Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants Number:7,112,665 from the United States Patent and Trademark Office (PTO) owispatent The invention relates to a DNA fragment containing a determined gene, the expression of which inhibits the antibiotic and herbicidal effects of Bialaphos and related products.It also relates to recombinant vectors, containing such DNA fragment, which enable this protective gene to be introduced and expressed into cells and plant cells.<H recombinants, Genetically, Genetically, eng, 7112665.html, Patent, pto, 7112665

Title: Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants

Abstract: The invention relates to a DNA fragment containing a determined gene, the expression of which inhibits the antibiotic and herbicidal effects of Bialaphos and related products.It also relates to recombinant vectors, containing such DNA fragment, which enable this protective gene to be introduced and expressed into cells and plant cells.

Patent Number: 7,112,665 Issued on 09/26/2006 to Leemans, et al.

Inventors: Leemans; Jan (Heusden, BE), Botterman; Johan (Zwijnaarde, BE), De Block; Marc (Gentbrugge, BE), Thompson; Charles (Grand Lancy/Geneve, CH), Mouva; Rao (Geneva, CH)
Assignee: Bayer BioScience N.V. (Ghent, BE)
Biogen, Inc. (Cambridge, MA)

Appl. No.: 08/465,219

Filed: June 5, 1995

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
07525300	May., 1990	5561236
07131140	Nov., 1987
PCT/EP87/00141	Mar., 1987

Foreign Application Priority Data


Mar 11, 1986 [GB]			86400521.0
Jan 21, 1987 [GB]			87400141.5

Current U.S. Class: 536/23.2 ; 536/23.7

Current International Class: C12N 15/52 (20060101); C12N 15/31 (20060101)

Field of Search: 435/172.3,172.1,240.1 800/205 536/27.1,23.4

References Cited [Referenced By]

U.S. Patent Documents


4769061	September 1988	Comai
5273894	December 1993	Strauch et al.
5276268	January 1994	Strauch et al.
5637489	June 1997	Strauch et al.
5648477	July 1997	Leemans et al.
5879903	March 1999	Strauch et al.

Foreign Patent Documents


0 173 327	Mar., 1986	EP
173327	Mar., 1986	EP
196375	Oct., 1986	EP
2007976	Oct., 1978	GB
84/02920	Aug., 1984	WO
84/20913	Aug., 1984	WO
86/02097	Apr., 1986	WO

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Primary Examiner: Kruse; David H
Attorney, Agent or Firm: Buchanan Ingersoll PC

Parent Case Text

This application is a divisional of application Ser. No. 07/525,300, filed May 17, 1990, now U.S. Pat. No. 5,561,236, which is a continuation of application Ser. No. 07/131,140, filed Nov. 5, 1987, abandoned, which is a continuation under 35 USC .sctn..sctn.363 and 120 of PCT/EP 87/00141, filed Mar. 11, 1987, published as WO 87/05629.

Claims

The invention claimed is:

1. An isolated DNA encoding a protein having phosphinothricin acetyltransferase activity, or a variant thereof retaining said activity, said protein comprising the amino acid sequence (SEQ ID No. 1): X Ser Pro Glu Arg Arg Pro Ala Asp Ile Arg Arg Ala Thr Glu Ala Asp MET Pro Ala Val Cys Thr Ile Val Asn His Tyr Ile Gln Thr Ser Thr Val Asn Phe Arg Thr Glu Pro Gln Glu Pro Gln Glu Trp Thr Asp Asp Leu Val Arg Leu Arg Gln Arg Tyr Pro Trp Leu Val Ala Glu Val Asp Gly Glu Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg Asn Ala Tyr Asp Trp Thr Ala Glu Ser Thr Val Tyr Val Ser Pro Arg His Gln Arg Thr Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys Ser Leu Glu Ala Gln Gly Phe Lys Ser Val Val Ala Val Ile Gly Leu Pro Asn Asp Pro Ser Val Arg Met His Glu Ala Leu Gly Tyr Ala Pro Arg Gly Met Leu Arg Ala Ala Gly Phe Lys His Gly Asn Trp His Asp Val Gly Phe Trp Gln Leu Asp Phe Ser Leu Pro Val Pro Pro Arg Pro Val Leu Pro Val Thr Glu Ile in which X is Met or Val, said DNA consisting of between 549 and 625 nucleotides.

2. The isolated DNA of claim 1, consisting of the nucleotide sequence (SEQ ID No. 2 from nucleotide 2 to nucleotide 549): NTG AGC CCA GAA CGA CGC CCG GCC GAC ATC CGC CGT GCC ACC GAG GCG GAC ATG CCG GCG GTC TGC ACC ATC GTC AAC CAC TAC ATC GAG ACA AGC ACG GTC AAC TTC CGT ACC GAG CCG CAG GAA CCG CAG GAG TGG ACG GAC GAC CTC GTC CGT CTG CGG GAG CGC TAT CCC TGG CTC GTC GCC GAG GTG GAC GGC GAG GTC GCC GGC ATC GCC TAC GCG GGC CCC TGG AAG GCA CGC AAC GCC TAC GAC TGG ACG GCC GAG TCG ACC GTG TAC GTC TCC CCC CGC CAC CAG CGG ACG GGA CTG GGC TCC ACG CTC TAC ACC CAC CTG CTG AAG TCC CTG GAG GCA CAG GGC TTC AAG AGC GTG GTC GCT GTC ATC GGG CTG CCC AAC GAC CCG AGC GTG CGC ATG CAC GAG GCG CTC GGA TAT GCC CCC CGC GGC ATG CTG CGG GCG GCC GGC TTC AAG CAC GGG AAC TGG CAT GAC GTG GGT TTC TGG CAG CTG GAC TTC AGC CTG CCG GTA CCG CCC CGT CCG GTC CTG CCC GTC ACC GAG ATC in which N is A or G.

Description

The invention relates to a process for protecting plant cells and plants against the action of glutamine synthetase inhibitors.

It also relates to applications of such process, particularly to the development of herbicide resistance into determined plants.

It relates further to non-biologically transformed plant cells and plants displaying resistance to glutamine synthetase inhibitors as well as to suitable DNA fragments and recombinants containing nucleotide sequences encoding resistance to glutamine synthetase inhibitors.

Glutamine synthetase (hereafter simply designated by GS) constitutes in most plants one of the essential enzymes for the development and life of plant cells. It is known that GS converts glutamate into glutamine. GS is involved in an efficient pathway (the only one known nowadays) in most plants for the detoxification of ammonia released by nitrate reduction, aminoacid degradation or photorespiration. Therefore potent inhibitors of GS are very toxic to plant cells. A particular class of herbicides has been developped, based on the toxic effect due to inhibit inhibition of GS in plants.

