CA1337597C - Phosphinotricin-resistance gene, and its use - Google Patents
Phosphinotricin-resistance gene, and its useInfo
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- CA1337597C CA1337597C CA000545037A CA545037A CA1337597C CA 1337597 C CA1337597 C CA 1337597C CA 000545037 A CA000545037 A CA 000545037A CA 545037 A CA545037 A CA 545037A CA 1337597 C CA1337597 C CA 1337597C
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- ptc
- gene
- resistance
- ptt
- acetylation
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
- C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
- C12N15/8277—Phosphinotricin
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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Abstract
Selection of Streptomyces viridochromogenes DSM 4112 for resistance to phosphinothricyl-alanyl-alanine (PTT) results in PTT-resistance selectants. The DNA fragment which carries the phosphinothricin(PTC)-resistance gene is obtained from the total DNA of these selectants by cutting with BamHI, cloning of a fragment 4.0 kb in size, and selection for PTT resistance. This gene is suitable for the production of PTC-resistant plants, and as a resistance marker and for the selective N-acetylation of the L-form of racemic PTC.
Description
, I--Specification Phosphinothricin-resistance gene, and its use Phosphinothricin (PTC, 2-amino-4-methylphosphinobutyric acid)~is an inhibitor of glutamine synthetase. PTC is a "structural unit" of the antibiotic phosphinothricyl-alanyl-alanine. This tripeptide (PTT) is active against Gram-positive and Gram-negative bacteria as well as against the fungus ~otrytis cinerea (Bayer et al., Helv.
Chim. Acta 55 (1972) 224). PTT is produced by the strain Streptomyces viridochromogenes Tu 494 (DSM 40736, DSM
4112).
German Patent 2,717,440 (issuQt April 5, 1984, Assigned to Hoechst AG) discloses that PTC acts as a total herbicide The published PCT
Application W0 86/02097 (published April 10, 1986, Applic~nt The General Hospital Corporation) describes plants whose resistance to PTC is attributable to o~erproduction of glutamine synth-tase C~eL~.oduction of this type, for example resulting from gene amplification, entsils th-risks of instability Hence, such an instability would be associated with a decreas- in th- O~eL~ Gduction of glutamin- synthetase, and th-competiti~e inhibitory action of PTC would reapp-ar In contrast, the invention, ~hich is defined in the patent claims, relates to a PTC-resistance gene and to its use for the production of PTC-resistant plants. In addition, this gene can also be used as a resistance marker.
Furthermore, the gene is suitable for the selective N-acetylation of the L-form of racemic PTC.
- lA - 1 337597 Further details of the invention are described below with the help of the examples illustrated in the accompanying drawings in which:
Figure 1 shows the restriction map of the DNA sequence wherein the PTC-resistance gene is located;
Figure 2 shows the restriction map of plasmid pEB2;
Figure 3 shows the restriction map of plasmid pPRl; and Figure 4 shows the restriction map of DNA sequence 1.
The PTC-resistance gene according to the invention can be obtained by cutting, with BamHI, the total DNA from Streptomyces viridochromogenes DSM 4112 which has been selected for PTT resistance, by cloning a fragment 4.0 kb in size, and by selection for PTT resistance. The restriction map (figure 1) details the characteristics of this 4.0 kb fragment.
Cloning experiments on sections of this 4 kb fragment were carried out to localize the position of the coding region more accurately. It emerged from this that the resistance gene is located on the 1.6 kb SstII-SstI fragment (positions 0.55 to 2.15 in Fig. 1). Digestion with BglII
resulted in the fragment which is 0.8 kb in size and which, after incorporation into a plasmid and transformation of S.
lividans, confers PTT resistance. This resistance is caused by N-acetylation of PTC.
Chim. Acta 55 (1972) 224). PTT is produced by the strain Streptomyces viridochromogenes Tu 494 (DSM 40736, DSM
4112).
German Patent 2,717,440 (issuQt April 5, 1984, Assigned to Hoechst AG) discloses that PTC acts as a total herbicide The published PCT
Application W0 86/02097 (published April 10, 1986, Applic~nt The General Hospital Corporation) describes plants whose resistance to PTC is attributable to o~erproduction of glutamine synth-tase C~eL~.oduction of this type, for example resulting from gene amplification, entsils th-risks of instability Hence, such an instability would be associated with a decreas- in th- O~eL~ Gduction of glutamin- synthetase, and th-competiti~e inhibitory action of PTC would reapp-ar In contrast, the invention, ~hich is defined in the patent claims, relates to a PTC-resistance gene and to its use for the production of PTC-resistant plants. In addition, this gene can also be used as a resistance marker.
Furthermore, the gene is suitable for the selective N-acetylation of the L-form of racemic PTC.
- lA - 1 337597 Further details of the invention are described below with the help of the examples illustrated in the accompanying drawings in which:
Figure 1 shows the restriction map of the DNA sequence wherein the PTC-resistance gene is located;
Figure 2 shows the restriction map of plasmid pEB2;
Figure 3 shows the restriction map of plasmid pPRl; and Figure 4 shows the restriction map of DNA sequence 1.
The PTC-resistance gene according to the invention can be obtained by cutting, with BamHI, the total DNA from Streptomyces viridochromogenes DSM 4112 which has been selected for PTT resistance, by cloning a fragment 4.0 kb in size, and by selection for PTT resistance. The restriction map (figure 1) details the characteristics of this 4.0 kb fragment.
Cloning experiments on sections of this 4 kb fragment were carried out to localize the position of the coding region more accurately. It emerged from this that the resistance gene is located on the 1.6 kb SstII-SstI fragment (positions 0.55 to 2.15 in Fig. 1). Digestion with BglII
resulted in the fragment which is 0.8 kb in size and which, after incorporation into a plasmid and transformation of S.
lividans, confers PTT resistance. This resistance is caused by N-acetylation of PTC.
Maxam and Gilbert sequencing of the 0.8 kb fragment reveals DNA
sequence I (Annex). The position of the resistance gene can be determined from the open reading frame of this sequence (from position 258). The end of the gene is located at the penultimate nucleotide shown (position 806), i.e. the last nucleotide (position 807) is the first of the stop codon.
The Shine-Dalgarno sequence in DNA sequence I is emphasized by underlining, as is the GTG acting as start codon. This GTG codon is expressed as methionine. Thus, the top line depicts the definitive reading frame.
DNA sequence II shows the restriction sites within the sequenced gene. Enzymes which cut the sequence more than six times are not indicated.
The antibiotic PTT is taken up by bacteria and broken down to PTC. The latter also inhibits glutamine synthetase in bacteria, so that the bacteria die of a lack of glutamine. Hence, PTT-producing bacteria ought to have a mechanism which protects them from the action of PTT, that is to say either prevents reuptake of the PTT which has been produced or permits a modification of the breakdown product PTC. However, surprisingly, the ~`
` - 3 - l 3 37 5 97 PTT producer S. viridochromogenes DSM 4112 is sensitive ~ to its own antibiotic. Unexpectedly, it proved possible, ho~ever, by selection for PTT resistance to find, at the surprisingly high rate of 10 5, selectants which are resistant to PTT and, moreover, suppress the background growth of adjacent colonies.
A gene bank was set up from the DNA of these selectants by isolating the DNA and cleaving it with BamHI and ligating it into a Streptomycetes vector. The ligation mixture was transformed into the commercially available strain S. lividans TK 23, resulting in about 5000 to 10000 transformants having an insert of about 1 to 5 kb per 1 ~9 of ligation mixture. Among the transformants there ~ere PTT-resistant S. lividans strains. It was possible, by isolation of the plasmids and retransform-ation into S. lividans, to show that the resistance is plasmid-coded. The gene responsible for the resistance is located on a 4 kb BamHI fragment (Figure 1). The coding region is located on the 0.8 kb BglII fragment.
The BamHI fragment contains no cleavage sites for the enzymes ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PvuI, PvuII and _ I.
Comparison with the restriction map of a resistance gene, which has not been characterized in detail, for S.
hygroscopicus FERM BP-130/ATCC 21705 (European Patent Application with the publication no. 0,173,327, Figure 7) shows that the resistance gene according to the invention differs from the known gene, which was found during the search for PTT biosynthesis genes.
It was possible to show, by incubation of cell extracts from S. viridochromogenes DSM 4112 and S. lividans TK 23 on the one hand, and the PTT-resistant S. viridochromogenes selectants and a plasmid-carrying S. lividans transform-ant, on the other hand, with PTC and acetyl-coenzyme A
that the latter cells have acetylating activity.
