NZ284494A - Transformed cells with streptothricin resistance - Google Patents

Description

New Zealand No. 284494 International No. PCT/FR95/00425 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 05.04.1994; Complete Specification Filed: 04.04.1995 Classification:^) C12N15/54.82; C12N5/10; A01H5/00 Publication date: 24 September 1998 Journal No.: 1432 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Method for selecting transformed eucaryotic cells and cells obtained thereby Name, address and nationality of applicant(s) as in international application form: L.V.M.H. RECHERCHE, 25, rue des Peupliers, F-92000 Nanterre, France 1 28 4 4 9 4 The present invention relates especially to a new process for selecting transformed eukaryotic, particularly animal or plant, cells.

This invention relates to the use of genes encoding 5 proteins capable of inactivating streptothricins in the selection of transformed plant tissues, in particular streptothricin acetyl transferases.

The streptothricins isolated from Streptomyces lavendulae are antimicrobial agents. They consist of three regions 10 represented by guiosamine, strep\olidin and a peptide chain of p-lysines. The length of this chain is variable from 1 to 7 residues and characterizes the type of streptothricin A, B, C and the like (Khoklov and Shutova, 1972; Carter et al., 1952). This class of antibiotic has both a bacteriostatic and bactericidal action, both on Gram-positive and 15 Gram-negative bacteria, in particular enterobacteria. This type of antibiotic binds to the 30s ribosome subunit, causing inhibition of protein syntheses.

Following the use of streptothricin in livestock feed, strains of Escherichia coli resistant to this antibiotic were isolated. It 20 was demonstrated that in most cases, this resistance to streptothricin was carried by plasmids of different compatibility groups which encoded a specific acetyl transferase called streptothricin acetyl transferase (SAT). Streptothricin acetylated on the amine group of the lysine residues is inactive.

Two genes satl and sat2 were characterized (Tietze et al., 1988). They are derived respectively from the transposons Tnl825 and Tnl826 which are related to the 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 exists 30 between these two sequences.

Another gene, sat4, encoding an acetyltransferase streptothricine was also characterized (Jacob et et al., FEMS Microbiol Lett.; 1994, Jul 1:120(1-2): 13-7). This gene was detected in Escherichia coli containing recombinant plasmid pAT132 which carries the sat4 35 gene as an insert. jjj_Z._PA*eNT OFFICE| 884 OCT 1996 2 28 A A 9 4 Within the framework of the present invention, it has been possible to demonstrate the existence of a sat3 gene which also encodes a streptothricin acetyl transferase but exhibits essentially no homology with the satl or sat2 genes.

More particularly, the present invention relates to a process for producing eukaryotic cells, isolated or otherwise, which are resistant to streptothricin or to its analogues, comprising the following stages: transformation of the said eukaryotic cells with the aid of an 10 expression vector for a DNA sequence encoding a streptothricin acetyl transferase (SAT) containing the DNA sequence encoding the said streptothricin acetyl transferase (SAT-DNA) situated in a suitable orientation, and one or more DNA sequences encoding the elements controlling the 15 expression of the said SAT-DNA sequence in the said cells, and selection of the cells resistant to streptothricin or to its analogues after transformation.

"Expression vector" designates any system allowing the incorporation and the expression of the SAT-encoding DNA 20 sequence; it may be a plasmid-type self-replicating system or a virus- or naked DNA- type integrative system.

Among the transformation techniques which can be used for implementing the present invention, there may be advantageously used, for plant cells, the so-called "Agro-infection" 25 technique using an Agrobacterium rhizogenes bacterium having a binary vector or a cointegrative vector, or the so-called "DNA bombardment" or "biolistic" technique, and for animal cells, sequences from a retrovirus may be advantageously used.

It is important to specify that among the sequences 30 encoding streptothricin acetyl transferase, there will be most particularly preferred the sequences chosen from: a) the satl, satl and sat3, sat4 genes, b) the DNA sequences which hybridize with the said genes and which encode a protein having a SAT activity, and c) the DNA sequences which are obtained by degeneracy from sequences a) and b) and which encode a protein [TZ. PATENT OFFICE] i :*4 OCT 1985 RECEIVED -3 - 28 4 4 having a SAT activity.

The sat3 sequence is attached to the present description, The satl sequence is present especially in Heim et al., 1989, The sat2 sequence is present especially in Tietze and Brevet, 1990.

The SAT-DNA sequences are placed under the control of at least one promoter which is functional in the transformed cell, the use of such promoters will be the subject of examples; they are nevertheless elements which are known by persons skilled in the art for eukaryotic cells, whether animal cells or plant cells.

It iB also possible to envisage processes using transposons.