These herbicides comprise as active ingredient a GS inhibitor.

There are at least two possible ways which might lead to plants resistant to the inhibitors of the action of glutamine synthetase; (1) by changing the target. It can be envisaged that mutations in the GS enzyme can lead to insensitivity towards the herbicide; (2) by inactivation of the herbicide. Breakdown or modification of the herbicide inside the plant could lead to resistance.

Bialaphos and phosphinothricin (hereafter simply designated by PPT) are two such inhibitors of the action of GS, (ref. 16, 17) and have been shown to possess excellent herbicidal properties (see more particularly ref. 2 as concerns Bialaphos).

Bialaphos has the following formula (I)

##STR00001##

PPT has the following formula (II)

##STR00002##

Thus the structural difference between PPT and Bialaphos resides in the absence of two alanine aminoacids in the case of PPT.

These two herbicides are non selective. They inhibit growth of all the different species of plants present on the soil, accordingly cause their total destruction.

Bialaphos was first disclosed as having antibiotic properties, which enabled it to be used as a pesticide or a fungicide. Bialaphos can be produced according to the process disclosed in U.S. Pat. No. 3,832,394, assigned to MEIJI SEIKA KAISHA LTD., which patent is incorporated herein by reference. It comprises cultivating Streptomyces hygroscopicus, such as the strain available at the American Type Culture Collection, under the ATCC number 21,705, and recovering Bialaphos from its culture medium. However, other strains, such as Streptomyces viridochromogenes, also produce this compound (ref. 1).

Other tripeptide antibiotics which contain a PPT moiety are or might be discovered in nature as well, e.g. phosalacin (ref. 15).

PPT is also obtained by chemical synthesis and is commercially distributed by the industrial Company HOECHST.

A number of Streptomyces species have been disclosed which produce highly active antibiotics which are known to incapacitate procaryotic cell functions or enzymes. The Streptomyces species which produce these antibiotics would themselves be destroyed if they had not a self defence mechanism against these antibiotics. This self defence mechanism has been found in several instances to comprise an enzyme capable of inhibiting the antibiotic effect, thus of avoiding autotoxicity for the Streptomyces species concerned. This modification is generally reversed when the molecule is exported from the cell.

The existence of a gene which encodes an enzyme able to modify the antibiotic so as to inhibit the antibiotic effect against the host has been demonstrated in several Streptomyces producing antibiotics, for example, in S. fradiae, S. azureus, S. vinaceus, S. erythreus, producing neomycin, thiostrepton, viomycin, and MLS (Macrolide Lincosamide Streptogramin) antibiotics respectively (ref. 4), (ref. 5), (ref. 6),(ref. 14 by CHATER et al., 1982 describes standard techniques which can be used for bringing these effects to light).

In accordance with the present invention, it has been found that Streptomyces hygroscopicus ATCC 21,705, also possesses a gene encoding an enzyme responsible of the inactivation of the antibiotic properties of Bialaphos. Experiments carried out by the applicants have lead to the isolation of such a gene and its use in a process for controlling the action of GS inhibitors, based on PPT or derived products.

An object of the invention is to provide a new process for controlling the action in plant cells and plants of GS inhibitors.

Another object of the invention is to provide DNA fragments and DNA recombinants, particularly modified vectors containing said DNA fragments, which DNA fragments contain nucleotide sequences capable, when incorporated in plant cells and plants, to protect them against the action of GS inhibitors.

A further object of the invention is to provide non-biologically transformed plant cells and plants capable of neutralizing or inactivating GS inhibitors.

A further object of the invention is to provide a process for selectively protecting plant species against herbicides of a GS inhibitor type.

More specifically an object of the invention is to provide a DNA fragment transferable to plant cells- and to whole plants--capable of protecting them against the herbicidal effects of Bialaphos and of structurally analogous herbicides.

A further object of the invention is to provide plant cells resistant to the products of the class examplified by Bialaphos, which products possess the PPT unit in their structure.

The process according to the invention for controlling the action in plant cells and plants of a GS inhibitor when contacted therewith, comprises providing said plants with a heterologous DNA fragment including a foreign nucleotide sequence, capable of being expressed in the form of a protein in said plant cells and plants, under condition such as to cause said heterologus DNA fragment to be integrated stably through generations in the cells of said plants, and wherein said protein has an enzymatic activity capable of inactivating or neutralization of said glutamine synthetase inhibitor.

A preferred DNA fragment is one derived from an antibiotic-producing-Streptomyces strain (or a sequence comprising a nucleotide sequence encoding the same activity) and which encodes resistance to a said GS inhibitors.

Preferred nucleotide sequences for use in this invention encode a protein which has acetyl tranferase activity with respect to said GS inhibitors.

A most preferred DNA fragment according to the invention comprises a nucleotide sequence coding for a polypeptide having a PPT acetyl transferase activity.

A particular DNA fragment according to the invention, for the subsequent transformation of plant cells, consists of a nucleotide sequence coding for at least part of a polypeptide having the following sequence (SEQ ID NO:1):

TABLE-US-00001 X SER PRO GLU 183 ARG ARG PRO ALA ASP ILE ARG ARG ALA THR GLU ALA ASP MET PRO 228 ALA VAL CYS THR ILE VAL ASN HIS TYR ILE GLU THR SER THR VAL 273 ASN PHE ARG THR GLU PRO GLN GLU PRO GLN GLU TRP THR ASP ASP 318 LEU VAL ARG LEU ARG GLU ARG TYR PRO TRP LEU VAL ALA GLU VAL 363 ASP GLY GLU VAL ALA GLY ILE ALA TYR ALA GLY PRO TRP LYS ALA 408 ARG ASN ALA TYR ASP TRP THR ALA GLU SER THR VAL TYR VAL SER 453 PRO ARG HIS GLN ARG THR GLY LEU GLY SER THR LEU TYR THR HIS 498 LEU LEU LYS SER LEU GLU ALA GLN GLY PHE LYS SER VAL VAL ALA 543 VAL ILE GLY LEU PRO ASN ASP PRO SER VAL ARG MET HIS GLU ALA 588 LEU GLY TYR ALA PRO ARG GLY MET LEU ARG ALA ALA GLY PHE LYS 633 HIS GLY ASN TRP HIS ASP VAL GLY PHE TRP GLN LEU ASP PHE SER 678 LEU PRO VAL PRO PRO ARG PRO VAL LEU PRO VAL THR GLU ILE 723

in which X represents MET or VAL, which part of said polypeptide is of sufficient length to confer protection against Bialaphos to plant cells, when incorportated genetically and expressed therein, i.e. as termed hereafter "plant-protecting capability" against Bialaphos.