Chromatography tests sho~ that the acetylation takes place on the amino group.
Since PTT-resistance has also been found in E. coli, and thus the resistance mechanism also functions in Gram-negative bacteria, it is possible to rule out resistancebased on transport phenomena. Thus, after coupling to plant promoters and using suitable vectors, the resistance gene according to the invention can be transformed into plants, and in this ~ay PIC-resistant plants can be produced.
The N-acetylation of PTC can also be used for racemate resolution of synthetic D,L-PTC since selective acetyl-ation of only the L-form takes place.
Thus the invention also relates to the use of the resis-tance gene for the selective N-acetylation of the L-form of racemic PTC.
The PTC acetyltransferase coded for by the resistance gene according to the invention can thus be used to separate racemic PTC, as can be obtained, for example, by the method of German Patent 2,717,440, into the optical antipodes by exposing the racemate to the acetylating action of this enzyme, since there is selective attack on the L-form vhile the D-form remains unchanged. The mixture thus obtained can then be fractionated in a manner knovn per se on the basis of the differences in properties.
The contacting of N-acyl-D,L-amino acids vith acylases, vhich are immobilised on carriers ~here appropriate, vith selective liberation of the L-amino acid, vhich can be extracted vith water-immiscible solvents from the mixture vith the N-acyl-D-amino acid after acidification, has be-n discloset (British Patent 1,369,462, p~bl~sh~d Octob-r 9, 1974, Inventors Nartin Cole and Renneth Utting). A correspQn~ng fractionation of N-acyl-D,L-PTC is tisclosed, for example, in Germ~n Offenlegungsschrift 2,939,269 (publishQd April 30, 1980, Applicant Nei~i Seika Raisha, Ltt.) 1 33~597 - or U S Patent No 4,226,941 (Issued October 7, 1980, Assignet to Mei~i Seika Raisha, Ltd ) The D-PCT which remains according to the invention can be racemized in kno~n manner (European Published Specification No 0 137 371, published April 17, 1983, Applicant Hoechst Aktiengesellgchaft, example 8), and then returned to the process.
It is possible, but not necessary, to isolate the enzyme, this also being intended to mean, here and hereinafter, always the enzymatically active part If the enzyme is isolated, it can be used in ths free form or the form immobilized on a carrier Examples of suitable carriers are described in European Patent Application No 84 111 208 9 (published unter No 0 141 223 on May 15, 1985, Applicant Hoechst AG) Hc~e~a., it is aApe~ent not to isolate the enzyme but to use any desired PTC-resistant cells which express the enzyme according to the invention. Thus, it is possible and expedient to use the PTT-resistant selectants of S. viridochromogenes DSM 4112.
Moreover, it is possible and advantageous to use any desired cell ~hich has been transformed ~ith the gene according to the invention and ~hich is able to express PTC acetyltransferase. In this connection, the gene according to the invention, this also being intended to mean active parts thereof, can be introduced into the host cell in plasmid-integrated form or by using other customary methods of gene manipulation, for example by transfection. For example, incorporation into a E. coli expression plasmid and transformation of E. coli with such a plasmid is expedient, for example by the methods ~noun from ~o~n Pllbl~R~d Specification No 0 163 249 (p~hl~shed April 12, 1985, Applicant Hoecbst AG) ant European Publishet Specification No 0 171 024 (ptlbl~shed February 12, 1986, Applicant Hoechst AG) For the N-acetylation, according to th- invention, of L-PTC in the racemate th-e cells which express PTC acetyltransferase can be uset in tbe free or immobilized form, with- the customary methods of immobilisation being used (for example German Offenlegungssc_rift No 32 37 341 (publish-d April 12, 1984, Applicant Hoechst AG) ant literatur- cit-t therein) - 6 - ~ 3~ 59~
he enzymatic acetylation, according to the invention, of L-PTC is carried out in the manner customary for enzymatic reactions, ~ith the conditions of the method being governed by the characteristics of the organism used. In principle, methods suitable for this are the same as for the abovementioned selective deacylation method.
The invention is illustrated in detail in the examples ~hich follow. Unless otherwise stated, parts and percentage data relate to weight.
Example 1: P~I-resistant selectants The strain S. viridochromogenes DSM 4112 was cultured on minimal medium (Hop~ood et al., Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Nor~ich, England (1985), page 233) and increas-ing concentrations of PTT ~ere added. At a concentration of 100 ~g/ml one resistant colony ~as found per 105 colonies, approximately.
Example 2: Preparation of the vector The plasmid pSVH1 (Eu~ops-n Patent 0,070,522, Issued Octob-r 29, 1986, Agsignee Hoechst AG; U S Patent 4,673,642, Issued June 16, 1987, Assignee Hoechst AG) is cut with BglII, ant th- fragm-nt about 7 1 kb in slze is isolatet ant ligatet ~ith th- 1 1 kb BclI fragment having thiostrepton- resistanc- (EuLop~an Patent Appl~c~ti~ 85 103 460 3 publishet October 16, 1985 unter Publ~at~on No 0 158 201) The plasmid pEB2 which is about 8 15 kb in siz- is obtain-d (Figure 2) Example 3: Isolation of the resistance gene The total DNA is isolated from the selectants obtained in example 1, and it is cleaved ~ith ~amHI. The plasmid pE~2 is like~ise opened ~ith ~amHI, and the t~o mixtures are combined and ligated. The ligation m;xture is trans-formed into S. lividans TK 23 (obtainable from the ,~ . .
John Innes Foundation), ~ith 5ûO0 to 10000 transformants having an insert of about 1 - 5 kb being obtained per 1 ~9 of ligation mixture. Selection for PTT-resistance produces t~o resistant S. lividans colonies. The plasmid ~hich has been taken up is isolated from the latter and is cut ~ith BamH~. A 4 kb BamHI fragment ~hich carries the gene responsible for resistance is found. This plasmid ~as called pPR1 (figure 3).
Retransformation ;nto S. lividans TK 23 shows, that the PTT-resistance is plasmid-coded, since the transformants gro~ on minimal medium containing 100 ~g/ml PTT.
Example 4: Demonstration of the inactivation of PTC by ; N-acetylation The follo~ing strains were examined to demonstrate the acetylating activity of the cloned fragment: S. viri-dochromogenes DSM 40736, S. viridochromogenes (PTT-resistant mutant), S. lividans TK23 and S. lividans TK 23 (pPR1).
This entails the strains being inoculated into lysis medium A (European Published Specification 0 158 872, published October 23, 1985, Applicant Hoechst AG, page 6) and incubated at 30C in an orbital shaker for 2 days. After harvesting, 1 mg of mycelium is disrupted with ultrasound in a suitable buffer (for example RS buffer: C.J. Thompson et al., J. sacteriol. 151 (1982), 678-685). The procedure for a typical experiment to measure PTC breakdown is as follows:
100 ~l of PTC solution (250 ~g/ml) and 50 ~l of acetyl-CoA (4 mg/ml) are added to 250 ~l of crude extract, and the mixture is incubated at 30C for 2 hours. The amounts of PTC which are still present after this time are measured by HPLC. The results of this are as - 8 - l 337 5~7 follows:
Strain unreacted PTC
introduced PTC
S. lividans TK23 100%
S. viridochromogenes72%
(DSM 40736) S. viridochromogenes7%
Selectant S. lividans TK23 (pPR1) 31%
A comparison with reference substances on thin-layer chromatography (no stain with ninhydrin) demonstrates that N-acetylation of the PTC has taken place.