The present invention is in particular intended to be applied to plant cells; accordingly, the vectors used are more particularly vectors derived from Agro-bacterium, especially derived from A. tumefaciens or A. rhizogenes, this technology also being known to persons skilled in the art.

The present invention also relates to the use of the genes encoding these streptothricin acetyl transferases as markers selective for transformed eukaryotic cells, isolated or otherwise, differentiated or otherwise, existing for example in the form of a cellular suspension, of cellular aggregates, of embryos, of tissues or of plants.

The present invention also relates to the use of the genes encoding streptothricin acetyl transferases as selective markers for trapping.a plant promoter. It has indeed been found that the said genes, especially when they are placed with the right border of a T-DNA, can be expressed by a promoter from the plant.

However, it is clearly understood that the expression of streptothricin acetyl transferase is only one selection element, that in most cases the genetic construct used for the transformation will contain, in addition, a set of DNA sequences allowing the transformed 28 4 4 to cn m n ) ; i ■ ^ I ! -~'J !£ ; o ] ;<4J i CL ' cell and the transformed tissues, organs or plants which would be derived therefrom/ to express a character of interest, especially, in the case of plaint cells, a character of agronomic interest.

It is of course not possible to make an exhaustive list of characters of interest which may be carried by the vectors; there will be simply mentioned the resistance to certain insects in the control of rodent species for crops, the resistance to certain herbicides in order to facilitate for example weed control in certain crops, also the resistance to certain types of disease such as fungal diseases or climatic conditions; there may also be mentioned characters such as the increase in content in certain active ingredients or products of secondary metabolism, or alternatively the possibility for certain plants to be able to do without certain supplies such as for example nitrogenous supplies, or alternatively the enhancement of the technological or food properties of certain plants.

The present invention also relates to the cells obtained by the process 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), chosen from: a) the safc3 sequence attached to the description, b) the DNA sequences which hybridize with the said gene and encode a protein having a SAT activity, c) the DNA sequences which are obtained by degeneracy from sequences a) and b) and which encode a protein having a SAT activity.

It should be noted that comparison of this sat3 sequence with that of satl and sat2 show no homology. This confirms the result of the tests previously carried out showing no hybridization between the sat3 sequence on the one hand and the satl and sat2 sequences on the other hand. The sat3 gene encodes a protein of 180 amino acids having a molecular weight of 20,335 daltons. The acetyl - 5 28 4 4 94 transferase activity of these three genes is specific for streptothricin and is inactive in other antibiotics such as chloramphenicol, gentamycin, kanamycin and neomycins (Tschape et al., 1984).

The present invention also relates to the cloning and expression vectors for the SAT-DNA sequences as well as the plasmid vectors, the integrative vectors, the transformed eukaryotic cells, isolated or otherwise, differentiated or otherwise, and the plants, which are 10 obtained using the process according to the present invention.

Other characteristics and advantages of the present invention will emerge on reading the examples below.

One of the advantages of streptothricins is that a large number of prokaryotic and eukaryotic cells are sensitive to them.

Before testing, the efficiency of the construct described above, the sensitivity of various organisms to 20 streptothricin was investigated. By way of example, the optimum inhibitory concentrations found for various cells are given in Table 1. The minimum concentration starting from which cell division is completely abolished will be called optimum inhibitory concentration. The experienced 25 research scientist will know that this optimum concentration varies according to the cells in question and the method of culture, and that it should be determined before carrying out the transformation of new cells.

OCT 1996 284 4 9 4 Table 1 : Optimum inhibitory concentration of streptothricin for various cells and tissues Cells Concentration in mg/1 Escherichia coli 100 Agrobacterivm tumefaciens 100 Saccharomyces cerevisiae 100 Schizosaccharomyces pombe 50 Nicotiana tabacum (foliar discs) 100 Caucus carota ("hairy root") 100 Lotus comiculatus (germination of seeds) 100 Arabidopsis thaiiana (root culture in vitro) Vine (suspension cell culture) Mouse mammary cells HC11 (in vitro culture) 100 Hamster ovary cells CHO (in vitro culture) 100 The following experiments are provided by way of example in order to understand more clearly the use of the present invention. It should be understood that these examples are provided to illustrate the advantage of such a selectable marker, but cannot in any case be considered as the only possible case of the use of this marker.

