mode of culture, and that it will have to be determined before proceeding to the transformation of new cells.

Table 1: Optimal Inhibitory Concentration of Streptothricin for Different Cells and Tissues

<tb><SEP> Cells <SEP> Concentration <SEP> in <SEP> mg / l
<tb> Escherichia <SEP> coli <SEP> 100
<tb> Agrobacterium <SEP> tum <SEP> efaciens <SEP> 100
<tb> Saccharomyces <SEP> cerevisia <SEP> e <SEP> 100
<tb><SEP> Schizosaccharomyces <SEP> pombe <SEP> 50
<tb><SEP> Nicotiana <SEP> Tobacco <SEP>(SEP> Leaf Disks) <SEP> 100
<tb><SEP> Daucus <SEP> carota <SEP>("hairy<SEP>root"<SEP> 100
<tb><SEP> Lotus <SEP> corniculatus <SEP> (germination <SEP> of <SEP> seeds <SEP> 100
<tb> Arabidopsis <SEP> thaliana <SEP>(SEP> culture) of <SEP> root <SEP> in <SEP> 25
<tb> vitro)
<tb> Vine <SEP>(<SEP> culture of <SEP><SEP> cells in <SEP> suspension) <SEP> 10
<tb><SEP> Mammary cells <SEP> of <SEP> mice <SEP> HC1 <SEP> l (culture <SEP> in <SEP> 100
<tb> vitro)
<tb> ovarian <SEP> cells <SEP> of <SEP> hamster <SEP> CHO <SEP> (culture <SEP> in <SEP> 100
<tb> vitro)
<Tb>
The following experiments are provided by way of example to better understand the use of the present invention. It should be understood that these examples are provided to illustrate the interest of such a selection marker, but can in no way be considered as the only possible cases of the use of this marker.

In the appended figures, FIG. 1 represents a DNA fragment containing the sat3 gene, and FIG. 2 represents the BglII-BglII fragment of the construction.
pJBJ 106 used for the transformations described in the examples
below.

Example 1
First experience
The construct comprising the essential elements for testing the use of the sat3 gene in the selection of transgenic plants was carried out in the vector pK18 (Pridmore, 1987). In this vector different DNA fragments were cloned sequentially. The final construction is called pJBJ106. Only the BgnI-BgflI fragment (4.7 kb), essential in this study, is represented (Fig. 2). Starting from the left to the right, between the BglII and Clan sites, is a BglII-DraI fragment that covers the left side of the T-DNA of pRi2659 (A. rhizogenes cucumopine plasmid).
NCPPB2659 (Brevet et al., 1988)). This fragment resulting from the digestion of the plasmid pN10AA (Brevet et al., 1988) contains the left border of the T-DNA of pRi2659. The Clal, HindIII and EcoRV sites come from the multiple cloning sequence (MCS) of the Bluescript KS plasmid (Stratagene). The sequence between XmnI and SstI contains the untranslated terminator portion of nopaline synthase (nos-ter) isolated as an EcoRI-SstI fragment of plasmid pBil21 (Clontech). The EcoRI site which served as a cloning site was then digested and filled with the Klenow enzyme to give rise to the XmnI site. The KpnI-BamHI fragment contains the coding sequence of the sat3 gene. This fragment originates from plasmid pJBsat3 constructed by cloning an Sspl-HindIII fragment from plasmid pIE928 (Tietze et al., 1989) to the unique SmaI site of pK18 (Pridmore, 1987). In the pJBJ106 construct, between the BamHI site and the SpeI is contained the CAMV 35S promoter sequence extracted from plasmid pBil21 (Clontech) following HindIII digestion.
Bam. The Spel-EcoRI segment, which follows, consists of the right part of the
T-DNA of plasmid pRi2659 from plasmid pMOA9 (Brevet et al., 1988). This segment contains the entire gene for functional cucumopine synthesis as well as the right border of T-DNA with its repeats (Hansen et al., 1992). The portion between EcoRI and BglII is a fraction of the vector plasmid pK18. The entire fragment between the two extreme sites BglII was then ligated to the BglII segment.
BgnI of pBin19 (Bevan, 1981) which contains the origin of replication of this plasmid with a broad host spectrum derived from the plasmid RK2 and which has as a bacterial selection marker the kanamycin resistance gene. This last construction is called pJBJ302.