_ _ 9 _ 1 33759~
DNA Sequence I
IleTrpSerAspVaiLeuGlyAlaGlyProUalLeuProGlyAspAspPhePheSerLeuGly61yThrSerIle AspLeuGluArgArgProGlyGlyAr~SerGlyAlaAlaArgGlyArgLeuLeuLeuProAroAr9Hi5LeuHls ArgSerGlyAlaThrSerTrpGlyProValArgCysCysProGlyThrThrSerSerProSerAlaAlaProPro AGATCTGGAGCGAC6TCCTbGGGGCC66TCCGGTGCTGCCCGGGGACGACTTCTTCTCCCTCGGCGGCACCTCCA 75 SerArgSerArgArgGlyProProArgAspProAlaAlaArgProArgSerArgArgGlyArgArgCysArgTrp AspProAlaValAspGlnProGlyThrArgHisGlnGlyProValValGluGluGlyGluAlaAlaGlyGlyAsp IleGlnLeuSerThrArgProAlaProGlyThrSerGlyProSerSerLysLysGluAroProProValGluMet SerAlaLeuArgValValSerArgIleArgLysGluLeuGlyValProLeuArgLeuAlaValIlePheGluThr LeuGlyValAlaGlyGlyLeuAlaHlsProGlnGlyThrArgAroAlaThrProAlaArgArgAspLeuArgAsp SerArgArgCysGlyTrpSerArgAlaSerAlaArgAsnSerAlaCysHisSerGlySerProOP SerSerArg ArgProThrAlaProProArgAlaCysGlyCysProValArgArgfllaValGlyAlaArgArgSerArgArgSer ArgArgGlnProHisAspArgAlaAspAlaLeuPheGluAlaHisTrpGluProGluGlyHisAspGluLeuArg 61uAlaAsnArgThrThrGluArgMetArgLeuSerSerProThrGlySerArgSerAlaThrIleLysSerVal ProSerLeuGluAlaValAlaGluSerValLeuArgGluLeuLysGlyThrAM QC ArgGlyAlaArgHisPro fllaValProGlySerGlyGlyArgIleArgThrProArgThrGluGlyAspValValLysArgCysProProPro ArgArgProTrpLysArgTrpProAsnProTyrSerAlaAsnOP ArgGlyArgSerLysGluValProAlaThr CGCCGTCCCTGGAAGCG6TGGCCGAflTCCGTACTCCGCGAACTGAAG5GGAC6TAGTAAAGAGGTGCCCGCCACC Z25GCGGCAGGGACCTTCGCCACCGGCTTAGGCATGAGGCGCTTGACTTCCCCTGCATCATTTCTCCACGGGCGGTGG
AlaThrGlyProLeuProProArgIleArgValGlyArgValSerProSerThrThrPheLeuHisGlyGlyGly ArgGlyGlnPheArgHisGlyPheGlyTyrGluAlaPheGlnLeuProArgLeuLeuSerThrGlyAlaValArg GlyAspArgSerAlaThrAlaSerAspThrSerArgSerSerPheProValTyrTyrLeuProAlaArgTrp61y LeuSerGlnAsnThrGluGlyArgProHisMetSerProGluflroArgProValGluIleArgProAlaThrAla AlaPheAlaGluHisArgArgLysThrThrArgGluProArgThrThrProGlyArgAspProSerArgHisArg ArgPheArgArgThrProLysGluAspHlsThrOP AlaGlnAsnAspAlaArgSerArgSerValProProPro CGCTTTCGCAGAACACCGAA6GAAGACCACAC~I~AGCCCAGAAC6ACGCCCGGTCGAGATCCGTCCCGCCACCG 300 GCGAAAGCGTCTtGTGGCTTCCTTCT6GT6TGCACTCGGGTCTT6CTGC6GGCCAGCTCTAGGCAGGGCGGTGGC
AlaLysAlaSerCysArgLeuPheValValArgSerGlyLeuValValGlyProArgSerGlyAspArgTrpArg LysArgLeuValGlyPheSerSerTrpValHisAlaTrpPheSerAlaArgAspLeuAspThrGlyGlyGlyGly SerGluCysPheValSerProLeuGlyCysThrLeuGlySerArgArgGlyThrSerIleArgGlyAlaValAla AlaAspMetAlaAlaValCysAspIleValAsnHisTyrIleGluThrSerThrValAsnPheArgThrGluPro ArgArgHisGlyGlyGlyLeuArgHisArg61nSerLeuHisArgAspGluHisGlyGlnLeuProTyrGlyAla ProProThrTrpArgArgSerAlaThrSerSerIleThrThrSerArgArgAlaArgSerThrSerValArgSer AroArgCysProProProArgArgCysArgOP AspSerCysArgSerSerCysProOP SerGlyTyrProAla 61yValHisArgAroAspAlaValAspAspIleValValAspLeuArgAlaAroAspValGluThrArgLeuArg AlaSerMetAlaAlaThrGlnSerMetThrLeuOP AM MetSerValLeuValThrLeuLysArgValSerGly 61nThrProGlnGluTrpIleAspAspLeu61uArgLeuGlnAspArgTyrProTrpLeuValAlaGluValGlu AlaAspSerAlaGlyValAspArgArgProGlyAlaProProGlyProLeuProLeuAlaArgAroArgGlyGly ArgArgLeuArgArgSerGlySerThrThrTrpSerAlaSerArgThrAlaThrProGlySerSerProArgTrp CGCAGACTCCGCAGGAGTGGATCGACGACCTGGAGCGCCTCCAG~ACCGCTACCCCT6GCTCGTCGCCGAGGT6G 450 AlaSerGluAlaProThrSerArgArgGlyProAlaGly61yPro61ySer61yArgAlaArgArgArgProPro LeuserArgLeuLeuproAsplJalval6lnLeuAlaGluLeuvalAlavalGlypro6luAspGlyLeuHisLeu CysvalGlycysserHisIleserserArg5erArgArgTrpserAroAM 61yGlnSerThrAlaSerThrSer A
_ - 10 - l 337~7 DNA Sequence I (Continuation) 6lyvalvalAla6lyIleAlaTyrAla6lyproTrpLysAlaArgA~nAlaTyrA~pTrpThrual6luserThr 61yAroAroArgArgHlsAroLeuAroAroproLeu6lu6lypro6lnAroLeuAr9LeuAspAroAroualA~p ArgAla5erSerProAlaSerProThrProAlaProGlyArgProAloThrProThrThr61yProSerSerArg A666C6TC6TC6CC66CATC6CCTAC5CC66CCCCT66AA66CCC6CAAC6CCTAC6ACT66ACC6TC6A6TC6A 5~5 ProAroArgArgArgCysArgArgArgArg61yArgSerPro61yCysArgArgArgSerSerAr9ArgThrSer AlaAspAsp61yAlaAsp61yVal61yAla61yProLeu61yAlaValGlyValValPro61yAspLeuArgAro ProThrThrAlaFroMetAlaA~ AlaPro61y61nPheAlaArgLeuAlaAM Ser61nValThrSerAspV~l ValTyrValSerHlsArgHis61nArgLeu61yLeu61ySerThrLeuTyrThrHisLeuLeuLy-SerMetGlu 61yValArgLeuProProAlaProAlaAlaArgThrGlyLeuHjsProLeuHi~ProProAla61uValHis61y ArgCysThrSerProThr61yThrSer61ySerA~pTrpAlaProProSerThrProThrCysOP SerProTrp ProThrArgArgGly61yAla61yAlaAlaAroValProSerTrp61yAroCy~61y61yAlaSerThrTrpPro HisUalAspGlyUalProVolLeuPro61uSer61nAla61y61y61uVal61yVal61n61nLeu61yHisLeu ThrTyrThrGluTrpAroCysTrpArgSerProSerPro61uValAroAM ValTrpAroSerPheA~p~letSer AlaGln61yPheLy~SerValValAlaValIleGlyLeuProAsnA~pProSerValAroLeuHis61ùAlaLeu 61yProGlyLeuGlnGluAr961yAroAroHlsAroThrAla61nAroPro61uAr9AlaProAlaArg61yAla ArgProArgAlaSerArgAlaTrpSerProSerSerAspCysProThrThrArgAlaCysAlaCysThrAroArg Pro61yProSerOP SerArgProArgArgOP ArgValAlaTrpArg61ySerAroAla61yAloAroProAla 61yLeuAla61uLeuAlaH~Asp61yAspAspSerGln61yValValArgAlaHi~AlaGlnValLeuArg61u AlaTrpProLysLeuLeuThrThrAlaThrMctProSer61yLeuSer61yLeuThrArgArgCy~SerAlaSer 61yTyrThrAlaArgGlyThrLeuArgAlaAlaGlyTyrLysHi~61yGlyTrpHisAspVal61yPheTrp61n ArgIleHlsArgAlaAroA~pAlaAla61ySerArgLeu61nAl-Arg61yLeuAlaArgArg61yValLeuAla SerAspThrProArgAla61yArgCys61y61nProAlaThrSerThr61yAla61yThrThrTrp61ySer61y ArgIleCysArgAlaAroSerAlaAlaProLeuAroSerCy~AlaAroProSerAlaA!oAroProThrArgAla SerUal61yArgAlaProArg61nProCys61yAlaValLeuValProAlaProUalValHlsPro61uProLeu ProTyrValAlaAroProValSerArgAlaAloProAM LeuCysProPro61nCysSerThrProAsn61nCys ArgAspPheGluLeuProAlaProProArgProValArgProvalThr6lnIle AlaArgLeuAroAlaAla61yProAlaProProAroProAlaArgHisThrAsp SerAlaThrSerSerCysArgProArgProAlaProScr61yProSerHisArgSer flGCGCGACTTCGAGCT6CC6GCCCC6CCCC6CCCC6TCC66CCCGTCACACA6ATCT 807 AlaArgSerAroAlaAlaPro61yAla61y61yArg61yAlaArgOP ValSerArg AlaVal61uLeu61nArg61yArgGlyAla61yA~pPro61yAspCysLeuAsp ArgSerLysSerSer61yAla61y61yAro61yThrAro61yThrValCysIle DNA Sequence II