In the accompanying figures, Figure 1 represents the BglZI-Bglll fragment of the construct pJBJ106. It constitutes the DNA segment which is essential in the transformations described in the examples below, Figure 2 represents the construction map produced in 284494 co cn CO CO CD 1 I "JJ J MJ •x order to obtain the plasmid pJBJ333 which allows the use of the sat3 gene to trap plant promoters. Example 1 First experiment The construct combining the elements essential for testing the use of the sat3 gene in the selection of transgenic plants was made in the vector pK18 (Pridmore, 1987) . In this vector, various DNA fragments were cloned sequentially. The final construct is called pJBJ106. Only the BglXX-BglXX fragment (4.7 kb) , which is essential in this study, is represented (Figure 1). From the left to the right, between the BglXX and Clal sites, is a Bglll-Dral fragment which covers the left portion of the T-DNA of pRi2659 (A. rhizogen.es NCPPB2659 cucumopine plasmid (Brevet et al., 1988)). This fragment, derived from the digestion of the plasmid pM0A4 (Brevet et al., 1988) contains the left border of the T-DNA of pRi2659. The Clal, Hindlll and EcoRV sites are all obtained from the multiple cloning sequence (MCS) of the plasmid Bluescript KS (Stratagene) . The sequence situated between Xmnl and Ssfcl contains the untranslated terminator portion of nopaline synthase (nos-ter) isolated in the form of an EcoRI-SstI fragment of the plasmid pBil21 (Clontech). The EcoRX site which served as cloning site was then digested and filled with the Klenow enzyme to produce the Xmnl site. The Xpnl-BamHI fragment contains the coding sequence of the sat3 gene. This fragment is obtained from the plasmid pJBsat3 construeted by cloning an Sspl-HindXI fragment derived from the plasmid pIE928 (Tietze et al., 1989) at the unique SmaX site of pK18 (Pridmore, 1987). The sequence of the CaMV 35S promoter extracted from the plasmid pBil21 (Clontech) following a HindXX-BamKX % digestion is contained in the construct pJBJ106, between the BamHI and SpeX site. The Spel-EcoRI fragment, which follows, consists of the right portion of the T-DNA of the plasmid pRi2659 derived from the plasmid pMOA9 (Brevet et al., 1988). This segment contains the entire functional gene for the synthesis of cucoomopine, as well as the right border of the T-DNA with its repeat 284 4 94 sequences (Hansen et al., 1992). The portion between 2?coRI and BglII is a fraction of the vector plasmid pK18. The entire fragment between the two terminal BglII sites was then ligated to the BglXT-BgllT segment of pBinl9 5 (Bevan, 1984) which contains the replication origin of this broad host spectrum plasmid derived from the plasmid RK2 and which has as bacterial selectable marker the kanamycin resistance gene. The latter construct is called pJBJ302.

The plasmid pJBJ3 02 was introduced into a strain of A. rhizogenes having 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/1. The transformants 15 were cultured on synthetic agar medium supplemented with mannopine as sole carbon source and with kanamycin (100 mg/1). A clone was selected and its plasmid content was checked by agarose gel electrophoresis, after a mini-extraction (on 5 ml of overnight culture at 28° in rich 20 medium supplemented with Km) according to the Kado method (Kado and Liu, 1981) but carried out at 37° in order to minimize breaks in the Ri plasmid. This plasmid pRi8196 serves both as donor of T-DNA inducing the formation of roots of the "hairy root" type, and provide the VIR 25 functions necessary for the transfer of the T-DNA construct carried by the binary vector. A bacterium carrying these two plasmids is capable, when it is inoculated into a plant, of causing differentiation of doubly transformed roots, containing both the "hairy root" T-DNA and that of 3 0 interest carried by the second plasmid. This clone was used to inoculate carrot discs (Petit et al., 1983). After two to three weeks, "hairy root" type roots appear. They are then excised from the carrot disc and cultured at 25° in the dark on MS sucrose (at 30 g/1) (Murashige and Skoog, 1962) agar medium free of growth hormone but supplemented with cephalosporin (250 mg/1) to inhibit any bacterial growth. On this medium, the roots have a growth of 2 to 5 mm in 24 hours and they show numerous ramifications. It is known that these roots constitute cell 284 4 94 , & 3 5 ' to CD an & I ; > ( <■&i ;u I CJC clones (David et al., 1984). Roots (about 1.5 cm in length) were transplanted on solid MS sucrose medium supplemented or otherwise with streptothricin (100 mg/1) . The roots containing only T-DNA from pRi8196 showed low growth over 24 hours and stopped growing. When replaced on streptothricin-free medium, their growth did not resume, showing the irreversible effect of the antibiotic. On the other hand, some roots showed substantial growth both on the medium supplemented with streptothricin and the antibiotic-free medium. These roots were considered to have integrated the construct containing the sat3 gene. Practically 70% to 80% of the roots proved to be capable of growing in the presence of streptothricin. This result is in good agreement with observations previously described (Petit et al., 1986).

