DK175254B1 - Phosphinothricin resistance gene sequence and its use - Google Patents

Abstract

Selection of Streptomyces viridochromogenes DSM 4112 using phosphinothricyl-alanyl-alanine (PTT) results in PTT-resistant selectants. Subjecting the total DNA of these selectants to cutting with Bam HI, cloning of a 4.0 kb fragment and selection for PTT resistance result in the DNA fragment which harbours the phosphinothricin (PTC)-resistance gene. The latter is suitable for producing PTC-resistant plants, and as resistance marker and for the selective N-acetylation of the L form of racemic PTC. <IMAGE>

Description

DK 175254 B1 i

Phosphinothricin (PTC, 2-amino-4-methylphosphinobutyric acid) is a glutamine synthetase inhibitor. PTC is a "building block" of the phosphinothricyl-alanyl-alanine antibiotic.

This tripeptide (PTT) is active against gram-positive and 5 gram-negative bacteria and also against the fungus Botrytis, cinerea (Bayer et al., Chim. Acta 55 (1972) 224). PTT

is produced by the strain Streptomyces viridochromogenes Tu 494 (DSM 40736, DSM 4112).

From German Patent No. 2,717,440 it is known that 10 PTC acts as total herbicide. In published PCT application WO 86 / 02.097, plants whose resistance to PTC is due to their overproduction of glutamine synthetase are described. However, such overproductions, for example as a result of a gene amplification, save the risk of instability itself. Thus, in the case of such instability, the overproduction of glutamine synthetase would decline and the competitive inhibitory effect of PTC would become effective again.

In contrast, the invention defined in the claims relates to a resistance gene sequence against PTC and its use in the production of PTC resistant plants. Furthermore, this gene sequence can also be used as a resistance marker. Furthermore, the gene is suitable for selective N-acetylation of the L-form of racemic PTC.

The resistance gene sequence of the invention against PTC can be obtained from the entire DNA of Streptomyces viridochromogenes <DSM 4112 selected for PTT resistance, by clipping with BamHI, cloning a 4.0 kb fragment and selection for PTT resistance. The restriction map 30 (Figure 1) further characterizes this 4.0 kb fragment.

By cloning subregions of this 4 kb fragment, the location of the code area is located closer. Hereby, it appears that the resistance gene is on the 1.6 kb SstII-SstI fragment (positions 0.55 - 2.15 in Fig. 1).

By digestion with BglII, the 0.8 kb fragment is obtained which, after incorporation into a plasmid and transformation of S.