TCTA6~CCTC6CT6CA6GACCCCC66CCA6GCCAC6ACG66CCCCTGCT6AA6AA6A6G6 1 B6LII XHOII, 2 DPNI SAU3A, 5 65UI, 1' AATII ACYI, 13 nAEII
, 17 APYI ECORII, Z6 RSRII, ,7 AVAII, 35 BBVI, 39 AVhI NCII
SMAI, 40 NCII, S' ~BOII, S9 ~NLI, 61 TC6GC66CACCTCCATCTC66C6TT6C666T66TCTC6C6CATCC6CA~66AACTC66C6 A6CC6CC6T66A66TA6A6CC6CAAC6CCCACCAGA6C6C6T~66C6TTCCTT6A6CC6C
^ ^ ^ ^ ^
66 H6ICI, 70 ~NLI, 97 FNUDII, 100 SFANI, 101 FO~I, 1'1 T6CCACTCC66CTC6CC6T6ATCTTC6A6AC6CC6TCCCT66AA6C66T66CC6AATCC6 AC66T6A66CC6A6C66CACTAGAA6CTCT6C66Ch666ACCTTC6CCACC6GCTTA6GC
122 B6LI, 140 DPNI SAU3A, 147 nBoII~ 149 ACYI H6AI TTHlllI, 158 APYI ECORII, 169 CFRI GDIII, 174 HINFI, IB0 RSAI, 186 FNUDII, 201 ~AEII, 211 ~NLI, L13 H6ICI, 214 SDUI, 66CTTCCTTCT66T6T6CACTC666TCtT6CT6C666CCA6CTCTA66CA666C66T66C
247 nBoII~ 254 AFLIII, 255 P~ACI, ZS6 nAEII~ 260 H6IJII SDUI
, 271 ACYI H6AI, :75 NCII, 283 XHOII, 284 BINI DPNI SAU3A, 303 BGLI, 308 NLAIII, 324 TTHlllI, 350 H6IAI SDUI, 357 HINCI
I, 367 RSAI, 380 HINFI, 394 8INI, 395 DPNI SAU3A, 404 APYI ECOR
II, 405 GSUI, 409 HAEII, 413 nNLI~ 414 65UI, 416 APYI ECORII
, 419 A~AII, 430 APYI ECORII, 444 nNLI~ 450 nNLI~ 453 ACYI, 454 HGAI, 462 NAEI~ 466 SFANI, 477 NAEI, C6666ACCTTCC666C6TT6CG6AT6CT6ACCT66CA6CTCA6CTGCC~CAT6CA6A66G
484 APYI ECORII, Sll A~AII, Sl9 HINFI, 521 ACCI HINCII SALI, 530 R5AI, 532 nAEII~
DNA Sequence 11 (Continuation) 1 337597 541 ACC6GCACCAGCGGCTC6GhCTGGGCTCCACCCTCTACACCCACCTGCT6AA6TCCATGG
5544 HGICI, 5~9 NSPBII, 563 HGIJII SDUI, 572 MNLI, 578 TAOII, 563 BSPMI, 595 NCOI 5TYI, 596 NLAIII, 600 MNLI, lO605 APYI ECORII, 650 A~AI, 669 MNLI, 671 HAEII, 6B6 FNUDII, 687 BSSHII, 6BB FNUDII, 690 15FNUDII, 695 HGAI, 698 BBVI, 705 BB~I, 70B NAEI, 716 TTHIIII
I, 7~1 ACGGG6GCTGGCACGACGTGGGGTTCTGGCAGCGCGACTTCGAGCTGCCGGCCCCGCCCC
TGCCCCCGACCGTGCTGCACCCChA6ACCGTCGCGCTGAAGCTC6ACG6CC6GG6CGGJG
20737 DRAIII, 736 MAEIl, 749 BB~I, 753 FNUDII, 763 ALUJ, 764 B
BVI, 767 NAEI, 781 6CCCC6TCCGGCCCGTCACACAGhTCT
CGGGGCAGGCCGGGCAGTGTGTCTAGA
25795 MAEIII, B02 BGLII XHOII, 803 DPNI SAU3A, P. .
~)
sequence I (Annex). The position of the resistance gene can be determined from the open reading frame of this sequence (from position 258). The end of the gene is located at the penultimate nucleotide shown (position 806), i.e. the last nucleotide (position 807) is the first of the stop codon.
The Shine-Dalgarno sequence in DNA sequence I is emphasized by underlining, as is the GTG acting as start codon. This GTG codon is expressed as methionine. Thus, the top line depicts the definitive reading frame.
DNA sequence II shows the restriction sites within the sequenced gene. Enzymes which cut the sequence more than six times are not indicated.
The antibiotic PTT is taken up by bacteria and broken down to PTC. The latter also inhibits glutamine synthetase in bacteria, so that the bacteria die of a lack of glutamine. Hence, PTT-producing bacteria ought to have a mechanism which protects them from the action of PTT, that is to say either prevents reuptake of the PTT which has been produced or permits a modification of the breakdown product PTC. However, surprisingly, the ~`
` - 3 - l 3 37 5 97 PTT producer S. viridochromogenes DSM 4112 is sensitive ~ to its own antibiotic. Unexpectedly, it proved possible, ho~ever, by selection for PTT resistance to find, at the surprisingly high rate of 10 5, selectants which are resistant to PTT and, moreover, suppress the background growth of adjacent colonies.
A gene bank was set up from the DNA of these selectants by isolating the DNA and cleaving it with BamHI and ligating it into a Streptomycetes vector. The ligation mixture was transformed into the commercially available strain S. lividans TK 23, resulting in about 5000 to 10000 transformants having an insert of about 1 to 5 kb per 1 ~9 of ligation mixture. Among the transformants there ~ere PTT-resistant S. lividans strains. It was possible, by isolation of the plasmids and retransform-ation into S. lividans, to show that the resistance is plasmid-coded. The gene responsible for the resistance is located on a 4 kb BamHI fragment (Figure 1). The coding region is located on the 0.8 kb BglII fragment.
The BamHI fragment contains no cleavage sites for the enzymes ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PvuI, PvuII and _ I.
Comparison with the restriction map of a resistance gene, which has not been characterized in detail, for S.
hygroscopicus FERM BP-130/ATCC 21705 (European Patent Application with the publication no. 0,173,327, Figure 7) shows that the resistance gene according to the invention differs from the known gene, which was found during the search for PTT biosynthesis genes.
It was possible to show, by incubation of cell extracts from S. viridochromogenes DSM 4112 and S. lividans TK 23 on the one hand, and the PTT-resistant S. viridochromogenes selectants and a plasmid-carrying S. lividans transform-ant, on the other hand, with PTC and acetyl-coenzyme A
that the latter cells have acetylating activity.
Chromatography tests sho~ that the acetylation takes place on the amino group.
Since PTT-resistance has also been found in E. coli, and thus the resistance mechanism also functions in Gram-negative bacteria, it is possible to rule out resistancebased on transport phenomena. Thus, after coupling to plant promoters and using suitable vectors, the resistance gene according to the invention can be transformed into plants, and in this ~ay PIC-resistant plants can be produced.
The N-acetylation of PTC can also be used for racemate resolution of synthetic D,L-PTC since selective acetyl-ation of only the L-form takes place.
Thus the invention also relates to the use of the resis-tance gene for the selective N-acetylation of the L-form of racemic PTC.