A preferred DNA fragment consists of the following nucleotide sequence (SEQ ID NO:2):

TABLE-US-00002 GTG AGC CCA GAA 183 CGA CGC CCG GCC GAC ATC CGC CGT GCC ACC GAG GCG GAC ATG CCG 228 GCG GTC TGC ACC ATC GTC AAC CAC TAC ATC GAG ACA AGC ACG GTC 273 AAC TTC CGT ACC GAG CCG CAG GAA CCG CAG GAG TGG ACG GAC GAC 318 CTC GTC CGT CTG CGG GAG CGC TAT CCC TGG CTC GTC GCC GAG GTG 363 GAC GGC GAG GTC GCC GGC ATC GCC TAC GCG GGC CCC TGG AAG GCA 408 CGC AAC GCC TAC GAC TGG ACG GCC GAG TCG ACC GTG TAC GTC TCC 453 CCC CGC CAC CAG CGG ACG GGA CTG GGC TCC ACG CTC TAC ACC CAC 498 CTG CTG AAG TCC CTG GAG GCA CAG GGC TTC AAG AGC GTG GTC GCT 543 GTC ATC GGG CTG CCC AAC GAC CCG AGC GTG CGC ATG CAC GAG GCG 588 CTC GGA TAT GCC CCC CGC GGC ATG CTG CGG GCG GCC GGC TTC AAG 633 CAC GGG AAC TGG CAT GAC GTG GGT TTC TGG CAG CTG GAC TTC AGC 678 CTG CCG GTA CCG CCC CGT CCG GTC CTG CCC GTC ACC GAG ATC 723

or of a part thereof expressing a polypeptide having plant-protecting capability against Bialaphos.

The invention also relates to any DNA fragment differing from the preferred one indicated hereabove by the replacement of any of its nucleotides by others, yet without modifying the genetic information of the preferred DNA sequence mentioned hereabove (normally within the meaning of the universal genetic code), and furthermore to any equivalent DNA sequence which would encode a polypeptide having the same properties, particularly a Bialaphos-resistance-activity.

It will be understood that the man skilled in the art should be capable of readily assessing those parts of the nucleotide sequences that could be removed from either side of any of the DNA fragments according to the invention, for instance by removing terminal parts on either side of said DNA fragment, such as by an exonucleolytic enzyme, for instance Bal31, by recloning the remaining fragment in a suitable plasmid and by assaying the capacity of the modified plasmid to transform appropriate cells and to protect it against the Bialaphos antibiotic or herbicide as disclosed later, whichever assay is appropriate.

For the easiness of language, these DNA fragments will be termed hereafter as "Bialaphos-resistance DNA". In a similar manner, the corresponding polypeptide will be termed as "Bialaphos-resistance enzyme".

While in the preceding discussion particular emphasis has been put on DNA fragments capable, when introduced into plant cells and plants, to confer on them protection against Bialaphos or PPT, it should be understood that the invention should in no way be deemed as limited thereto.

In a same manner, the invention pertains to DNA fragments which, when introduced into such plant cells, would also confer on them a protection against other GS inhibitors, for instance of intermediate products involved in the natural biosynthesis of phosphinotricin, such as the compounds designated by the abbreviations MP101 (III), MP102 (IV), the formula of which are indicated hereafter:

##STR00003##

##STR00004##

More generally, the invention has opened the route to the production of DNA fragments which, upon proper incorporation into plant cells and plants, can protect them against GS inhibitors when contacted therewith, as this will be shown in a detailed manner in relation to Bialaphos and PPT in the examples which will follow.

This having been established, it will be appreciated that any fragment encoding an enzymatic activity which would protect plant cells and plants against said GS inhibitors, by inactivationg, should be viewed as an equivalent of the preferred fragments which have been disclosed hereabove. This would apply especially to any DNA fragments that would result from genetic screening of the genomic DNAs of strains, particularly of antibiotic-producing strains, likely to possess genes which, even-though structurally different, would encode similar activity with respect to Bialaphos or PPT, or even with respect to other GS inhibitors. This applies to any gene in other strains producing a PPT derivative.

Therefore, it should be understood that the language "Bialaphos-resistance DNA" or "Bialaphos-resistance enzyme" used thereafter as a matter of convenience is intended to relate not only to the DNAs and enzymes specifically concerned with resistance to PPT or most directly related derivatives, but more generally with other DNAs and enzymes which would be capable, under the same circumstances, of inactivating the action in plants of GS inhibitors.

The invention also relates to DNA recombinants containing the above defined Bialaphos-resistance DNA fragments recombined with heterologous DNA, said heterologous DNA containing regulation elements and said Bialaphos-resistance DNA being under the control of said regulation elements in such manner as to be expressible in a foreign cellular environment compatible with said regulation elements. Particularly the abovesaid Bialaphos-resistance-DNA fragments contained in said DNA recombinants are devoid of any DNA region involved in the biosynthesis of Bialaphos, when said Bialaphos-resistance-DNA fragment originate themselves from Bialaphos-producing strains.

By "heterologous DNA" is meant a DNA of an other origin than that from which said Bialaphos-resistance-DNA originated, e.g. is different from that of a Streptomyces hygroscopicus or Streptomyces viridochromogenes or even more preferably a DNA foreign to Streptomyces DNA. Particularly said regulation elements are those which are capable of controlling the transcription and translation of DNA sequences normally associated with them in said foreign environment. "Cellular" refers both to microorganisms and to cell cultures.

This heterologous DNA may be a bacterial DNA, particularly when it is desired to produce a large amount of the recombinant DNA, such as for amplification purposes. In that respect a preferred heterologous DNA consists of DNA of E. coli or of DNA compatible with E. coli. It may be DNA of the same origin as that of the cells concerned or other DNA, for instance viral or plasmidic DNA known as capable of replicating in the cells concerned.

Preferred recombinant DNA contains heterologous DNA compatible with plant cells, particularly Ti-plasmid DNA.

Particularly preferred recombinants are those which contain GS inhibitor inactivating DNA under the control of a promoter recognized by plant cells, particularly those plant cells on which inactivation of GS inhibitors is to be conferred.

Preferred recombinants according to the invention further relate to modified vectors, particularly plasmids, containing said GS-inhibitor-inactivating DNA so positioned with respect to regulation elements, including particularly promoter elements, that they enable said GS inhibitor-inactivating DNA to be transcribed and translated in the cellular environment which is compatible with said heterologous DNA. Advantageous vectors are those so engineered as to cause stable incorporation of said GS inhibitor inactivating DNA in foreign cells, particularly in their genomic DNA. Preferred modified vectors are those which enable the stable transformation of plant cells and which confer to the corresponding cells, the capability of inactivating GS inhibitors.