I DK 175254 B1 I

I 2 I

In lividans, PTT provides resistance. This resistance is conditional

I of the N-acetylation of PCT. IN

In the sequencing according to Maxam and Gilbert of the 0.8 kb I

In long fragments, the DNA sequence yields I (appendix). From the open I

In 5 reading frames in this sequence one can find the location of '' I

In the resistance gene (from position 258). End of gene sequence I

You are at the penultimate of the rendered nucleotides (style I

In length 806), i.e., the last nucleotide (position 807) is I

At the beginning of the stop codon. IN

In the 10 I DNA sequence I, the "Shine-Dalgarno Sequence" is produced

You raised by underlining, just as it does as a start codon

You know GTG. The defining reading frame is also expressed in I

In the top line. IN

In the DNA sequence II, the restriction cutting sites I show

In 15 of the sequenced gene. Enzymes that cut the sequence more

More than six times, not specified. IN

In the antibiotic PTT is absorbed by bacteria and degraded

I to form PTC. This also inhibits bacteria

In the glutamine synthetase, so that the bacteria die from glutamine-I

In 20 shortages. Accordingly, PTT-producing bacteria must possess an I

In mechanism that protects them from the effect of PTT, that is to say

Either hinder the resumption of the produced PTT or I

You enable a change of the degradation product PTC. Surprise- I

In the known PTT manufacturer S. viridochromogenes DSM 4112 is imid

For 25 years sensitive to its own antibiotic. Unexpectedly, you are

However, it succeeded in selecting PTT-I

In-resistance with the surprisingly high rate of approx. 10 “^ at ^ I

You will find selectants that are resistant to PTT and equal

You are led to suppress the background growth of the nearby colonies

In 30 kidneys. IN

I From DNA of these selectants, a gene bank I is created

You know that the DNA is isolated, cleaved with BamHI and ligated to I

In a streptomycet vector. The ligation mixture is transformed into I

In the commercially available strain S. lividans TK 23, where I

I 35 for each 1 mg ligation mixture is obtained from ca. 5000 to 10,000 I

In transformants with a built-in of approx. 1 to approx. 5 kb. IN

3 DK 175254 B1

Among the transformants are PTT-resistant S. livi-dance strains. Upon isolation of the plasmid and retransformation in S. lividans, it may be shown that the resistance is plasmid encoded.

The gene responsible for resistance resides on a 4 kb Bam HI-5 fragment (Fig. 1). The code area is located on the 0.8 kb BglII fragment. The BamHI fragment does not contain any intersection sites for the enzymes Clal, EcoRI, EcoRV, Hind-III, Hpal, KpnI, PvuI, PvuII and XhoI.

Comparison with the restriction map of an unspecified resistance gene from S. hygroscopicus FERM BP-130 / ATCC 21705 (European Patent Application Publication No. 173,327, Fig. 7) shows that the resistance gene sequence of the invention is different from the known gene found by the search for the PTT-15 biosynthetic genes.

By incubating cell extracts of S. viridochromogenes DSM 4112 and S. lividans TK 23 on the one hand and the PTT-resistant S. viridochromogenes selectants and a plasmid bearing S. lividans transformant on the other side with 20 PTC and acetyl-coenzyme A, it can be shown that the latter cells show an acetylating activity. Chromatographic examination results show that acetylation occurs on the amino group.

Also, since in E. coli a PTT-25 resistance can be detected, and the resistance mechanism also functions in gram-negative bacteria, a resistance due to transport phenomena can be excluded. Accordingly, upon coupling to vegetable promoters with suitable vectors, the resistance sequence of the invention can be transformed into plants, and PTT-resistant plants can thus be produced.

The N-acetylation of PTC can also be used for racial separation of synthetic D, L-PTC since selectively only acetylation of the L-form occurs.

Accordingly, the invention also relates to the use of the resistance gene sequence for selective N-acetylation of the L-form of racemic PTC.