In order to check that the roots growing in the presence of streptothricin indeed contained the construct, analyses, by paper electrophoresis, of the opine content of the roots, were carried out (Petit et al., 19 86). The roots incapable of growing on streptothricin contained only mannopine, a pRi8106 T-DNA marker, whereas all the roots capable of growing on streptothricin (20 independent roots analysed) contained both mannopine and cucumopine, the latter being a marker contained in the construct. This result indicates that the roots resistant to streptothricin integrated the construct. In order to confirm that these roots which synthesize cucumopine indeed had the sat 3 gene, analysis of genomic DNA extracted from the roots was undertaken by the PCR (Polymerase Chain Reaction) technique. Two oligonucleotides were synthesized. One* of the 20 bases has the sequence 5'-TCAATGCGTGAATTGGTCAT-3' situated at position 233-252 on the sequence, the other of 18 bases has the sequence 5'-GGATGCAGGCCACGATAC-3' situated at position 720-703 on the sequence. The use of these oligonucleotides in a PCR reaction makes it possible to amplify a DNA sequence of 488 base pairs covering practically the entire sat3 gene which can be detected after agarose gel electrophoresis, of the mixture subjected to the PCR I.

•~R i ^ !* rn (* o UJ oc 284494 reaction. The root genomic DNA was extracted by grinding 1 cm of root in 0.1 ml of sterile distilled water in Eppendorf type tubes and the tubes were incubated for 5 minutes on a boiling waterbath. After centrifugation, 1 fil of supernatant was added to 50 /il of conventional reaction mixture for PCR. The desired DNA was amplified over 3 0 cycles (1 cycle comprises the following incubations : 1 min at 94°, 1 min at 55° and 1.5 min at 72°). The results showed that only the genomic DNAs extracted from the roots synthesizing cucumopine made it possible to obtain the amplification of a fragment of 488 base pairs, confirming the presence of the sat3 gene.

In conclusion, the roots capable of growing on 100 mg/1 of streptothricin proved to be roots which were really transformed by the construct.

Example 2 Second experiment The plasmid pJBJ302 was introduced into the A. tumefaciens LBA4404 strain containing the plasmid pAi4404 (Ooms et al., 1982). This disarmed plasmid provides the VIR functions necessary for the transfer of any T-DNA. The transformed bacteria were selected on rich agar medium supplemented with kanamycin. The clo; es obtained were analysed as in the first experiment and a selected clone was used to inoculate foliar discs of Nicotiana tabacum va.. Xanthi according to the method of Horsch et al., 1985. 24 hours after the inoculation, the discs were transferred onto solid MS medium supplemented with 6-benzylaminopurine (BAP) (1 mg/1), naphthalene-acetic acid (NAA) (0.1 mg/1), cephalosporin (250 mg/1) and streptothricin (100 mg/1) to obtain plantlets derived from the transformed cells. Plantlets appeared after four ! weeks. They were transplanted on MS medium containing i .streptothricin. Their roots developed. After three weeks, the plants were individually propagated by cuttings on solid MS medium in a Magenta pot in order to obtain development of the plants. A leaf disc one centimetre in diameter was removed from each plant in order to evaluate its cucumopine content by electrophoresis (Petit et al., 284494 1986). Out of 50 plants analysed, 46 clearly showed the presence of cucumopine, which represents a percentage of 92%. The four plants which were apparently negative may represent false positives or plants in which cucumopine 5 synthase is too weakly expressed. In any case, a proportion of 10% of false positive plants would be perfectly acceptable and would reflect what is often observed with most selectable markers used to select transformed tissues. In order to confirm that the plants containing 10 cucumopine had also integrated the sat3 gene, their genomic DNA was extracted (Edwards et al., 1991) and analysed by the PCR technique as in the first experiment. DNA extracted from normal tobacco gives no signal whereas with the DNAs extracted from plants synthesizing cucumo-15 pine, the amplification of a DNA fragment of 488 base pairs was always found, confirming the presence of the sat3 gene.

In conclusion, the selection of transgenic tobaccos using the sat3 selectable marker proved to be 20 very efficient in at least 90% of the cases.

Example 3 Third experiment The A. tumefaciens strain used in the second experiment (strain LBA4404 containing the plasmid 25 pJBJ3 02) was used to transform the roots of Arabidopsis thaliana according to the method of Valvekens et al., (1988) but using streptothricin as selective agent in place of kanamycin. Two antibiotic concentrations, 25 mg/1 and 40 mg/1 were tested in parallel. After three 3 0 weeks, green plantlets appeared and were transplanted on GM medium (Vanvekens et al. , . 1988) supplemented with streptothricin (25 or 40 mg/1) in order to allow them to develop individually. Analysis of the opine content of these plantlets was carried out. Out of 10 plantlets to [77; 3jS~-i have grown on 25 mg/1 of streptothricin, 5 showed the presence of cucumopine whereas 14 out of 15 plantlets to have grown on 40 mg/1 of antibiotic were found to contain cucumopine. This result underlines the importance of using an optimum antibiotic concentration in order to —L id r" ' en Q en ' 'r— }— f— c__.