It seems that, as described later, the initiation codon of the Bialaphos-resistance-gene of the Streptomyces hygroscopicus strain used herein is a GTG codon. But in preferred recombinant DNAs or vectors, the Bialaphos-resistance-gene is modified by substitution of an ATG initiation codon for the initiation codon GTG, which ATG enables translation initiation in plant cells.

In the example which follows, the plant promoter sequence which has been used was constituted by a promoter of the 35 S cauliflower mosaic virus. Needless to say that the man skilled in the art will be capable of selecting other plant promoters, when more appropriate in relation to the plant species concerned.

According to an other preferred embodiment of the invention, particularly when it is desired to achieve transport of the enzyme encoded by the Bialaphos-resistance-DNA into the chloroplasts, the heterologous DNA fragment is fused to a gene or DNA fragment encoding a transit peptide, said last mentioned fragment being then intercalated between the GS inhibitor inactivating gene and the plant promoter selected.

As concerns means capable of achieving such constructions, reference can be made to the following British applications 84 32757 filed on Dec. 28, 1984 and 85 00336 filed on Jan. 7, 1985 and to the related applications filed in the United-States of America (No. 06/755,173, filed on Jul. 15, 1985), in the European Patent Office (No. 85 402596.2, filed on Dec. 20, 1985), in Japan (No. 299 730, filed on Dec. 27, 1985), in Israel (No. 77 466 filed on Dec. 27, 1985) and in Australia (No. 5 165 485, filed on Dec. 24, 1985), all of which are incorporated herein by reference.

Reference can also be made to the scientific literature, particularly to the following articles:

VAN DEN BROECK et al., 1985, Nature, 313, 358 363;

SCHREIER and al., Embo. J., vol. 4, No. 1, 25 32.

These articles are also incorporated herein by reference.

For the sake of the record, be it recalled here that under the expression "transit peptide", one refers to a polypeptide fragment which is normally associated with a chloroplast protein or a chloroplast protein sub-unit in a precursor protein encoded by plant cell nuclear DNA. The transit peptide then separates from the chloroplast protein or is proteolitically removed, during the translocation process of the latter protein into the chloroplasts. Examples of suitable transit peptides are those associated with the small subunit of ribulose-1,5 biphosphate (RuBP) carboxylase or that associated with the chlorophyl a/b binding proteins.

There is thus provided DNA fragments and DNA recombinants which are suitable for use in the process defined hereafter.

More particularly the invention also relates to a process, which can be generally defined as a process for producing plants and reproduction material of said plants including a heterologous genetic material stably integrated therein and capable of being expressed in said plants or reproduction material in the form of a protein capable of inactivating or neutralizing the activity of a glutamine synthetase-inhibitor, comprising the non biological steps of producing plants cells or plant tissue including said heterologous genetic material from starting plant cells or plant tissue not able to express that inhibiting or neutralizing activity, regenerating plants or reproduction material of said plants or both from said plant cells or plant tissue including said genetic material and, optionally, biologically replicating said last mentioned plants or reproduction material or both, wherein said non-biological steps of producing said plant cells or plant tissue including said heterologous genetic material, comprises transforming said starting plant cells or plant tissue with a DNA-recombinant containing a nucleotide sequence encoding said protein, as well as the regulatory elements selected among those which are capable of enabling the expression of said nucleotide sequence in said plant cells or plant tissue, and to cause the stable integration of said nucleotide sequence in said plant cells and tissue, as well as in the plant and reproduction material processed therefrom throughout generations.

The invention also relates to the cell cultures containing Bialaphos-resistance-DNA, or more generally said GS-inhibitor-inactivating DNA, which cell cultures have the property of being resistant to a composition containing a GS inhibitor, when cultured in a medium containing a such composition at dosages which would be destructive for non transformed cells.

The invention concerns more particularly those plant cells or cell cultures in which the Bialaphos-resistance DNA is stably integrated and which remains present over successive generations of said plant cells. Thus the resistance to a GS inhibitor, more particularly Bialaphos or PPT, can also be considered as a way of characterizing the plant cells of this invention.

Optionally one may also resort to hybridization experiments between the genomic DNA obtained from said plant cells with a probe containing a GS inhibitor inactivating DNA sequence.

More generally the invention relates to plant cells, reproduction material, particularly seeds, as well as plants containing a foreign or heterologous DNA fragment stably integrated in their respective genomic DNAs, said fragments being transferred throughout generations of such plant cells, reproduction material, seeds and plants, wherein said DNA fragment encodes a protein inducing a non-variety-specific enzymatic activity capable of inactivating or neutralizing GS inhibitors, particularly Bialaphos and PPT, more particularly to confer on said plant cells, reproduction material, seeds and plants a corresponding non-variety-specific phenotype of resistance to GS inhibitors.

"Non-variety-specific" enzymatic activity or phenotype aims at referring to the fact that they are not characteristic of specific plant genes or species as this will be illustrated in a non-limitative way by the examples which will follow. They are induced in said plant materials by essentially non-biological processes applicable to plants belonging to species normally unrelated with one another and comprising the incorporation into said plant material of heterologous DNA, e.g. bacterial DNA or chemically synthesized DNA, which does not normally occur in said plant material or which normally cannot be incorporated therein by natural breeding processes, and which yet confers a common phenotype (e.g. herbicide resistance) to them.

The invention is of particular advantageous use in processes for protecting field-cultivated plant species against weeds, which processes comprise the step of treating the field with an herbicide, e.g. Bialaphos or PPT in a dosage effective to kill said weeds, wherein the cultivated plant species then contains in their genome a DNA fragment encoding a protein having an enzymatic activity capable of neutralizing or inactivating said GS inhibitor.

By way of illustration only, effective doses for use in the abovesaid process range from about 0.4 to about 1.6 kg/Hectare of Bialaphos or PPT.

There follows now a disclosure of how the preferred DNA fragment described hereabove was isolated starting from the Streptomyces hygroscopicus strain available at the American Type Culture Collection under deposition number ATCC 21 705, by way of exemplification only.

The following disclosure also provides the technique which can be applied to other strains producing compounds with a PPT moiety.

The disclosure will then be completed with the description of the insertion of a preferred DNA fragment conferring to the transformed cells the capability of inactivating Bialaphos and PPT. Thus the Bialaphos-inactivating-DNA fragment designated thereafter by Bialaphos-resistance gene or "sfr" gene, isolated by the above described technique into plasmids which can be used for transforming plant cells and conferring to them a resistance against Bialaphos, also merely by way of example for non-limitative illustration purposes.