I DK 175254 B1 I

i 4 I

In the I gene encoded by the resistance gene sequence of the invention

Thus, in PCT acetyltransferase can be used to separate I

In racemic PCT such as may be prepared, for example, according to I

In German Patent No. 2,717,440, in the optical antipodes, I

In that the racemate is exposed to the acetylating effect of this

In enzyme, the L-form being selectively attacked, while the D-form ^ I

You remain unchanged. The mixture thus obtained can i

You are then separated on the basis of its various properties

In a manner known per se. IN

I It is known to contact N-acyl-D, L-amino acids

I with optionally carrier-fixed acylases, the L-amino acid I

I selectively releases which acid can be extracted from the mixture

In gene with N-acyl-D-amino acid after acidification with non-water mixture

In solvents only (British Patent No. 1,369,462). IN

For example, a corresponding separation of N-acyl-D, L-PTC is I

Known from German Publication No. 2,939,269 or I

In U.S. Patent No. 4,226,941. IN

In the D-PCT remaining in accordance with the invention, on I

In a known way, European patent application is racemized with of- I

In the publication number (EP-A) 137,371, Example 8) and I

You are then returned to the process. IN

In the isolation of the enzyme, by which here and in it I

In the following also always understand the enzymatically active I

In part, is possible, but not necessary. If the enzyme is isolated, I

In 25, it can be used in free or carrier-fixed form. Suitable I

For example, carriers are described in EP-A No. 141,223. Her- I

Preferably, however, the enzyme is not isolated, but one is used

In any PTC-resistant cells expressing the enzyme I

In accordance with the invention. Thus, one can conveniently use. IN

In the PTT-resistant selectants of S. viridochromogenes DSM I

I 4112. However, advantageous may also be any, I

I, transformed with the gene sequence of the invention into I

In use capable of expressing PTC-acetyl-I

In the transferase. The gene according to the invention, whereby

I must also understand active parts of this, in the for- I

In binding in plasmid integrated form or with other common I

5 GB 175254 B1 genetic engineering methods, for example by transfection. Convenient, for example, is the incorporation into an E. coli expression plasmid and transformation of E. coli with such a plasmid, for example, according to the methods known from EP-A Nos. 163,249 and 5,171,024.

For N-acetylation according to the invention of the L-PTC in the racemate, the cells expressing the PTC-acetyl transferase can be used in free or fixed form, with the usual fixation methods used (e.g. German Publication No. 3.237 .341 and literature cited therein).

The enzymatic acetylation according to the invention of L-PTC is carried out in the manner customary for enzymatic reactions, the process conditions according to the given conditions of the organism used. In principle, the same methods as for the aforementioned selective afacylation method are considered.

In the following examples, the invention is further elucidated.

Parts and percentages are by weight, unless otherwise stated.

Example 1: PTT resistant selectants

The strain S. viridochromogenes DSM 4112 is grown on minimal medium (Hopwood et al., Genetic Manipulation of Strep-25 tomyces, A Laboratory Manual, The John Innes Foundation,

Norwich, England (1985), p. 233) and PTT is added in increasing concentrations. At a concentration of 100 µg / ml, one resistant colony per ca. 105 colonies. 1 2 3 4 5 6

Example 2: Preparation of the vector 2

The plasmid pSVH1 (European Patent No. 070,522) 3 is cut with BglII, the approx. 7.1 kb fragment is isolated and 4 ligated with the 1.1 kb Bell fragment with thiostrepton resistance (European patent application with publication number 158,201). The 8.15 large plasmid pEB2 is obtained (FIG.

2).

I DK 175254 B1 I

I 6 I

In Example 3: Isolation of the resistance gene I

I From the selectants of Example 1, I is isolated

Throughout the DNA and cleave it with BamHI. The plasmid pEB2 is opened I

In the same way with BamHI, the two mixtures are combined and ligated. IN

In 5 The ligation mixture is transformed by S. lividans TK 23 'I

I (available from the John Innes Foundation), as per 1 // g I

In ligation mixture, from 5000 to 10,000 transformants are obtained

With a built-in from approx. 1 to approx. 5 kb. By selection with I

For PTT resistance, two resistant S. lividans colo- I are obtained

In 10 kidneys. From these, the recorded plasmid is isolated and cut

In with BamHI. A 4 kb BamHI fragment is obtained which carries it

In the gene responsible for resistance. This plasmid is designated I

In pPR1 (Fig. 3). IN

I By retransformation to S. lividans TK 23, I can

In Figure 15, PTT resistance is shown to be plasmid-encoded since transformants I

I grow on minimal medium containing 100 μg PTT per ml. ml. IN

In Example 4: Detection of the inactivation of PTC by N-acetyl-I

In honor I

I 20 To detect the acetylated activity of the I

In cloned fragments, the following strains are examined:

I S. viridochromogenes DSM 40736, S. viridochromogenes (PTT-I

I-resistant mutants), S. lividans TK23 and S. lividans TK I

I 23 (pPR1). IN

In addition, the strains are seeded in light medium A (European I

In patent application with publication number 158,872, p

6) and incubated for 2 days at 30 ° C in a washing machine. IN

After harvest, 1 g of mycelium is dissolved in a suitable buffer (Example I

In pelvis RS buffer: C. J. Thompson et al., J. Bacteriol. 151 I

In 30 (1982), 678-685) with ultrasound. A typical experiment for I

The measurement of PTT degradation proceeds as follows *. IN

To 250 µl of crude extract is added 100 filter PTC-I

I solution (250 µg / ml) and 50 μϋίθΓ (4 mg / ml) acetyl-CoA, I

Incubate for 2 hours at 30 ° C. They then added more

In 35 PTC amounts present are measured by HPLC. Thereby I

You get the following result:

7 DK 175254 B1

Strain untranslated PTC

applied PTC

5 S. lividans TK23 100% S. viridochromogenes 72% (DSM 40736) S. viridochromogenes 7% selectants 10 S. lividans TK 23 (pPRl) 31%

The presence of N-acetylation of PTC can be demonstrated by comparison with reference substances by thin-layer chromatography (no staining with ninhydrin).