U.J Lj i CD rj -v' . r~> ; l_» _ 1 : 1 Si LU CC "1 Q I £i ; m i w i ill n 284 4 94 obtain a high probability of selecting only, or at least predominantly, transformed plants. With 40 mg/1 of streptothricin, 93% of the plants obtained had integrated the construct. In order to check that these plants indeed had the sat3 gene, their genomic DNA was extracted and served as template in PCR reactions as in the first experiment. The DNA of all the plants synthesizing cucumopine made it possible to amplify the typical DNA fragment of the sat3 gene of 488 base pairs whereas the DNA of the non-transformed plants gave no signal.

These results confirm that it is important to determine the optimum streptothricin concentration for each type of cells which it is desired to transform and that it is possible to use, under these conditions, the sat 3 gene to select tissues transformed with a high frequency.

Example 4 Fourth experiment Foliar discs one centimetre in diameter, sterilely cut with the aid of a metal punch, were removed either from normal tobaccos, or from tobaccos transformed with the sat3 gene, cultured in vitro. These discs were placed in Petri dishes containing MS20 agar medium supplemented with NAA (1 g/1) in order to induce the formation of roots. A batch of Petri dishes was supplemented with streptothricin (100 mg/1). The foliar discs were incubated at 248 under 16 hours of light per day. After fifteen days, roots appeared from the normal and trs-nsgenic 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 Bhowed no cell 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 sat3 gene and those lacking it. 28 4 4 9 4 - 13 c"! I I ' 1 —. to 0"5 CD T""« ! ; a : !JJ i*2 •' / CD 1 i i o V 1 UJ ! oz fv i < I LJ Example 5 Yeast cells and mammalian cells Other experiments intended to verify the efficiency of the sat3 gene for the selection of transformed cells of the yeast Schizosaccharomyces pombe were undertaken, according to a conventional transformation procedure with the construct described in Figure 2 in which the sat3 gene is placed under the control of the promoter of the cauliflower mosaic virus 35S RNA, which is known to be active in this yeast. These experiments made it possible to show the efficiency of the sat3 gene in Schizosaccharomyces pombe. The trains formation 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 early genes of the human cytomegalovirus (CMV) (used in the experiments described) , of the SV40 virus or of the Rous sarcoma virus (RSV) . It can also be placed under the control of the promoter of a gene which is expressed only in a given cell type. Experiments were carried out with a construct where the sat3 gene was placed under the control of the CMV promoter. Mouse mammary cells HC11 and hamster ovary cells CHO were transformed with this construct by the liposome technique described by Feigner et al. (1987). In both cases, it was possible to obtain a large number of transformed clones by selection in the presence of streptothricin.

These experiments show that the sat3 gene can be used to select transformed yeast and mammalian cells. Example 6 The aim of this experiment is to check that the sat3 gene is capable of being used to trap plant promoters . The strategy developed is based on that previously described (Koncz et al., 1989) where the aph(3')H (aminoglycoside phosphotransferase II) gene was used as selectable marker. The principle of the strategy is to place the sat3 gene, free of promoter, between the borders of an Agrobacterium rh.izogen.es T-DNA, the ATG 284494 codon for the initiation of translation of the selectable gene being placed in juxtaposition with the inner end of the right border of the T-DNA. If the integration of this T-DNA into the plant genome occurs in a promoter region, 5 the sat3 gene will be expressed. The transformed cells will acquire resistance to streptothricin and only they will be able to regenerate plantlets in the presence of the antibiotic. To test the hypothesis, the plasmid pJBJ332 was constructed. Only the essential part is 10 represented (Pig. 2). The remainder of the plasmid is no other than the pBinl9 type replicon part (Bevan, 1984), as in the case of the plasmid pJBJ3 02 which is used in the preceding experiments. The Bglll-Clal DNA segment, which contains the left border of the T-DNA of the 15 plasmid pRi2 659 is found on reading Figure 2 from the left to the right. It is derived from the plasmid pJBJ10 6 described above (Fig. 1) . The segment between Hindlll and EcoRI is no other than the multiple cloning site (MCS) obtained from the plasmid pUC19 (Yanisch-Perron et al. 20 19 85) . The region between EcoRI and Xhol contains the untranslated terminator part of nopaline synthase (nos-ter) , the sat3 gene, free of the promoter to which a XhoX site was added just upstream of the ATG for the initiation of translation. The Xhol-Bglll right region 25 contains the right border of the T-DNA of the plasmid pRi2659. The plasmid pUC18 (Yanish-Perron et al., 1985) was cloned into the plasmid pJBJ332, at the EcoRI site, to give the construct pJBJ333 (Fig. 2). pJBJ333 is a plasmid with a broad host spectrum 30 which was introduced, by electroporation, into the A. tumefaciens LBA4404 strain containing the plasmid Vir pAL4404 (Ooms et al., 1982). The transformed bacteria were selected on rich agar medium in the presence of Kanamycin. The clones obtained were analyzed for their i ,35 plasmid content and a clone was selected for the rest of ?. the experiment. It was used to infect 2 00 tobacco foliar cd ' -j i. discs. The regeneration and selection of the transformed u- o plantlets were carried out as in the second experiment ££[ described above. After 8 weeks, 13 well developed I I I P —J O LU CC 28 A A plantlets appeared among very numerous small plantlets with very weak development. They were transplanted on antibiotic-free MS medium in order to allow them to root rapidly. Foliar discs were removed in order to extract the genomic DNA (Edwards et al., 1991) and an analysis by the PCR technique was performed as in the first experiment. Nine plants gave a PCR signal which corresponds to the amplification expected for the sat3 gene.