The following disclosure is made with reference to the drawings in which:

FIG. 1 is a restriction map of a plasmid containing a Streptomyces hygroscopicus DNA fragment encoding Bialaphos-resistance, which plasmid, designated hereafter as pBG1 has been constructed according to the disclosure which follows;

FIG. 2 shows the nucleotide sequence (SEQ ID NO:12) of a smaller fragment obtained from pBG1, subcloned into another plasmid (pBG39) and containing the resistance gene;

FIG. 3 shows the construction of a series of plasmids given by way of example, which plasmids aim at providing suitable adaptation means for the insertion therein of the Bialaphos-resistance gene or "sfr" gene;

FIGS. 4A and 4B show the construction of a series of plasmids given by way of example, which plasmids contain suitable plant cell promoter sequences able to initiate transcription and expression of the foreign gene inserted under their control into said plasmids;

FIG. 5A shows a determined fragment of the nucleotide sequence (SEQ ID NO:13) of the plasmid obtained in FIG. 3;

FIG. 5B shows the reconstruction of the first codons of a Bialaphos-resistance gene, from a FokI/BglII fragment obtained from pBG39 and the substitution of an ATG initiation codon for the GTG initiation codon of the natural "sfr" gene (SEQ ID NO:14);

FIG. 5C shows the reconstruction of the entire "sfr" gene, namely the last codons thereof (SEQ ID NO:15), and its insertion into a plasmid obtained in FIGS. 4A and 4B;

FIG. 6A shows an expression vector containing the "sfr" gene placed under the control of a plant cell promoter;

FIG. 6B shows another expression vector deriving from the one shown in FIG. 6A, by the substitution of some nucleotides.

FIG. 7 shows the construction of a series of plasmids given by way of examples, to ultimately produce plasmids containing the promoter region and the transit peptide sequence of a determined plant cell gene, for the insertion of the "sfr" gene under the control of said promoter region and downstream of said transit peptide sequence (SEQ ID NOS:16 and 17).

FIGS. 8 to 11 will be referred to hereafter.

The following experiment was set up to isolate a Bialaphos-resistance-gene from S. hygroscopicus, according to standard techniques for cloning into Streptomyces.

2.5 .mu.g of S. hygroscopicus genomic DNA and 0.5 .mu.g of Streptomyces vector pIJ61 were cleaved with PstI according to the method described in ref. 6. The vector, fragments and genomic fragments were mixed and ligated (4 hours at 10.degree. C. followed by 72 hours at 4.degree. C. in ligation salts which contain 66 mM Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM MgCl.sub.2, 10 mM 2-mercaptoethanol and 0.1 mM ATP) at a total DNA concentration of 40 .mu.g ml.sup.-1 with T4 DNA ligase. Ligation products were introduced into 3.times.10.sup.9 S. lividans strain 66 protoplasts by a transformation procedure mediated by polyethylene-glycol (PEG) as described hereafter. These protoplasts gave rise to 5.times.10.sup.7 colonies and 4.times.10.sup.4 pocks after regeneration on 20 plates of R2 agar containing 0.5% of Difco yeast extract (R2 YE). Preparation and composition of the different mediums and buffers used in the disclosed experiments are described hereinafter. When these lawns were replica-plated on minimal medium plates containing 50 .mu.g ml.sup.-1 Bialaphos, drug resistant colonies appeared at a frequency of 1 per 10.sup.4 transformants. After purification of the drug resistant colonies, there plasmid DNA was isolated and used to retransform S. lividans protoplasts. Non selective regeneration followed by replication to Bialaphos-containing-medium demonstrated a 100% correlation between pocks and Bialaphos resistant growth. The recombinant plasmids of several resistant clones all contained a 1.7 Kb PstI insert (see FIG. 1).

Subcloning of the Herbicide Resistance Gene

The 1.7 Kb PstI insert was then subcloned into the high copy number streptomycete vector pIJ385 to generate plasmid pBG20. S. lividans strains which contained pBG20 were more than 500 times more resistant to Bialaphos. S. lividans growth is normally inhibited in minimal medium containing 1 .mu.g/ml Bialaphos; growth of transformants containing pBG20 was not noticeably inhibited in a medium containing 500 .mu.g/ml Bialaphos. The PstI fragment was also subcloned in either orientation into the PstI site of the plasmid pBR322, to produce plasmids pBG1 and pBG2, according to their orientation. A test on minimal M9 medium demonstrated that E. coli E8767 containing pBG1 or pBG2 was resistant to Bialaphos.

A .+-.1.65 Kb PstI-BamHI fragment was subcloned from pBG1 into the plasmid pUC19 to produce the plasmid pBG39, and conferred Bialaphos resistance to E. coli, W3110, C600 and JM83.

Using an in vitro coupled transcription-translation system (ref. 5) from S. lividans extracts, the 1,65 Kb PstI-BamHI fragment in pBG39 was shown to direct the synthesis of a 22 Kd protein. In the following, this 1,65 Kb insert includes a fragment coding for a 22 Kd protein and will be called "sfr" gene.

Fine Mapping and Sequencing of the Gene

A 625 bp Sau3A fragment was subcloned from pBG39 into pUC19 and still conferred Bialaphos resistance to a E. coli W3110 host. The resulting clones were pBG93 and pBG94, according to the orientation.

The orientation of the gene in the Sau3A fragment was indicated by experiments which have shown that Bialaphos resistance could be induced with IPTG from the pUC19 lac promoter in pBG93. In the presence of IPTG (0.5 mM) the resistance of pBG93/W3110 increased from 5 to 50 .mu.g/ml on a M9 medium containing Bialaphos. The W3110 host devoid of pBG93, did not grow on M9 medium containing 5 .mu.g/ml Bialaphos. These experiments demonstrated that the Sau3A fragment could be subcloned without loss of activity. They also provided for the proper orientation as shown in the FIG. 2, enclosed thereafter. The protein encoded by these clones was detected by using coupled transcription-translation systems derived from extracts of S. lividans (ref. 7). Depending on the orientation of the Sau3A fragment, translation products of different sizes were observed; 22 Kd for pBG94 and .+-.28 Kd for pBG93. This indicated that the Sau3A fragment did not contain the entire resistance gene and that a fusion protein was formed which included a polypeptide sequence resulting from the translation of a pUC19 sequence.

In order to obtain large amounts of the protein, a 1.7 Kb PstI fragment from pBG1 was cloned into the high copy number Streptomycete replicon pIJ385. The obtained plasmid, pBG20, was used to transform S. hygroscopicus. Transformants which contained this plasmid had more than 5 times as much PPT acetylating activity and also had increased amounts of a 22 kd protein on sodium dodecylsulfate gels (SDS gels). Furthermore, both the acetyl transferase and the 22 kd protein appeared when the production of Bialaphos begun. The correlation of the in vitro data, kinetics of synthesis, and amplified expression associated with pBG20 transformants strongly implied that this 22 Kd band was the gene product.

The complete nucleotide sequence of the 625 bp Sau3A fragment was determined as well as of flanking sequences. Computer analysis revealed the presence of an open reading frame over the entire length of the Sau3A fragment.