I DK 175254 B1 I

I I

In DNA Sequence I I

I IleTrpSerAspValLeuGlyAIaGlyProValLeuProGlyAspAspPhePheSerLeuGlyGlyThrSerlle I

I A5pLeu61uArgArgPro61y61yArgSerGlyAleAlaArgGlyArgLeuLeuLeuProArgArgHi5LeuHi5 I

I ArgSerSlyAIaThpSerTrpGlyProValArgCysCysProGlyThrThrSerSerProSerAlaAlaProPro I

I AGATCTG6AGCGACGTCCTG & GGGCCGGTCCGSTGCTGCCCGG6SAC6ACTTCTTCTCCCTCGSCGGCACCTCCA 75 I

I TCTA6ACCTC6CTGCAGGACCCCC6GCCAGGCCACGAC5G6CCCCTSCTGAAGAAGA6G & A5CC6CC5TGGAGGT _ I

I SerArgSerArgArgGlyProProArgAspProAlaAlaArgProArgSerArgArgGlyArgArgCysArgTrp I

I AspProAlaUalAspGlnPpoGlyThrArgHisGlnGlyPpoValUalGluGluGlyGluAlaAla61yGlyA5P I

I IleGlnLeuSepThrArgProAlaProGlyThrSerGlyProSerSerLysLysGluArgProPpoyalGluttei I

I SerAlaLeuArgUaiyalSerApglleArgLysGluLeuGly ^ JalPpoLeuArgLeuAlayalllePheGluThr I

I LeuGly'JalAlaGlyGlyLeuAlaHisProGlnGlyThpArgApgAlaThpPpoAlaArgApgAapLeuApgAsp I

I SerArgArgCys & lyTrpSerArgAlaSerAlaArgAsnSerAlaCysHisSerGlySerProOP Ser5erApg. IN

I TCTC & GCGTTGCGGGTG5TCTCGC6CATCCGCAA6GAACTC6GC6TGCCACTCCG & CTCGCC6T5ATCTTCGAGA 150 I

I A6AGCCGCAACGCCCACCAGAGCGCSTA6SCGTTCCTTGAGCCGCACGGTGA6SCCGASCGGCACTAGAAGCTCT I

I ArgProThpAlaProProArgAlaCysGlyCysProUalArgArgAlaUalGlyAlaArgArgSerArgArgSer I

I ArgApgGlnProHi 5AspArgAlaAspAlaLeuPhe61uAlaHisTrp61uProGluSlyHisAspGluLeuArg I

I 51uAlaAsnArgThrThr61uArgf1etArgLeu5erSerProThrGly5erArgSerAlaThrIleLys5erVal I

I ProSerleuGluAlaValAlaGluSer-ValLeuArgGluLeuLysGlyThrAM OC Arg & lyAlaArgHisPro I

I AlaValProGlySerGlyGlyArglleArgThrProArgThrSluGlyAspVelValLysApgCysProProPro I

I ArgArgProTrpLysArgTppProAsnPpoTyrSerAlaAsnOP ArgGlyArgSerLysGluValProAlaThr I

I CGCCGTCCCTGGAASCGGTGGCCGAATCCGTACTCCGCGAACTGAAG5G6ACSTA6TAAAGAG6TGCCCGCCACC 225 I

I gcggcagggaccttcgccaccsgcttaggcatgaggcgcttgacttcccctgcatcatttctccacgggcggtgg I

I AlaThrGlyProLeuProProArgIleArgyalGlyArgValSerPrD5erThrThrPheLeuHi5Gly61yGly I

I ApgGlyGlnPheArgHisGlyPheGlyTyrGluAlaPheGlnLeuProArgLeuLeuSerThrSlyAlaUalArg I

I GlyAspArgSepAlaThrAlaSerAapThrSerArgSerSerPheProyalTyrTyrLeuProAiaArgTppGYy I

I LeuSerGlnAsnThrG1uGIyArgProHi sUa] 5erPro61uArgArgProya1 GIul1eArgProA] aThpAla I