In order to check that the sat3 gene is transcribed in the selected plants, the total RNA was extracted from 200 mg of leaf (Lokemann et al. 1987) and treated by digestion with RNase-free DNasel (Pharmacia) in order to remove any trace of DNA. DNAs complementary (cDNAs) to messenger RNAs were synthesized in vitro from 1 ng of total RNA, using a poly dT primer said the Boehringer kit called "First strand cDNA synthesis". A fraction of the reaction medium was used as template in a PCR reaction using the two oligonucleotides described above for the amplification of the sat3 gene. This method for searching for transcribed RNAs carries the abbreviated name of RT-PCR. The nine plants selected above gave amplification of the DNA fragment typical of the sat3 gene whereas the plants judged to be negative gave no DNA signal.

In order to check that the transformed plants expressed resistance to streptothricin, the 13 plants selected during the first stage were propagated by cutting in vitro on MS20 agar medium containing the antibiotic. Only the transformed plants developed roots in the presence of the selective agent, within a period of eight days.

The conclusion of this experiment is that the promoter-free sat3 gene, placed in juxtaposition with the right border of a T-DNA, can be expressed by a plant promoter following its integration into the plant genome and constitutes a marker capable of being used to trap such a promoter. Moreover, the pJBJ333 construct used contains, between the two borders, the origin of replication of the plasmid pUC18 and the gene for resistance 28 4 4 to ampicillin. This structure should make it possible to clone into E. coli, the plant DNA segment .containing the integration site as proposed by Koncz et al., (1989). 17 SEQUENCE LISTING 28449 (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: LVMH RECHERCHE (B) STREET: 25 RUE DES PEUPLIERS (C) CITY: NANTERRE (E) COUNTRY: FRANCE (F) POSTAL CODE (ZDP): 92000 (ii) TITLE OF INVENTION: PROCESS FOR SELECTING TRANSFORMED EUKARIOTIC CELLS AND CELLS OBTAINED (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: FR 9403969 (B) FILING DATE: 05-APR-1994 (2) INFORMATION FOR SEQ ID NO: 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1074 base pairs (B) TYPE: nucleotide 3 5 (C) STRANDEDNESS; double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) 40 (iv) ANTI-SENSE: YES (ix) CHARACTERISTIC : (A) NAME/KEY: CDS (B) LOCATION: 221.,760 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGCAGGAGC AGAAGAGCW ACATCTGGAA GCAAAGCCAG GAAAGCGGCC TATGGAGCTG 60 TGCGGCAGCG CTCAGTAGGC AATTTTTCAA AATATTGTTA AGCCTTTTCT GAGCATGGTA 120 TTTTTCATGG CATTGCTATG ATTTATTTTT TAAAACTATT GAAAATAAAG AAAATTATTA 180 TATTATTTAT AATAAAGGTC TGTGACAAGG AATCCCCGTC ATG ACG CCA CAG TCA 235 Met Thr Pro Gin Ser 1 5 l^f^ENToFfK^ L J*4 °cr m i ATG CGT GAA TTG GTC h7C TGT CGT GCA AGC GAT GCC GAC GTT CTT CAG Met Arg Glu Leu Val lie Cys Arg Ala Ser