Characterization of the sfr Gene Product

A series of experiments were performed to determine that the open reading frame of the "sfr" gene indeed encoded the Bialaphos resistance enzyme. To determine the 5' end of the resistance gene, the NH.sub.2-terminal sequence of the enzyme was determined. As concerns more particularly the technique used to determine the said sequence, reference is made to the technique developed by J. VANDEKERCKHOVE, Eur. J. Bioc. 152, p. 9 19, 1985, and to French patent applications No. 85 14579 filed on Oct. 1, 1985 and No. 85 13046 filed on September 2, 1985, all of which are incorporated herein by reference.

This technique allows the immobilization on glass fibre sheets coated with the polyquaternary amine commercially available under the registered trademark POLYBRENE of proteins and of nucleic acids previously separated on a sodium dodecylsulfate containing polyacrylamide gel. The transfer is carried out essentially as for the protein blotting on nitrocellulose membranes (ref. 8). This allows the determination of amino-acid composition and partial sequence of the immobilized proteins. The portion of the sheet carrying the immobilized 22 kd protein produced by S. hygroscopicus pBG20 was cut out and the disc was mounted in the reaction chambre of a gas-phase sequenator to subject the glass-fibre bound 22 Kd protein to the Edman degradation procedure. The following amino-acid sequence was obtained SEQ ID NO:4):

Pro-Glu-Arg-Arg-Pro-Ala-Asp-Ile-Arg-Arg

This sequence matched an amino-acid sequence which was deduced from the open reading frame of the 625 bp Sau3A fragment. It corresponded to the stretch from codon 3 to codon 12.

Thus, the NH.sub.2-terminus of the 22 Kd protein was upstream of this sequence. It was determined that translation of the actual protein was likely to be initiated 2 amino-acids earlier at a GTG initiation codon. GTG is often used as initiator codon in Streptomyces and translated as methionine. The protein translated from the GTG initiation codon would be 183 amino-acids long and would have a molecular weight of 20 550. This was in good agreement with the observed approximate molecular weight of 22 000.

Furthermore, the termination codon, TGA, was located just downstream of the Sau3A site. Cloning of the 625 bp Sau3A fragment in a BamHI site digested pUC19 did not result in the reconstruction of the termination codon. This explained the fusion proteins which were observed in the in vitro transcription-translation analysis.

Mechanism of PPT-Resistance

Having defined a first phenotype and some of the physical characteristics of the resistance gene and its gene product, a series of experiments was then carried out to understand the mechanism by which it confers resistance. As described hereabove, PPT is the portion of Bialaphos which inhibits glutamine synthetase (GS) and that N-acetyl PPT is not an inhibitor. Using a standard assay (ref. 9), S. hygroscopicus ATCC 21 705 derivates were shown to contain a PPT acetyl transferase which was not found in S. lividans. The activity does not acetylate the Bialaphos tripeptide. S. lividans carrying the resistance gene cloned in pBG20 or pBG16 (a plasmid containing the 625 bp Sau3A fragment cloned into another streptomycete vector, pIJ680) also contained the activity which could acetylate PPT but not Bialaphos. The PPT derived reaction product produced by extracts of pBG20/S. lividans was isolated in order to confirm that it was indeed acetyl-PPT. Analysis by mass spectroscopy showed that the molecular weight had increased relative to PPT by the equivalent of one acetyl group. It was thus concluded that the 625 bp Sau3A fragment contained sequences which code for PPT acetyl transferase.

The experimental conditions and reagents used in the techniques disclosed hereabove were as follows:

Preparation and Composition of the Mediums and Buffers Above Used

1.degree. P medium: 10.3 g of sucrose, 0.025 g of K.sub.2SO.sub.4, 0.203 g of MgCl.sub.2.6H.sub.2O and 0.2 ml of a trace element solution are dissolved in 80 ml of distilled water and autoclaved. Then in order, 1 ml of KH.sub.2PO.sub.4 (0.5%), 10 ml of CaCl.sub.2, 2H.sub.2O (3.68%), and 10 ml of TES buffer (0.25 M), pH: 7.2) are added. Trace element solution (per liter): ZnCl.sub.2, 40 mg; FeCl.sub.3.6H.sub.2O, 200 mg; CuCl.sub.2.2H.sub.2O, 10 mg; MnCl.sub.2.4H.sub.2O, 10 mg; Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 10 mg; (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O, 10 mg.

2.degree. R2YE: 10.3 g of sucrose, 0.025 g of K.sub.2SO.sub.4, 1.012 g of MgCl.sub.2.6H.sub.2O, 1 g of glucose, 0.01 g of Difco 25 casamino acids, and 2.2 g of Difco agar are dissolved in 80 ml distilled water and autoclaved. 0.2 ml of trace element solution, 1 ml of KH.sub.2PO.sub.4 (0.5%), 8.02 ml of CaCl.sub.2.2H.sub.2O (3.68%), 1.5 ml of L-proline (20%), 10 ml of TES buffer (0.25 M) (pH: 7.2), 0.5 ml of (1 M) NaOH, 5 ml of yeast extract (10%) are sequentially added.

3.degree. TE: 10 mM TRIS HCl, 1 mM EDTA, pH 8.0.

4.degree. YEME: Difco yeast extract (0.3%), Difco peptone (0.5%), oxoid malt extract (0.3%), glucose (1%).

Transformation of S. lividans Protoplasts

1. A culture composed of 25 ml YEME, 34% sucrose, 0.005 M MgCl.sub.2, 0.5% glycine, in a 250 ml baffled flask, is centrifuged during 30 to 36 hours.

2. The pellet is suspended in 10.3% sucrose and centrifuged. This washing is repeated once.

3. The mycelium is suspended in 4 ml lysozyme solution (1 mg/ml in P medium with CaCl.sub.2 and MgCl.sub.2 concentrations reduced to 0.0025 M) and incubated at 30.degree. C. for 15 to 60 minutes.

4. The solution is mixed by pipetting three times in a 5 ml pipette and incubated for further 15 minutes.

5. P medium (5 ml) is added and mixed by pipetting as in step 4.

6. The solution is filtered through cotton wool and protoplasts are gently sedimented in a bench centrifuge at 800.times.G during 7 minutes.

7. Protoplasts are suspended in 4 ml P medium and centrifuged again.

8. Step 7 is repeated and protoplasts are suspended in the drop of P medium left after pouring off the supernatant (for transformation).

9. DNA is added in less than 20 .mu.l TE.

10. 0.5 ml PEG 1 000 solution (2.5 g PEG dissolved in 7.5 ml of 2.5% sucrose, 0.0014 K.sub.2SO.sub.4, 0.1 M CaCl.sub.2, 0.05 M TRIS-maleic acid, pH 8.0, plus trace elements) is immediately added and pipetted once to mix the components.