The PTC acetyltransferase coded for by the resistance gene according to the invention can thus be used to separate racemic PTC, as can be obtained, for example, by the method of German Patent 2,717,440, into the optical antipodes by exposing the racemate to the acetylating action of this enzyme, since there is selective attack on the L-form vhile the D-form remains unchanged. The mixture thus obtained can then be fractionated in a manner knovn per se on the basis of the differences in properties.
The contacting of N-acyl-D,L-amino acids vith acylases, vhich are immobilised on carriers ~here appropriate, vith selective liberation of the L-amino acid, vhich can be extracted vith water-immiscible solvents from the mixture vith the N-acyl-D-amino acid after acidification, has be-n discloset (British Patent 1,369,462, p~bl~sh~d Octob-r 9, 1974, Inventors Nartin Cole and Renneth Utting). A correspQn~ng fractionation of N-acyl-D,L-PTC is tisclosed, for example, in Germ~n Offenlegungsschrift 2,939,269 (publishQd April 30, 1980, Applicant Nei~i Seika Raisha, Ltt.) 1 33~597 - or U S Patent No 4,226,941 (Issued October 7, 1980, Assignet to Mei~i Seika Raisha, Ltd ) The D-PCT which remains according to the invention can be racemized in kno~n manner (European Published Specification No 0 137 371, published April 17, 1983, Applicant Hoechst Aktiengesellgchaft, example 8), and then returned to the process.
It is possible, but not necessary, to isolate the enzyme, this also being intended to mean, here and hereinafter, always the enzymatically active part If the enzyme is isolated, it can be used in ths free form or the form immobilized on a carrier Examples of suitable carriers are described in European Patent Application No 84 111 208 9 (published unter No 0 141 223 on May 15, 1985, Applicant Hoechst AG) Hc~e~a., it is aApe~ent not to isolate the enzyme but to use any desired PTC-resistant cells which express the enzyme according to the invention. Thus, it is possible and expedient to use the PTT-resistant selectants of S. viridochromogenes DSM 4112.
Moreover, it is possible and advantageous to use any desired cell ~hich has been transformed ~ith the gene according to the invention and ~hich is able to express PTC acetyltransferase. In this connection, the gene according to the invention, this also being intended to mean active parts thereof, can be introduced into the host cell in plasmid-integrated form or by using other customary methods of gene manipulation, for example by transfection. For example, incorporation into a E. coli expression plasmid and transformation of E. coli with such a plasmid is expedient, for example by the methods ~noun from ~o~n Pllbl~R~d Specification No 0 163 249 (p~hl~shed April 12, 1985, Applicant Hoecbst AG) ant European Publishet Specification No 0 171 024 (ptlbl~shed February 12, 1986, Applicant Hoechst AG) For the N-acetylation, according to th- invention, of L-PTC in the racemate th-e cells which express PTC acetyltransferase can be uset in tbe free or immobilized form, with- the customary methods of immobilisation being used (for example German Offenlegungssc_rift No 32 37 341 (publish-d April 12, 1984, Applicant Hoechst AG) ant literatur- cit-t therein) - 6 - ~ 3~ 59~
he enzymatic acetylation, according to the invention, of L-PTC is carried out in the manner customary for enzymatic reactions, ~ith the conditions of the method being governed by the characteristics of the organism used. In principle, methods suitable for this are the same as for the abovementioned selective deacylation method.
The invention is illustrated in detail in the examples ~hich follow. Unless otherwise stated, parts and percentage data relate to weight.
Example 1: P~I-resistant selectants The strain S. viridochromogenes DSM 4112 was cultured on minimal medium (Hop~ood et al., Genetic Manipulation of Streptomyces, A Laboratory Manual, The John Innes Foundation, Nor~ich, England (1985), page 233) and increas-ing concentrations of PTT ~ere added. At a concentration of 100 ~g/ml one resistant colony ~as found per 105 colonies, approximately.
Example 2: Preparation of the vector The plasmid pSVH1 (Eu~ops-n Patent 0,070,522, Issued Octob-r 29, 1986, Agsignee Hoechst AG; U S Patent 4,673,642, Issued June 16, 1987, Assignee Hoechst AG) is cut with BglII, ant th- fragm-nt about 7 1 kb in slze is isolatet ant ligatet ~ith th- 1 1 kb BclI fragment having thiostrepton- resistanc- (EuLop~an Patent Appl~c~ti~ 85 103 460 3 publishet October 16, 1985 unter Publ~at~on No 0 158 201) The plasmid pEB2 which is about 8 15 kb in siz- is obtain-d (Figure 2) Example 3: Isolation of the resistance gene The total DNA is isolated from the selectants obtained in example 1, and it is cleaved ~ith ~amHI. The plasmid pE~2 is like~ise opened ~ith ~amHI, and the t~o mixtures are combined and ligated. The ligation m;xture is trans-formed into S. lividans TK 23 (obtainable from the ,~ . .
John Innes Foundation), ~ith 5ûO0 to 10000 transformants having an insert of about 1 - 5 kb being obtained per 1 ~9 of ligation mixture. Selection for PTT-resistance produces t~o resistant S. lividans colonies. The plasmid ~hich has been taken up is isolated from the latter and is cut ~ith BamH~. A 4 kb BamHI fragment ~hich carries the gene responsible for resistance is found. This plasmid ~as called pPR1 (figure 3).
Retransformation ;nto S. lividans TK 23 shows, that the PTT-resistance is plasmid-coded, since the transformants gro~ on minimal medium containing 100 ~g/ml PTT.
Example 4: Demonstration of the inactivation of PTC by ; N-acetylation The follo~ing strains were examined to demonstrate the acetylating activity of the cloned fragment: S. viri-dochromogenes DSM 40736, S. viridochromogenes (PTT-resistant mutant), S. lividans TK23 and S. lividans TK 23 (pPR1).
This entails the strains being inoculated into lysis medium A (European Published Specification 0 158 872, published October 23, 1985, Applicant Hoechst AG, page 6) and incubated at 30C in an orbital shaker for 2 days. After harvesting, 1 mg of mycelium is disrupted with ultrasound in a suitable buffer (for example RS buffer: C.J. Thompson et al., J. sacteriol. 151 (1982), 678-685). The procedure for a typical experiment to measure PTC breakdown is as follows:
100 ~l of PTC solution (250 ~g/ml) and 50 ~l of acetyl-CoA (4 mg/ml) are added to 250 ~l of crude extract, and the mixture is incubated at 30C for 2 hours. The amounts of PTC which are still present after this time are measured by HPLC. The results of this are as - 8 - l 337 5~7 follows:
Strain unreacted PTC
introduced PTC
S. lividans TK23 100%
S. viridochromogenes72%
(DSM 40736) S. viridochromogenes7%
Selectant S. lividans TK23 (pPR1) 31%
A comparison with reference substances on thin-layer chromatography (no stain with ninhydrin) demonstrates that N-acetylation of the PTC has taken place.