Asp Ala Asp Val Leu Gin 10 15 20 283 18 284 4 94 CTT GCG CGG TGC GAT TTC TCT TTC GAG GTC ACA GCT GAG CTC GAA GAG 331 Leu Ala Arg Cys Asp Phe Ser Phe Glu Val Thr Ala Glu Leu Glu Glu 25 30 35 CCG TTC GAT GAC ATG CGG TCC GTT CCA GTC AAG CCG CCC TAC CTC AAG 379 Pro Phe Asp Asp Met Arg Ser Val Pro Val Lys Pro Pro Tyr Leu Lys 40 45 50 AAC TAT GGC TTT GAT GCC GAT GAG TTG GTC GAG CAT ATG AAC AAC TCT 427 Asn Tyr Gly Phe Asp Ala Asp Glu Leu Val Glu His Met Asn Asn Ser 55 60 65 GCT GGG GCG TTG TTT GTG GCT CGG GCG GAC AAT TGC CTT GTT GGC TAC 475 Ala Gly Ala Leu Phe Val Ala Arg Ala Asp Asn Cys Leu Val Gly Tyr 70 75 80 85 TTG GCC GTG TCT CAA AGC TGG AAC GAA TAT GCC GTC ATC GAT GAT ATC 523 Lau Ala Val Ser Gin Ser Trp Asn Glu Tyr Ala Val lie Asp Asp lie 90 95 100 GCG GTC GAT GTG CCC TAT CGG GGG AGT GGC GTT TCG CGC TTG CTG ATG 571 Ala Val Asp Val Pro Tyr Arg Gly Ser Gly Val Ser Arg Leu Leu Met 105 110 115 GAT GCA GCT GTG GAC TGG GCA CGA AAT GTG CCG TCG GCA GGC GTA CGT 619 Asp Ala Ala Val Asp Trp Ala Arg Asn Val Pro Ser Ala Gly Val Arg 120 125 130 CTG GAG ACG CAG TCC GTT AAT CTC GCC GCA TGT CGC TTT TAC CGA CGA 667 Leu Glu Thr Gin Ser Val Asn Leu Ala Ala Cys Arg Phe Tyr Arg Arg 135 140 145 TAC GGT TTC CGG TTA GGT GGT TAT GAT CGC TAC CTG TAT CGT GGC CTG 715 Tyr Gly Phe Arg Leu Gly Gly Tyr Asp Arg Tyr Leu Tyr Arg Gly Leu 150 155 160 165 CAT CCG GGC AGC CGA GAG GTA GCT CTG TTC TGG TAT TTG AGT TTT TAA 763 His Pro Gly Ser Arg Glu Val Ala Leu Phe Trp Tyr Leu Ser Phe 170 175 180 ATGACAAACT TTGGCCGTCC GGGAAACGGC ACTCGGCCAG ATGAGCGGAG TTATGAATGA 823 GTGAGTGGCG ACGACAGCAA GTCTGTGCAC CTATTCCCAT GCCTGCGAGC ATGGCAACCA 883 GTCTGGAAGG ATACCAATGG GCGCCTATCA CAATTGGTGA ATCCGGCAGC AATGTTTATC 943 GACTTTATGG GAAACCAAAA GCTCCTGATT TGTTTTTGAA GCGAGGTAAG TACGACGTTG 1003 CTGATGATGT GACCGATGAA ATGGTCAGGC TACGCTGGCT TGCCGAACGT ATCCCTGTGC 1063 CAACCGTCGT C 1074 19 284 4 94 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 180 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Thr Pro Gin Ser Met Arg Glu Leu Val lie Cys Arg Ala Ser Asp 15 10 15 Ala Asp Val Leu Gin Leu Ala Arg Cys Asp Phe Ser Phe Glu Val Thr 20 25 30 Ala Glu Leu Glu Glu Pro Phe Asp Asp Met Arg Ser Val Pro Val Lys 35 40 45 Pro Pro Tyr Leu Lys Asn Tyr Gly Phe Asp Ala Asp Glu Leu Val Glu 50 55 60 His Met Asn Asn Ser Ala Gly Ala Leu Phe Val Ala Arg Ala Asp Asn 65 70 75 80 Cys Leu Val Gly Tyr Leu Ala Val Ser Gin Ser Trp Asn Glu Tyr Ala 85 90 95 Val lie Asp Asp lie Ala Val Asp Val Pro Tyr Arg Gly Ser Gly Val 100 105 110 Ser Arg Leu Leu Met Asp Ala Ala Val Asp Trp Ala Arg Asn Val Pro 115 120 125 Ser Ala Gly Val Arg Leu Glu Thr Gin Ser Val Asn Leu Ala Ala Cys 130 135 140 Arg Phe Tyr Arg Arg Tyr Gly Phe Arg Leu Gly Gly Tyr Asp Arg Tyr 145 150 155 160 Leu Tyr Arg Gly Leu His Pro Gly Ser Arg Glu Val Ala Leu Phe Trp 165 170 175 Tyr Leu Ser Phe 180 j"£f7 r'. r >•:.M f OFHvE 34 OCT 1996 I B£ejBVEp 2 8 4 4 i&T REFERENCES Bevan M. (1984). Nucl. Acida Res. 12:8711t8721.