 Plasmid pJBJ302 was introduced into an A strain. rhizogenes possessing the mannopine plasmid pRi8196 (Chilton et al., 1982) by electroporation and the transformed cells were selected on rich agar medium supplemented with kanamycin (Km) at 100 mg / l. The transformants were subcultured on synthetic agar medium supplemented with mannopine as the sole source of carbon and kanamycin (100 mg / l).

A clone was selected and its plasmid content was verified by agarose gel electrophoresis, after a mini-extraction (on 5 ml of overnight culture at 28 in rich medium supplemented with Km) according to the Kado method (Kado and Liu, 1981) but performed at 37 to minimize breaks in the Ri plasmid. This plasmid pRi8196 serves both as a T-DNA donor inducing the root formation of hairy root type, and provides the VIR functions necessary for the transfer of the T-DNA construct carried by the binary vector A bacterium carrying these two plasmids is able, when inoculated to a plant, to cause the differentiation of doubly transformed roots, containing both the T-DNA of hairy root "and that of interest carried by the second plasmid. This clone was used to inoculate carrot discs (Petit et al., 1983). After two to three weeks roots of "hairy root" type appear. They were excised from the carrot disc and cultured in the dark on MS sucrose medium (30 g / l) (Murashige and Skoog, 1962) agar not containing growth hormone but supplemented with cephalosporin. (250 mg / l) to inhibit any bacterial growth. On this medium, the roots have a growth of 2 to 5 mm in 24 hours and they have many ramifications. These roots are known to be cell clones (David et al., 1984). Roots (approximately 1.5 cm in length) were transplanted onto solid MS sucrose medium with or without streptothricin (100 mg / l). Roots containing only pRi8196 T-DNA showed low growth in 24 hours and stopped growing. Replaced on streptothricin-free medium their growth did not resume, showing the irreversible effect of the antibiotic.

On the other hand, certain roots showed a significant growth as well on medium supplemented with streptothricin that deprived of this antibiotic. These roots were considered to have integrated the construct containing the sat3 gene. Almost 70% to 80% of the roots proved to be able to grow in the presence of streptothricin. This result is in good agreement with previously described observations (Petit et al., 1986).

To verify that the roots growing in the presence of streptothricin did contain the construct, analyzes by paper electrophoresis of the opine content of the roots were performed (Petit et al., 1986). The roots unable to grow on streptothricin contained only mannopine, a marker of pRi8106 T-DNA, while all roots capable of growing on streptothricin (20 independent roots analyzed) contained both mannopine and cucumopine, the latter being a marker contained in the construction. This result indicates that streptothricin-resistant roots have integrated the construct. To confirm that these cucumopin-synthesizing roots did indeed have the sat3 gene, a genomic DNA analysis extracted from the roots was undertaken using the
PCR ("Polymerase Chain Reaction"). Two oligonucleotides were synthesized. One of 20 bases has the sequence 5 'TCAATGCGTGAATTGGTCAT-3' located at position 233-252 on the sequence, the other 18 bases has the sequence 5'-GGATGCAGGCCACGATAC-3 'located at position 720-703 on the sequence (Fig. 1). The use of these oligonucleotides in a PCR reaction makes it possible to amplify a 488 base pair DNA sequence covering almost all of the sat3 gene which can be detected after electrophoresis, of the mixture subjected to the PCR reaction, on agarose gel. The genomic root DNA was extracted by grinding 1 cm of root in 0.1 ml of sterile distilled water in Eppendorf tubes and the tubes were incubated for 5 minutes in a boiling water bath. After 1 μl centrifugation of supernatant was added to 50 μl of the standard PCR reaction mixture The desired DNA was amplified over 30 cycles (1 cycle includes the following incubations: 1 min at 94, 1 min at 55 ° and 1.5 min to 72 "). The results showed that only genomic DNAs extracted from cucumopin-synthesizing roots allowed amplification of a 488 base pair fragment confirming the presence of the sat3 gene.