_ _ 9 _ 1 33759~
DNA Sequence I
IleTrpSerAspVaiLeuGlyAlaGlyProUalLeuProGlyAspAspPhePheSerLeuGly61yThrSerIle AspLeuGluArgArgProGlyGlyAr~SerGlyAlaAlaArgGlyArgLeuLeuLeuProAroAr9Hi5LeuHls ArgSerGlyAlaThrSerTrpGlyProValArgCysCysProGlyThrThrSerSerProSerAlaAlaProPro AGATCTGGAGCGAC6TCCTbGGGGCC66TCCGGTGCTGCCCGGGGACGACTTCTTCTCCCTCGGCGGCACCTCCA 75 SerArgSerArgArgGlyProProArgAspProAlaAlaArgProArgSerArgArgGlyArgArgCysArgTrp AspProAlaValAspGlnProGlyThrArgHisGlnGlyProValValGluGluGlyGluAlaAlaGlyGlyAsp IleGlnLeuSerThrArgProAlaProGlyThrSerGlyProSerSerLysLysGluAroProProValGluMet SerAlaLeuArgValValSerArgIleArgLysGluLeuGlyValProLeuArgLeuAlaValIlePheGluThr LeuGlyValAlaGlyGlyLeuAlaHlsProGlnGlyThrArgAroAlaThrProAlaArgArgAspLeuArgAsp SerArgArgCysGlyTrpSerArgAlaSerAlaArgAsnSerAlaCysHisSerGlySerProOP SerSerArg ArgProThrAlaProProArgAlaCysGlyCysProValArgArgfllaValGlyAlaArgArgSerArgArgSer ArgArgGlnProHisAspArgAlaAspAlaLeuPheGluAlaHisTrpGluProGluGlyHisAspGluLeuArg 61uAlaAsnArgThrThrGluArgMetArgLeuSerSerProThrGlySerArgSerAlaThrIleLysSerVal ProSerLeuGluAlaValAlaGluSerValLeuArgGluLeuLysGlyThrAM QC ArgGlyAlaArgHisPro fllaValProGlySerGlyGlyArgIleArgThrProArgThrGluGlyAspValValLysArgCysProProPro ArgArgProTrpLysArgTrpProAsnProTyrSerAlaAsnOP ArgGlyArgSerLysGluValProAlaThr CGCCGTCCCTGGAAGCG6TGGCCGAflTCCGTACTCCGCGAACTGAAG5GGAC6TAGTAAAGAGGTGCCCGCCACC Z25GCGGCAGGGACCTTCGCCACCGGCTTAGGCATGAGGCGCTTGACTTCCCCTGCATCATTTCTCCACGGGCGGTGG
AlaThrGlyProLeuProProArgIleArgValGlyArgValSerProSerThrThrPheLeuHisGlyGlyGly ArgGlyGlnPheArgHisGlyPheGlyTyrGluAlaPheGlnLeuProArgLeuLeuSerThrGlyAlaValArg GlyAspArgSerAlaThrAlaSerAspThrSerArgSerSerPheProValTyrTyrLeuProAlaArgTrp61y LeuSerGlnAsnThrGluGlyArgProHisMetSerProGluflroArgProValGluIleArgProAlaThrAla AlaPheAlaGluHisArgArgLysThrThrArgGluProArgThrThrProGlyArgAspProSerArgHisArg ArgPheArgArgThrProLysGluAspHlsThrOP AlaGlnAsnAspAlaArgSerArgSerValProProPro CGCTTTCGCAGAACACCGAA6GAAGACCACAC~I~AGCCCAGAAC6ACGCCCGGTCGAGATCCGTCCCGCCACCG 300 GCGAAAGCGTCTtGTGGCTTCCTTCT6GT6TGCACTCGGGTCTT6CTGC6GGCCAGCTCTAGGCAGGGCGGTGGC
AlaLysAlaSerCysArgLeuPheValValArgSerGlyLeuValValGlyProArgSerGlyAspArgTrpArg LysArgLeuValGlyPheSerSerTrpValHisAlaTrpPheSerAlaArgAspLeuAspThrGlyGlyGlyGly SerGluCysPheValSerProLeuGlyCysThrLeuGlySerArgArgGlyThrSerIleArgGlyAlaValAla AlaAspMetAlaAlaValCysAspIleValAsnHisTyrIleGluThrSerThrValAsnPheArgThrGluPro ArgArgHisGlyGlyGlyLeuArgHisArg61nSerLeuHisArgAspGluHisGlyGlnLeuProTyrGlyAla ProProThrTrpArgArgSerAlaThrSerSerIleThrThrSerArgArgAlaArgSerThrSerValArgSer AroArgCysProProProArgArgCysArgOP AspSerCysArgSerSerCysProOP SerGlyTyrProAla 61yValHisArgAroAspAlaValAspAspIleValValAspLeuArgAlaAroAspValGluThrArgLeuArg AlaSerMetAlaAlaThrGlnSerMetThrLeuOP AM MetSerValLeuValThrLeuLysArgValSerGly 61nThrProGlnGluTrpIleAspAspLeu61uArgLeuGlnAspArgTyrProTrpLeuValAlaGluValGlu AlaAspSerAlaGlyValAspArgArgProGlyAlaProProGlyProLeuProLeuAlaArgAroArgGlyGly ArgArgLeuArgArgSerGlySerThrThrTrpSerAlaSerArgThrAlaThrProGlySerSerProArgTrp CGCAGACTCCGCAGGAGTGGATCGACGACCTGGAGCGCCTCCAG~ACCGCTACCCCT6GCTCGTCGCCGAGGT6G 450 AlaSerGluAlaProThrSerArgArgGlyProAlaGly61yPro61ySer61yArgAlaArgArgArgProPro LeuserArgLeuLeuproAsplJalval6lnLeuAlaGluLeuvalAlavalGlypro6luAspGlyLeuHisLeu CysvalGlycysserHisIleserserArg5erArgArgTrpserAroAM 61yGlnSerThrAlaSerThrSer A
_ - 10 - l 337~7 DNA Sequence I (Continuation) 6lyvalvalAla6lyIleAlaTyrAla6lyproTrpLysAlaArgA~nAlaTyrA~pTrpThrual6luserThr 61yAroAroArgArgHlsAroLeuAroAroproLeu6lu6lypro6lnAroLeuAr9LeuAspAroAroualA~p ArgAla5erSerProAlaSerProThrProAlaProGlyArgProAloThrProThrThr61yProSerSerArg A666C6TC6TC6CC66CATC6CCTAC5CC66CCCCT66AA66CCC6CAAC6CCTAC6ACT66ACC6TC6A6TC6A 5~5 ProAroArgArgArgCysArgArgArgArg61yArgSerPro61yCysArgArgArgSerSerAr9ArgThrSer AlaAspAsp61yAlaAsp61yVal61yAla61yProLeu61yAlaValGlyValValPro61yAspLeuArgAro ProThrThrAlaFroMetAlaA~ AlaPro61y61nPheAlaArgLeuAlaAM Ser61nValThrSerAspV~l ValTyrValSerHlsArgHis61nArgLeu61yLeu61ySerThrLeuTyrThrHisLeuLeuLy-SerMetGlu 61yValArgLeuProProAlaProAlaAlaArgThrGlyLeuHjsProLeuHi~ProProAla61uValHis61y ArgCysThrSerProThr61yThrSer61ySerA~pTrpAlaProProSerThrProThrCysOP SerProTrp ProThrArgArgGly61yAla61yAlaAlaAroValProSerTrp61yAroCy~61y61yAlaSerThrTrpPro HisUalAspGlyUalProVolLeuPro61uSer61nAla61y61y61uVal61yVal61n61nLeu61yHisLeu ThrTyrThrGluTrpAroCysTrpArgSerProSerPro61uValAroAM ValTrpAroSerPheA~p~letSer AlaGln61yPheLy~SerValValAlaValIleGlyLeuProAsnA~pProSerValAroLeuHis61ùAlaLeu 61yProGlyLeuGlnGluAr961yAroAroHlsAroThrAla61nAroPro61uAr9AlaProAlaArg61yAla ArgProArgAlaSerArgAlaTrpSerProSerSerAspCysProThrThrArgAlaCysAlaCysThrAroArg Pro61yProSerOP SerArgProArgArgOP ArgValAlaTrpArg61ySerAroAla61yAloAroProAla 61yLeuAla61uLeuAlaH~Asp61yAspAspSerGln61yValValArgAlaHi~AlaGlnValLeuArg61u AlaTrpProLysLeuLeuThrThrAlaThrMctProSer61yLeuSer61yLeuThrArgArgCy~SerAlaSer 61yTyrThrAlaArgGlyThrLeuArgAlaAlaGlyTyrLysHi~61yGlyTrpHisAspVal61yPheTrp61n ArgIleHlsArgAlaAroA~pAlaAla61ySerArgLeu61nAl-Arg61yLeuAlaArgArg61yValLeuAla SerAspThrProArgAla61yArgCys61y61nProAlaThrSerThr61yAla61yThrThrTrp61ySer61y ArgIleCysArgAlaAroSerAlaAlaProLeuAroSerCy~AlaAroProSerAlaA!oAroProThrArgAla SerUal61yArgAlaProArg61nProCys61yAlaValLeuValProAlaProUalValHlsPro61uProLeu ProTyrValAlaAroProValSerArgAlaAloProAM LeuCysProPro61nCysSerThrProAsn61nCys ArgAspPheGluLeuProAlaProProArgProValArgProvalThr6lnIle AlaArgLeuAroAlaAla61yProAlaProProAroProAlaArgHisThrAsp SerAlaThrSerSerCysArgProArgProAlaProScr61yProSerHisArgSer flGCGCGACTTCGAGCT6CC6GCCCC6CCCC6CCCC6TCC66CCCGTCACACA6ATCT 807 AlaArgSerAroAlaAlaPro61yAla61y61yArg61yAlaArgOP ValSerArg AlaVal61uLeu61nArg61yArgGlyAla61yA~pPro61yAspCysLeuAsp ArgSerLysSerSer61yAla61y61yAro61yThrAro61yThrValCysIle DNA Sequence II
TCTA6~CCTC6CT6CA6GACCCCC66CCA6GCCAC6ACG66CCCCTGCT6AA6AA6A6G6 1 B6LII XHOII, 2 DPNI SAU3A, 5 65UI, 1' AATII ACYI, 13 nAEII
, 17 APYI ECORII, Z6 RSRII, ,7 AVAII, 35 BBVI, 39 AVhI NCII
SMAI, 40 NCII, S' ~BOII, S9 ~NLI, 61 TC6GC66CACCTCCATCTC66C6TT6C666T66TCTC6C6CATCC6CA~66AACTC66C6 A6CC6CC6T66A66TA6A6CC6CAAC6CCCACCAGA6C6C6T~66C6TTCCTT6A6CC6C
^ ^ ^ ^ ^