I AiaPheAlaGluHisArgArgLysThrThrArgGluProArgThrThrProGlyArgAspPpoSerArgHisArg I

I ArgPheArgApgThrProLysGluAspHisThrOP AlaGlnAsnAspAlaArgSerArgSepyalProProPro I

I C6CTTTCSCAGAACACCGAA6SAAGACCACAC5IÉA6CCCA6AAC & ACSCCCG6TC6A6ATCC6TCCCGCCACC & 300 I

I BCGAAAGCSTCTT & TS & CTTCCTTCT65TGTGCACTCG65TCTT5CT & C & GGCCÅ & CTCTAGGCA &&& CG6TGGC I

I AlaLysAlaSerCysArgLeuPheyelValArQ5erGlyLeuValVal61yProArgSer61yAspArgTrpArg I

I LysArgLeu ^ alGlyPhyserSerTrpyalHisAIaTrpPheSerAlaArgAspLauAspThrGiyGlyGlyGly I

I Ser61uCy5PheyalSerProLeuGlyCysThrLeuGlySerArgArg6lyThrSerIleArgGlyAlaWalAla I

I AlaAspMetAlaAlaValCysAspIleyalAsnHisTyrlleGluThrSerThrValAsnPheArgThrGluPro I

I ArgArgHxsGlySlyGlyLeuArgHisArgGlnSerLeuHisArgAspGluHisGlyGlnLeuProTyrGlyAla I

I ProProThrTrpArgArgSerAlaThrSerSerlleThrThrSerArgArgAlaArgSerThrSerValArgSer I

I CCSCCGACATS5CG5CGGTCT6CGACATCSTCAATCACTACATCGASACSASCACGGTCAACTTCCSTACGGAGC 375 I

I SGC5GCTGTACCSCC & CCAGAC6CT6TA6CAGTTA5T6AT6TAGCTCT6CTC6TGCCA6TTGAA & SCAT6CCTCG I

I ArgArgCysProProProArgArgCysArgOP AspSerCysArgSerSerCysProOP SerGlyTyrProAla I

I SlyUalHisArgArgAspAlayalAspAspIleUaiyalAspLeuArgAlaArgAspyelGluThrArgLeuArg I

I AiaSerMetAlaAlaThrSlnSerMetThrLeuOP AM MetSerValLeuValThrLeuLysArgVaiSerGly I

I GlnThrProSlnGIuTppIleAspAspLeuGIuArgLeuSlnAspArgTyrProTrpLeuValAlaGIuVaiGlu I

I AlaAspSerAla61yUalAspArgArgPro61yAlaProProGlyProLeuProLeuAlaArgArgArgGly61y I

I ArgArgLeuArgArg5er61ySerThrThrTrpSerAlaSerArgThrAlaThrProGlySerSerProArgTrp I

I C6CAGACTCCGCA9GAGTGGATCGAC5ACCTG6AGC6CCTCCAGGACCGCTACCCCT6GCTC6TC6CCGAG6TGG 450 I

I 6CGTCTGA6 & C5TCCTCACCTASCTGCT6GACCTC6CGGAG6TCCT66C6AT6GG6ACCGA6CAGC66CTCCACC I

I Ala5er61uAlaProThrSerArgArg61yProAla61y61yProGlySerGlyArgAlaArgArgArgProPrD I

I LeuSerArgLeuLeuProA5pValUal61nLeuAla51uLeuValAlaValSlyPrD61uA5p61yLeuHisLeu I

I CysUalGlyCysSerHisIleSerSerArgSerArgArgTrpSerArgAM SlyGlnSerThrAlaSerThrSer I

9 DK 175254 B1 DNA Sequence I (continued)