 In conclusion, the roots capable of growing on 100 mg / l of streptothricin turned out to be roots actually transformed by the construction.

Example 2
Second Experience
Plasmid pJBJ3O2 was introduced into the A strain. LBAL tumefaciens containing plasmid pA14404 (Ooms et al., 1982). This disarmed plasmid provides the VIR functions needed to transfer everything
T-DNA. the transformed bacteria were selected on rich agar medium supplemented with kanamycin. The clones obtained were analyzed as in the first experiment and a selected clone was used to inoculate leaf disks of Nicotiana ta bac um va .. Xanthi according to the method of Horsch et al., 1985. 24 hours after inoculation, the disks were transferred to MS solid medium supplemented with 6-benzylaminopurine (BAP) (1 mg / l), acetic-naphthalene (NAA) (0.1 mg / l), cephalosporin (250 mg / l) and streptothricin (100 mg / l) to obtain seedlings from the transformed cells. After four weeks seedlings appeared. They were transplanted into MS medium containing streptothricin. Their roots have grown. After three weeks, the plants were individually cuttured on solid MS medium in Magenta pot for plant development. A one centimeter diameter leaf disc was taken from each plant to search for its cucumopine content by electrophoresis (Petit et al., 1986). Of 50 plants analyzed, 46 clearly showed the presence of cucumopine, which represents a percentage of 92%. The four apparently negative plants may represent false positives or plants in which cucumopine synthase is too weakly expressed. In any case, a proportion of 10% false positive plants would be perfectly acceptable and would reflect what is often observed with most selection markers used to select transformed tissues. To confirm that plants containing cucumopine had also integrated the sat3 gene, their
Genomic DNA was extracted (Edwards et al., 1991) and analyzed by the PCR technique as in the first experiment. The DNA extracted from normal tobacco gives no signal whereas with the DNAs extracted from plants synthesizing cucumopine, it has always been found the amplification of a DNA fragment of 488 base pairs, confirming the presence of sat3 gene.

 In conclusion, the selection of transgenic tobaccos using the sat3 selection marker was found to be very effective in at least 90% of cases.

Example 3
Third Experience
The strain of A. tumefaciens used in the second experiment (strain LBA4404 containing the plasmid pJBJ302) was used to transform Arabidopsis thaliana roots according to the method of
Valvekens et al. (1988) but using streptothricin as a selective agent instead of kanamycin. Two antibiotic concentrations, 25 mg / l and 40 mg / l, were tested in parallel. After three weeks, green seedlings appeared and were transplanted into GM medium (Vanvekens et al., 1988) supplemented with streptothricin (25 or 40 mg / l) to allow them to grow individually. Analysis of the opine content of these seedlings has been undertaken. On 10 seedlings grown on 25 mg / l of streptothricin, 5 showed the presence of cucumopine while 14 of 15 seedlings grown on 40 mg / l of antibiotic were found to contain cucumopine. This result emphasizes the importance of using an optimal antibiotic concentration to obtain a high probability of selecting only or at least a majority of transformed plants. With 40 mg / l streptothricin, 93% of the plants obtained had integrated the construction. To verify that these plants did have the sat3 gene, their genomic DNA was extracted and served as a template in PCR reactions as in the first experiment. DNA from all cucumopin-synthesizing plants allowed amplification of the 488 base pair sat3 gene-like DNA fragment, whereas the non-transformed plant DNA gave no signal.

 These results confirm that it is important to determine the optimal concentration of streptothricin for each type of cell that is to be transformed and that it is possible under these conditions to use the sat3 gene to select tissues transformed with high frequency.

Example 4
Fourth ex-experience
Leaf disks of one centimeter in diameter, sterilely cut with a metal punch, were taken either on normal tobacco or on tobacco transformed by the gene sat3, grown in vitro. These disks were placed in petri dishes containing MS20 agar medium supplemented with NAA (1 g / l) to induce root formation. One batch of petri dishes was streptothricin (100 mg / l) added. Leaf discs were incubated at 24 under 16 hours of light per day. After fifteen days roots appeared from the normal and transgenic tobacco discs on the streptothricin-free medium. On the other hand, on the medium containing the antibiotic, only the transgenic tobacco discs gave rise to roots whereas the normal tobacco discs showed no cellular or root development.