66 H6ICI, 70 ~NLI, 97 FNUDII, 100 SFANI, 101 FO~I, 1'1 T6CCACTCC66CTC6CC6T6ATCTTC6A6AC6CC6TCCCT66AA6C66T66CC6AATCC6 AC66T6A66CC6A6C66CACTAGAA6CTCT6C66Ch666ACCTTC6CCACC6GCTTA6GC
122 B6LI, 140 DPNI SAU3A, 147 nBoII~ 149 ACYI H6AI TTHlllI, 158 APYI ECORII, 169 CFRI GDIII, 174 HINFI, IB0 RSAI, 186 FNUDII, 201 ~AEII, 211 ~NLI, L13 H6ICI, 214 SDUI, 66CTTCCTTCT66T6T6CACTC666TCtT6CT6C666CCA6CTCTA66CA666C66T66C
247 nBoII~ 254 AFLIII, 255 P~ACI, ZS6 nAEII~ 260 H6IJII SDUI
, 271 ACYI H6AI, :75 NCII, 283 XHOII, 284 BINI DPNI SAU3A, 303 BGLI, 308 NLAIII, 324 TTHlllI, 350 H6IAI SDUI, 357 HINCI
I, 367 RSAI, 380 HINFI, 394 8INI, 395 DPNI SAU3A, 404 APYI ECOR
II, 405 GSUI, 409 HAEII, 413 nNLI~ 414 65UI, 416 APYI ECORII
, 419 A~AII, 430 APYI ECORII, 444 nNLI~ 450 nNLI~ 453 ACYI, 454 HGAI, 462 NAEI~ 466 SFANI, 477 NAEI, C6666ACCTTCC666C6TT6CG6AT6CT6ACCT66CA6CTCA6CTGCC~CAT6CA6A66G
484 APYI ECORII, Sll A~AII, Sl9 HINFI, 521 ACCI HINCII SALI, 530 R5AI, 532 nAEII~
DNA Sequence 11 (Continuation) 1 337597 541 ACC6GCACCAGCGGCTC6GhCTGGGCTCCACCCTCTACACCCACCTGCT6AA6TCCATGG
5544 HGICI, 5~9 NSPBII, 563 HGIJII SDUI, 572 MNLI, 578 TAOII, 563 BSPMI, 595 NCOI 5TYI, 596 NLAIII, 600 MNLI, lO605 APYI ECORII, 650 A~AI, 669 MNLI, 671 HAEII, 6B6 FNUDII, 687 BSSHII, 6BB FNUDII, 690 15FNUDII, 695 HGAI, 698 BBVI, 705 BB~I, 70B NAEI, 716 TTHIIII
I, 7~1 ACGGG6GCTGGCACGACGTGGGGTTCTGGCAGCGCGACTTCGAGCTGCCGGCCCCGCCCC
TGCCCCCGACCGTGCTGCACCCChA6ACCGTCGCGCTGAAGCTC6ACG6CC6GG6CGGJG
20737 DRAIII, 736 MAEIl, 749 BB~I, 753 FNUDII, 763 ALUJ, 764 B
BVI, 767 NAEI, 781 6CCCC6TCCGGCCCGTCACACAGhTCT
CGGGGCAGGCCGGGCAGTGTGTCTAGA
25795 MAEIII, B02 BGLII XHOII, 803 DPNI SAU3A, P. .
~)
Claims (11)
1. A process for preparing a phosphinothricin(PTC)-resistance gene which comprises selecting Streptomyces viridochromogenes DSM 4112 for resistance to phosphinothricyl-alanyl-alanine (PTT), cutting with BamHI the total DNA from the resistant strains, cloning a fragment 4.0 kb in size, and selecting for PTT resistance.
2. A PTC-resistance gene which is located within the DNA-sequence mapped in Figure 1.
3. A PTC resistance gene which has the DNA sequence I as shown in Figure 4, positions 258-806.
4. A process for the production of PTC-resistant plants which comprises incorporating into the genome of the plant a gene as claimed in claim 2.
5. A process for the production of PTC-resistant plants which comprises incorporating into the genome of the plant a gene as claimed in claim 3.
6. A PTT-resistance marker in bacteria comprising the gene as claimed in claim 2.
7. A PTT-resistance marker in bacteria comprising the gene as claimed in claim 3.
8. A PTC-resistance marker in plant cells comprising the gene as claimed in claim 2.
9. A PTC-resistance marker in plant cells comprising the gene as claimed in claim 3.
10. A process for the selective N-acetylation of the L-form of racemic PTC which comprises contacting racemic PTC with a cell expressing the gene as claimed in claim 2, or with the enzyme coded by this gene.
11. A process for the selective N-acetylation of the L-form of racemic PTC which comprises contacting racemic PTC with a cell expressing the gene as claimed in claim 3, or with the enzyme coded by this gene.
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3628747.4 | 1986-08-23 | ||
| DE19863628747 DE3628747A1 (en) | 1986-08-23 | 1986-08-23 | Phosphinothricin resistance gene and its use |
| DE3637307 | 1986-11-03 | ||
| DEP3637307.9 | 1986-11-03 | ||
| DE19863642829 DE3642829A1 (en) | 1986-08-23 | 1986-12-16 | Phosphinothricin-resistance gene |
| DEP3642829.9 | 1986-12-16 | ||
| DEP3700313.5 | 1987-01-08 | ||
| DE19873700313 DE3700313A1 (en) | 1986-08-23 | 1987-01-08 | Use of a phosphinothricin-resistance gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1337597C true CA1337597C (en) | 1995-11-21 |
Family
ID=27433686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000545037A Expired - Lifetime CA1337597C (en) | 1986-08-23 | 1987-08-21 | Phosphinotricin-resistance gene, and its use |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0257542B1 (en) |
| JP (4) | JPH0797994B2 (en) |
| CN (2) | CN1040772C (en) |
| AT (1) | ATE75776T1 (en) |
| AU (1) | AU604743B2 (en) |
| CA (1) | CA1337597C (en) |
| DK (1) | DK175254B1 (en) |
| ES (1) | ES2038631T3 (en) |
| FI (1) | FI100251B (en) |
| GR (1) | GR3005200T3 (en) |
| HU (1) | HU217208B (en) |
| IL (1) | IL83604A (en) |
| NZ (1) | NZ221526A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7407913B2 (en) | 1996-09-05 | 2008-08-05 | Syngenta Crop Protection, Inc. | Process for the control of weeds |
Families Citing this family (30)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3765449D1 (en) * | 1986-03-11 | 1990-11-15 | Plant Genetic Systems Nv | PLANT CELLS RESISTED BY GENE TECHNOLOGY AND RESISTANT TO GLUTAMINE SYNTHETASE INHIBITORS. |
| CN87100603A (en) * | 1987-01-21 | 1988-08-10 | 昂科公司 | Vaccines against melanoma |
| DE3716309A1 (en) * | 1987-05-15 | 1988-11-24 | Hoechst Ag | RESISTANCE TO PHOSPHINOTHRICIN |
| DE3732972A1 (en) * | 1987-07-02 | 1989-01-12 | Hoechst Ag | RESISTANCE GENES TO PHOSPHINOTHRICIN AND ITS USE |
| DE4126414A1 (en) * | 1991-08-09 | 1993-02-11 | Hoechst Ag | DEACETYLASE GENES FOR PRODUCING PHOSPHINOTHRICIN OR PHOSPHINOTHRICYL-ALANYL-ALANINE, METHOD FOR THEIR INSULATION AND THEIR USE |
| DE4222407C1 (en) * | 1992-07-08 | 1993-10-07 | Max Planck Gesellschaft | Modular promoter construct |
| AR008158A1 (en) * | 1996-09-05 | 1999-12-09 | Syngenta Participations Ag | PROCESS FOR THE CONTROL OF BAD HERBS IN USEFUL PLANTS CROPS THAT ARE RESISTANT TO A PHOSPH-HERBICIDE AND A HERBICIDAL COMPOSITION FOR SUCH USE. |
| DE19836684A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of a synergistic herbicidal combination including a glufosinate- or glyphosate-type, imidazolinone or protoporphyrinogen oxidase to control weeds in rice |
| DE19836700A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of a synergistic herbicide combination including a glufosinate- or glyphosate-type, imidazolinone or protoporphyrinogen oxidase inhibitory azole herbicide to control weeds in cereals |