11. After 60 seconds, 5 ml of P medium are added and the protoplasts are sedimented by gentle centrifugation.

12. The pellet is suspended in P medium (1 ml).

13. 0.1 ml is plated out on R2YE plates (for transformation dry plates to 85% of their fresh weigh e.g. in a laminar flow cabinet).

14. Incubation at 30.degree. C.

A--Construction of a "sfr" Gene Cassette

A "sfr" gene cassette was constructed to allow subsequent cloning in plant expression vectors.

Isolation of a FokI-BglII fragment from the plasmid pBG39 containing a "sfr" gene fragment led to the loss of the first codons, including the initiation codon, and of the last codons, including the stop codon.

This fragment of the "sfr" gene could be reconstructed in vitro with synthetic oligonucleotides which encode appropriate amino-acids.

The complementary synthetic oligonucleotides were (SEQ ID NOS:5 6) 5'-CATGAGCCCAGAAC and 3'-TCGGGTCTTGCTGC.

By using such synthetic oligonucleotides, the 5' end of the "sfr" gene could be reformed and the GTG initiation codon substituted for a codon well translated by plant cells, particularly an ATG codon.

The DNA fragment containing the oligonucleotides linked to the "sfr" gene was then inserted into an appropriate plasmid, which contained a determined nucleotide sequence thereafter designated by an "adapter" fragment.

This adapter fragment comprised:

a TGA termination codon which enabled the last codons of the "sfr" gene to be reformed;

appropriate restriction sites which enabled the insertion of the fragment of the nucleotide sequence comprising the "sfr" gene partially reformed with the synthetic oligonucleotides; this insertion resulted in the reconstruction of an intact "sfr" gene;

appropriate restriction sites for the isolation of the entire "sfr" gene.

The "sfr" gene was then inserted into another plasmid, which contained a suitable plant promoter sequence. The plant promoter sequence consisted of the cauliflower mosaic virus promoter sequence (p35S). Of course the invention is not limited to the use of this particular promoter. Other sequences could be chosen as promoters suitable in plants, for example the TR 1' 2' promoter region and the promoter fragment of a Rubisco small subunit gene from Argbidopsis thaliana hereafter described.

1.degree. Construction of the Plasmid pLK56.2 (FIG. 3)

The construction of plasmid pLK56.2 aimed at obtaining a suitable adaptor including the following sequence of restriction sites: SmaI, BamHI, NcoI, KpnI, BglII, MluI, BamHI, HindIII and XbaI.

The starting plasmids used for this construction, pLK56, pJB64 and pLK33 were those disclosed by BOTTERMAN (ref. 11).

The DNA fragments hereafter described were isolated and separated from low melting point agarose (LGA).

The plasmid pLK56 was cleaved by the enzymes BamHI and NdeI. A NcoI-NdeI fragment (referred to in the drawings by arc "a" in broken line) obtained from plasmid pJB64 was substituted in pLK56 for the BamHI-NdeI fragment shown at "b". Ligation was possible after filling in the BamHI and NcoI protruding ends with the DNA polymerase I of E. coli (Klenow's fragment).

Particularly recircularization took place by means of a T4 DNA ligase. A new plasmid pLK56.3 was obtained.

This plasmid was cleaved by the enzymes XbaI and PstI.

The BamHI-PstI fragment of pLK33 (c) (on FIG. 3) was substituted for the XbaI-PstI fragment (d) of pLK56.3, after repairing of their respective ends by Klenow's fragment.

After recircularization by means of the T4 DNA ligase, the obtained plasmid pLK56.2 contained a nucleotide sequence which comprised the necessary restriction sites for the subsequent insertion of the "sfr" gene.

2.degree. Construction of the Plasmid pGSH150 (FIG. 4A)

Parallel with the last discussed construction, there was produced a plasmid containing a promoter sequence recognized by the polymerases of plant cells and including suitable restriction sites, downstream of said promoter sequence in the direction of transcription, which restriction sites are then intented to enable the accomodation of the "sfr" gene then obtainable from pLK56.2, under the control of said plant promoter.

Plasmid pGV825 is described in DEBLAERE et al. (ref. 10). Plasmid pJB63 is from BOTTERMAN (ref. 11).

pGV825 was linearized with PvuII and recircularized by the T4 DNA ligase, resulting in the deletion of an internal PvuII fragment shown at (e), (plasmid pGV956).

pGV956 was then cleaved by BamHI and BglII.

The BamHI-HindIII fragment (f) obtained from pJB63 was dephosphorylated with calf intestine phosphatase (CIP) and substituted for the BamHI-BglII fragment of pGV956.

Plasmid pGV1500 was obtained after recircularization by means of T4 DNA ligase.

An EcoRI-HindIII fragment obtained from plasmid pGSH50 was purified. The latter plasmid carried the dual TR 1' 2' promoter fragment described in VELTEN et al., (ref. 13). This fragment was inserted in pGV1500, digested with HpaI and HindIII and yielded pGSH150.

This plasmid contains the promoter fragment in front of the 3' end of the T-DNA transcript 7 and a BamHI and ClaI sites for cloning.

3.degree. Construction of the Plasmid pGSJ260 (FIG. 4B)

CP3 is a plasmid derived from pBR322 and which contains the 35S promoter region of cauliflower mosaic virus within a BamHI fragment.

pGSH150 was cut by BamHI and BglII.

The BamHI-BglII fragment (h) of CP3, which contained the nucleotide sequence of p35S promoter, was substituted for the BamHI-BglII fragment (i) in pGSH150 to form plasmid pGSJ250. pGSJ250 was then opened at its BglII restriction site.

A BamHI fragment obtained from mGV2 (ref. 12) was inserted in pGSJ250 at the BglII site to form plasmid pGSJ260.

However prior to inserting the "sfr" gene obtainable from pLK56.2 into plasmid pGSJ260, it was still desirable to further modify the first in order to permit insertion in a more practical manner. Thus pLK56.2 was further modified as discussed below to yield pGSR1.

Starting from plasmid pGSJ260, two plasmid constructions for subsequent transformations of plant cells were made:

a first plasmid permitting the expression of the "sfr" gene in the cytoplasm of plant cells, and

a second plasmid so modified as to achieve transport of the Bialaphos-resistance enzymes to the chloroplasts of plant cells.

First Case: Plasmid Enabling the Expression of the "sfr" Gene in the Cytoplasm of Plant Cells

Cloning of the sfr Gene Cassette in a Plant Expression Vector (pGSR2) (FIG. 5)

On FIG. 5A (SEQ ID NO:13), the nucleotide sequence of the adapter of pLK56.2 is shown. In particular, the locations of BamHI, NcoI, BglII restriction sites are shown.