 This result shows that the expression of the resistance to streptothricin is maintained during the development of the plant and that it is very easy to make, a posteriori, the distinction between plants containing the gene sat3 and those which do not .

Example 5
Yeast cells and mammalian cells
Other experiments aimed at verifying the efficacy of the sat3 gene for the selection of transformed cells of yeast
Schizosaccharomyces pombe were undertaken, following a standard transformation procedure with the construct described in Figure 2 in which the sat3 gene is placed under the control of the ART 355 promoter of cauliflower mosaic virus, which is known that it is active in this yeast. These experiments made it possible to show the efficacy of the sat3 gene in Schizosaccharomyces pombe. The transformation was carried out using the technique of Ito et al (1983).

 For use in animal cells, the streptothricin resistance gene can be placed under the control of a promoter allowing ubiquitous expression such as that of the precystic genes of the human cytomegalo virus tCMV (used in the described experiments), SV40 virus or Rous sarcoma virus (RSV). It may just as well be under the control of the promoter of a gene expressing only in a given cell type. Experiments were performed with a construct where the Sat3 gene was placed under the control of the CMV promoter.

Mouse cells of HC1 1 mice and hamster ovary cells
CHO were transformed with this construct by the liposome technique described by Felgner et al. (1987). In both cases it was possible to obtain a large number of clones transformed by selection in the presence of streptothricin.

 These experiments show that the sat3 gene can be used to select transformed yeast and mammalian cells.

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Claims (15)

 A method for obtaining eukaryotic cells resistant to streptothricin or its analogs comprising the following steps - transformation of said eukaryotic cells using a vector  of Expression of a DNA Sequence Encoding a Streptothricin  acetyl transferase (SAT) having the DNA sequence coding for  said streptothricin acetyl transferase (DNA-SAT) located in  suitable orientation and one or more coding DNA sequences  for the elements controlling the expression of said DNA sequence  SAT in said cells, and - selection of cells resistant to streptothricin or its  analogs after transformation.  2. Method according to claim 1, characterized in that the sequence coding for streptothricin acetyl transferase is chosen from: a) the genes sat1, sat2, sat3, b) the DNA sequences which hybridize with said genes and which encode  for a protein with SAT activity, and c) the DNA sequences that are obtained by degeneration from  sequences a) and b) and which encode a protein having a  SAT activity.  3. Method according to one of claims 1 or 2, characterized in that the DNA-SAT sequence is placed under the control of a functional promoter in the transformed cell.  4. Method according to one of claims 1 to 3, characterized in that the eukaryotic cell is a plant cell.  5. Method according to claim 4, characterized in that the vector is derived from Agrobacterium.  6. Method according to claim 5, characterized in that the vector is derived from Agrobacterium tumefaciens or Agrobacterium rhizogen es.  7. Method according to one of claims 1 to 6, characterized in that the vector further comprises a set of DNA sequences allowing the transformed cell to express a character of interest.  8. Method according to claim 7, characterized in that the character of interest is a function of agronomic interest for plant cells.  9. Method according to one of claims 1 to 8, characterized in that the vector is an integration vector.  10. Eukaryotic cell obtained by the implementation of the method according to one of claims 1 to 9.  Plants obtained from cells according to claim 10.  12. Use as a selective marker in the transformation of plant tissues and subsequent plants of genes encoding streptothricin acetyl transferase.  13. DNA sequence encoding streptothricin acetyl transferase (SAT-DNA) selected from a) the sat3 sequence shown in Figure 1, b) DNA sequences that hybridize with said gene and encode  a protein with SAT activity, c) DNA sequences that are obtained by degeneration from  sequences a) and b) and which encode a protein having a  SAT activity.  14. Vector for cloning and expression of a SAT in a eukaryotic cell, characterized in that it comprises the DNA sequence coding for said streptothricin acetyl  transferase (SAT-DNA) located in a suitable orientation and one or  several DNA sequences coding for the controlling elements  expressing said DNA-SAT sequence in said cells.  15. Vector according to claim 14, characterized in that the elements controlling the expression are elements of the host organism.