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Claims (17)

CLAIMS I V"-">

1. Process for producing eukaryotic cells which are resistant to streptothricin or to its analogues, comprising the 5 following stages: transformation of the said eukaryotic cells with the aid of an expression vector for a DNA sequence encoding a streptothricin acetyl transferase (SAT) containing the DNA sequence encoding the said streptothricin acetyl transferase 10 (SAT-DNA) situated in a suitable orientation, and one or more

DNA sequences encoding the elements controlling the expression of the said SAT-DNA sequence in the said cells, and selection of the cells resistant to streptothricin or to its analogues after transformation. 15 2. Process according to Claim 1, characterized in that the sequence encoding streptothricin acetyl transferase is chosen from: a) the satl, satl and sat3, sat4 genes, b) the DNA sequences which hybridize under stringent conditions with the said genes and which encode a protein hiving a SAT activity, and 20 c) the DNA sequences which are obtained by degeneracy from sequences a) and b) and which encode a protein having a SAT activity.

3. Process according to either of Claims 1 and 2, characterized in that the SAT-DNA sequence is placed under the 25 control of a promoter which is functional in the transformed cell.

4. Process according to one of Claims 1 to 3, characterized in that the eukaryotic cell is a plant cell.

5. Process according to Claim 4, characterized in that the vector is derived from Agrobacterium. 30

6. Process according to Claim 5, characterized in that the vector is derived from Agrobacterium lumefaciens or Agrobacterium rhizogenes.

7. Process according to one of Claims 1 to 6, characterized in that the vector comprises, in addition, intellectual property office OF N.Z. 2 9 JUL 1998 RECEIVED - 23 284494 TTT •• i I ji ! ° i " i UJ j < Q_ K CO CO CF> \— CJ> cz> a set of DMA sequences allowing the transformed cell to express a character of interest.

8. Process according to Claim 7, characterized in that the character of interest is a function of agronomic 5 interest for the plant cells.

9. Process according to one of Claims 1 to 8, characterized in that the vector is an integrative vector.

10. Eukaryotic cell obtained by carrying out the 10 process according to one of Claims 1 to 9.

11. Plants obtained from cells according to Claim 10.

12. Use of the genes encoding a streptothricin acetyl transferase as selective marker in the transformation of plant tissues and subsequent plants. 15

13. Use of the genes encoding streptothricin acetyl transferase as selective marker for trapping a plant promoter.

14. Use according to Claim 13, characterized in that the gene is the sat3 gene. 20

15. DNA sequence encoding streptothricin acetyl transferase (SAT-DNA), chosen from: a) the sa t3 sequence attached to the present description, b) the DNA sequences which hybridize with the said gene 25 and encode a protein having a SAT activity, c) the DNA sequences which are obtained by degeneracy from sequences a) and b) and which encode a protein having a SAT activity.

16. Vector for the cloning and expression of a SAT in 3 0 an eukaryotic cell, characterized in that it contains: the DNA sequence encoding, the said streptothricin acetyl transferase (SAT-DNA) situated in a suitable orientation, and one or more DNA sequences encoding the elements controlling the expression of the said SAT-DNA sequence in the said cells. r-. ;

17. Vector according to Claim 16, characterized in ! that the elements controlling the expression are elements I of the host organism. jit I®* Vector according to Claim 17, characterized in 35 - 24 . 28 it is the plasmid PJBJ333. iN.Z. PATENT OFFICE} ^4 OCT 1996 RECEIVE' 284 4 PATENT OF INVENTION PROCESS FOR SELECTING TRANSFORMED EUKARYOTIC CELLS AND CELLS OBTAINED LVMH RECHERCHE DESCRIPTIVE ABSTRACT The invention, relates to a process for producing cells or clusters of eukaryotic, animal or plant, cells which are resistant to streptothricin or to its analogues, comprising the following stages: transformation of the said eukaryotic cells by a plasmid containing an exogenous DNA sequence encoding a streptothricin acetyl transferase (SAT) containing the DNA sequence encoding the said streptothricin acetyl transferase (SAT-DNA) situated in a suitable orientation, and one or more DNA sequences encoding the elements controlling the expression of the said SAT-DNA sequence in the said cells, and selection of the cells resistant to streptothricin or to its analogues after transformation. 04 OCT 1996 ■ ' •" ~-rT