This adapter fragment was cleaved by the enzymes NcoI and BglII.

FIG. 5B (SEQ ID NO:14) shows the FokI-BglII fragment (j) obtained from pBG39. The locations of these two restriction sites are shown on FIG. 2.

Using synthetic oligonucleotides, the first codons of the "sfr" gene were reformed, particularly the 5' end of the gene in which a ATG initiation codon was substituted for the initial GTG codon.

This FokI-BglII fragment completed with the synthetic oligonucleotides was then substituted in pLK56.2 for the NcoI-BglII fragment of the adapter. The 3' end of the gene was thus reformed too, after recircularization with T4 DNA ligased. The plasmid obtained, pGSR1, thus contained the entire "sfr" gene inserted in its adapter.

The plasmid pGSJ260 was then opened by BamHI (FIG. 5C) and the BamHI fragment obtained from pGSR1, which contained the entire "sfr" gene, was inserted into pGSJ260.

The obtained plasmid, pGSR2 (see FIG. 6A) contained a pBR322 replicon, a bacterial streptomycin resistance gene (SDM-SP-AD-transferase) and an engineered T-DNA consisting of:

the border fragments of the T-DNA;

a chimeric kanamycin gene which provided a dominant selectable marker in plant cells; and

a chimeric "sfr" gene.

The chimeric "sfr" gene consisting of:

the promoter region of the cauliflower mosaic virus (p35S);

the "sfr" gene cassette as described in FIG. 5;

the 3' untranslated region, including the polyadenylation signal of T-DNA transcript 7.

pGSR2 was introduced into Agrobacterium tumefaciens recipient C58ClRif.RTM. (pGV2260) according to the procedure described by DEBLAERE et al. (ref. 10).

This strain was used to introduce the chimeric "sfr" gene in N. tabacum SR.sub.1 plants.

Two variant plasmids deriving from pGSR2, namely pGSFR280 and pGSFR281, have been constructed. They differ in the untranslated sequence following the transcription initiation site. In pGSR2, this fragment consists of the following sequence (SEQ ID NO:7):

GAGGACACGCTGAAATCACCAGTCTCGGATCCATG;

while it consists of (SEQ ID NO:8):

GAGGACACGCTGAAATCACCAGTCTCTCTACAAATCGATCCATG

in pGSR280 and of (SEQ ID NO:9)

GAGGACACGCTGAAATCACCAGTCTCTCTACAAATCGATG

in pGSFR281, with an ATG codon being the initiation codon of the "sfr" gene. The "sfr" gene is also fused to the TR1' 2' promoter in the plasmid pGSH150 (FIG. 4A) yielding pGSFR160 and pGSFR161 (FIG. 6B). These plasmids contain slight differences in the pTR2 "sfr" gene configuration: the "sfr" gene is correctly fused to the endogenous gene 2' ATG in pGSFR161 (for sequences see ref. 13), whereas 4 extra base pairs (ATCC) are present just ahead of the ATG codon in pGSFR160. Otherwise, plasmids p65FR161 and p65FR160 are completely identical.

All plasmids are introduced in Agrobacterium by cointegration in the acceptor plamids pGV2260 yielding the respective plasmids pGSFR1280, pGSFR1281, pGSFR1160 and pGSFR1161.

Second Case: Construction of a Plasmid Containing the "sfr" Gene Downstream of a DNA Sequence Encoding a Transit Peptide and Suitable for Achieving Subsequent Translocation of the "sfr" Gene Expression Product into Plant-Cell-Chloroplasts

In another set of experiments, the nucleotide sequence which contained the "sfr" gene was fused to a DNA sequence encoding a transit peptide so as to enable its transport into chloroplasts.

A fragment of the "sfr" gene was isolated from the adapter fragment above described and fused to a transit peptide. With synthetic oligonucleotides, the entire "sfr" gene was reconstructed and fused to a transit peptide.

The plasmid (plasmid pATS3 mentioned below) which contained the nucleotide sequence encoding the transit peptide comprised also the promoter sequence thereof.

Construction of the Plasmid pGSR4 which Contains the "sfr" Gene Fused to a DNA Sequence Encoding Transit Peptide (FIG. 7)

Plasmid pLK57 is from BOTTERMAN, (ref. 11). Plasmid pATS3 is a pUC19 clone which contains a 2 Kb EcoRI genomic DNA fragment from Arabidopsis thaliana comprising the promoter region and the transit peptide nucleotide sequence of the gene, the expression thereof is the small subunit of ribulose biphosphate carboxylase (ssu). The A. thaliana small subunit was isolated as a 1 500 bp EcoRI-SphI fragment. The SphI cleavage site exactly occurs at the site where the coding region of the mature ssu protein starts.

Plasmids pLK57 and pATS3 were opened with EcoRI and SphI. After recircularization by means of the T4 DNA ligase, a recombinant plasmid pLKAB1 containing the sequence encoding the transit peptide (Tp) and its promoter region (Pssu) was obtained.

In order to correctly fuse the "sfr" gene at the cleavage site of the signal peptide, the N-terminal gene sequence was first modified. Since it was observed that N-terminal gene fusions with the "sfr" gene retain their enzymatic activity, the second codon (AGC) was modified to a GAC, yielding an NcoI site overlapping with the ATG initiator site. A new plasmid, pGSSFR2 was obtained. It only differs from pGSR1 (FIG. 5B), by that mutation. The NcoI-BamHI fragment obtained from pGSFR2 was fused at the SphI end of the transit peptide sequence. In parallel, the "sfr" gene fragment was fused correctly to the ATG initiator of the ssu gene (not shown in figures).

Introduction of the "sfr" Gene into a Different Plant Species

The Bialaphos-resistance induced in plants by the expression of chimeric genes, when the latter have been transformed with appropriate vectors containing said chimeric genes, has been demonstrated as follows. The recombinant plasmids containing the "sfr" gene were introduced separately by mobilization into Agrobacterium strain C58C.sub.1 Rif.RTM. (pGV2260) according to the procedure described by DEBLAERE and al., Nucl. Acid. Res., 13, p. 1 477, 1985. Recombinant strains containing hybrid Ti plasmides were formed. These strains were used to infect and transform leaf discs of different plant species, according to a method essentially as described by HORSH and al., 1985, Science, vol. 227. Transformation procedure of these different plant species given by way of example, is described thereafter.

1. Leaf Disc Transformation of Nicotiana tabacum

Used Media are described thereafter:

TABLE-US-00003 A.sub.1 MS salt/2 +1% sucrose 0.8% agar pH 5.7 A.sub.10 B5-medium +250 mg/l NH.sub.4NO.sub.3 750 mg/l CaCl.sub.2 2H.su