| DE19836659A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of synergistic herbicide combination including glufosinate- or glyphosate-type, imidazolinone, protoporphyrinogen oxidase inhibitory azole or hydroxybenzonitrile herbicide, to control weeds in cotton |
| DE19836660A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of a synergistic herbicide combination including a glufosinate- or glyphosate-type, imidazolinone or protoporphyrinogen oxidase inhibitory azole herbicide to control weeds in soya |
| PL218413B1 (en) | 1998-08-13 | 2014-12-31 | Bayer Cropscience Ag | Use of herbicide combinations for controlling harmful plants in crops of maize and a method for controlling harmful plants in tolerant crops of maize |
| CA2475485C (en) | 2002-02-26 | 2012-08-28 | Syngenta Limited | A method of selectively producing male or female sterile plants |
| CA2586241C (en) | 2004-11-17 | 2013-07-30 | Hokko Chemical Industry Co., Ltd. | Herbicide-resistance gene and utilization thereof |
| KR100743611B1 (en) * | 2006-03-28 | 2007-08-01 | 최광술 | Cable control system industrial robot |
| AR074941A1 (en) | 2009-01-07 | 2011-02-23 | Bayer Cropscience Sa | TRANSPLASTOMIC PLANTS EXEMPTED FROM SELECTOR MARKER |
| CN102869769A (en) | 2009-12-23 | 2013-01-09 | 拜尔知识产权有限公司 | Plants tolerant to HPPD inhibitor herbicides |
| EP2516630B1 (en) | 2009-12-23 | 2017-11-15 | Bayer Intellectual Property GmbH | Plants tolerant to hppd inhibitor herbicides |
| EP2516631B1 (en) | 2009-12-23 | 2018-02-14 | Bayer Intellectual Property GmbH | Plants tolerant to hppd inhibitor herbicides |
| BR112012015697A2 (en) | 2009-12-23 | 2015-08-25 | Bayer Intelectual Property Gmbh | Herbicide tolerant plants of hppd inhibitors. |
| WO2011076889A1 (en) | 2009-12-23 | 2011-06-30 | Bayer Cropscience Ag | Plants tolerant to hppd inhibitor herbicides |
| WO2011144684A1 (en) | 2010-05-21 | 2011-11-24 | Bayer Cropscience Ag | Herbicidal agents for tolerant or resistant rice cultures |
| WO2011144683A1 (en) | 2010-05-21 | 2011-11-24 | Bayer Cropscience Ag | Herbicidal agents for tolerant or resistant rape cultures |
| BR112012029616A2 (en) | 2010-05-21 | 2015-10-20 | Bayer Ip Gmbh | herbicidal agents for tolerant or resistant cereal crops |
| BR112012029621A2 (en) | 2010-05-21 | 2015-09-22 | Bayer Ip Gmbh | herbicidal agents for tolerant or resistant maize crops |
| WO2012004013A2 (en) | 2010-07-08 | 2012-01-12 | Bayer Bioscience N.V. | Glucosinolate transporter protein and uses thereof |
| US20140090102A1 (en) | 2011-01-24 | 2014-03-27 | Stéphane Pien | Use of the rd29 promoter or fragments thereof for stress-inducible expression of transgenes in cotton |
| EP2524602A1 (en) | 2011-05-20 | 2012-11-21 | Bayer CropScience AG | Herbicide agent for tolerant or resistant soya cultures |
| UA117816C2 (en) | 2012-11-06 | 2018-10-10 | Байєр Кропсайєнс Акцієнгезелльшафт | Herbicidal combinations for tolerant soybean cultures |
| DE202014101590U1 (en) | 2014-04-03 | 2014-04-29 | Igus Gmbh | Guidance system for supply lines and robots with guidance system |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3765449D1 (en) * | 1986-03-11 | 1990-11-15 | Plant Genetic Systems Nv | PLANT CELLS RESISTED BY GENE TECHNOLOGY AND RESISTANT TO GLUTAMINE SYNTHETASE INHIBITORS. |
-
1987
- 1987-08-19 ES ES198787112023T patent/ES2038631T3/en not_active Expired - Lifetime
- 1987-08-19 AT AT87112023T patent/ATE75776T1/en not_active IP Right Cessation
- 1987-08-19 EP EP87112023A patent/EP0257542B1/en not_active Expired - Lifetime
- 1987-08-20 FI FI873610A patent/FI100251B/en not_active IP Right Cessation
- 1987-08-21 CA CA000545037A patent/CA1337597C/en not_active Expired - Lifetime
- 1987-08-21 IL IL8360487A patent/IL83604A/en not_active IP Right Cessation
- 1987-08-21 AU AU77318/87A patent/AU604743B2/en not_active Expired
- 1987-08-21 NZ NZ221526A patent/NZ221526A/en unknown
- 1987-08-21 DK DK198704378A patent/DK175254B1/en not_active IP Right Cessation
- 1987-08-22 CN CN87105764A patent/CN1040772C/en not_active Expired - Lifetime
- 1987-08-22 JP JP62209123A patent/JPH0797994B2/en not_active Expired - Lifetime
- 1987-08-23 HU HU727/87A patent/HU217208B/en unknown
-
1990
- 1990-02-28 JP JP2049140A patent/JP2749424B2/en not_active Expired - Lifetime
-
1992
- 1992-07-16 GR GR920401371T patent/GR3005200T3/el unknown
-
1994
- 1994-10-14 JP JP6249627A patent/JPH07147985A/en active Pending
-
1996
- 1996-10-09 JP JP8268910A patent/JP2815847B2/en not_active Expired - Lifetime
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1998
- 1998-06-19 CN CN98115017A patent/CN1124347C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7407913B2 (en) | 1996-09-05 | 2008-08-05 | Syngenta Crop Protection, Inc. | Process for the control of weeds |
Also Published As
| Publication number | Publication date |
|---|---|
| HU217208B (en) | 1999-12-28 |
| DK437887A (en) | 1988-02-24 |
| JPS6371183A (en) | 1988-03-31 |
| CN1225393A (en) | 1999-08-11 |
| FI873610A0 (en) | 1987-08-20 |
| EP0257542A2 (en) | 1988-03-02 |
| JPH03103193A (en) | 1991-04-30 |
| IL83604A (en) | 2004-02-19 |
| CN1040772C (en) | 1998-11-18 |
| JP2749424B2 (en) | 1998-05-13 |
| CN1124347C (en) | 2003-10-15 |
| JPH0797994B2 (en) | 1995-10-25 |
| FI100251B (en) | 1997-10-31 |
| AU604743B2 (en) | 1991-01-03 |
| NZ221526A (en) | 1989-03-29 |
| AU7731887A (en) | 1988-05-19 |
| IL83604A0 (en) | 1988-01-31 |
| HUT44621A (en) | 1988-03-28 |
| FI873610A (en) | 1988-02-24 |
| JPH07147985A (en) | 1995-06-13 |
| EP0257542B1 (en) | 1992-05-06 |
| DK175254B1 (en) | 2004-07-26 |
| CN87105764A (en) | 1988-11-30 |
| JP2815847B2 (en) | 1998-10-27 |
| GR3005200T3 (en) | 1993-05-24 |
| JPH09107981A (en) | 1997-04-28 |
| ATE75776T1 (en) | 1992-05-15 |
| ES2038631T3 (en) | 1993-08-01 |
| EP0257542A3 (en) | 1990-03-07 |
| DK437887D0 (en) | 1987-08-21 |
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