GlyUalUalAlaGlylleAlaTyrAlaGlyProTrpLysAlaArgAsnAlaTypAspTrpThpUalGluSerThr GlyArQArgApgArgHiaArgLeuArgArgProLeuGluGlyProGlnArgLeuApgLeuAspArgArgValAsp * ArgAla5erSerProAla5erProThrProA] aProSlyArgProAlaThrProThrThr61yPro5er5erArg AGG & C6TC6TCGCC66CATC6CCTACGCC6GCCCCT66AA6GCCCGCAACGCCTAC6ACT6GACCSTCGA6TCGA B25

TCCC & CAGCAGCGGCCGTAGCGGATGCGGCCGGGGACCTTCCGGSCGTTGCGGATGCTGACCTGGCAGCTCAGCT

ProArgArgArgArgCysArgArgArgArgSlyArgSerProSlyCysArgArgArgSerSerApgArgThrSer AIaAspA5pGlyAIaAsp5IyVei61yAla61yProLeuSIyAiaVal61yVeU / aiPro61yA3pLeuArgArg ProThrThrAiaProMetAlaAM AlaProGlyGlnPheAiaArgLeuAlaAM SerSlnyalThrSerAspUal

ValTyrValSerHisArgHisSlnArgLeuGlyLeuGlySerThrLeuTyrThrHisLeLiLeuLysSerfletGlu GlyValArgLeuPrDProAlaProAlaAlaArgThrGlyLeuHisProLeuHisPrePpoAlaGluUalHisGly ArgCysThrSepProThr61yThrSer61ySerA5pTppAlaProPro5erThrPpoThrCysOP SerProTrp cggtgtacgtctcccaccggcaccagcgsctcggactgggctccaccctctacacccacctgctgaagtccatgg see

6CCACATGCAGAGGGTGGCCGTG6TCGCCGAGCCT6ACCCGAGSTSSGAGATGTGGGTGSACGACTTCASGTACC

ProThrApgArgSlySlyAlaGlyAlaAlaApgUalPpoSepTppGlyApgCysGlyGlyAlaSerThpTrpPpo

Hi.5ValA5pGlyValProValLeuPro61uSerSlnAla61y61y61uVal61yVal61nGlnLeuGlyHi5Leu ThpTyrThr61uTppArgCy5TppApgSerProSerPpo61uValArgS ValTrpArgS

AlaGlnGlyPheLysSerVelValAlaVaIIleGlyLeuPpoAsnAspProSerValArgLeuHisGluAlaLeu 61yPraGlyLeu61n61uArg61yArgArgHi sArgThpAlaGlnArgProGluArgAlaPpoAlaApgGlyAla ApgProArgAleSerArgAlaTppSerPpoSepSerAspCysProThrThrArgAlaCysAlaCysThpArgApg ASGCCCAGGGCTTCAA6A & CGTGGTCGCCGTCATCGGACT6CCCAAC6ACCCGAGC6T6CGCCT6CACSAGGC6C G7 TCCSSGTCCCSAAGTTCTC5CACCAGCGGCAGTAGCCTGACGGGTTGCTGG & CTCGCACGCGGAC6T6CTCCGCG Pro61yProSerOP SepArgPpoApgArgOP ApgValAlaTppApgSlySerApgAlaGlyAlaArgProAla

GlyLeuAlaGluLeuAlaHiaAspGlyAspAspSerGlnGlyValValArgAlaHisAlaGlnValLeuApg & lu

AlaTrpProLy3LeuLeuThrThrAlaThrMetPro5er61yLeu5ep61yLeuThrArgArgCysSepAlaSer

SlyTypThpAlaArgGlyThrLeuArgAleAlaGlyTypLysHisGlyGlyTrpHisAspValSlyPheTppGln ArglleHisApgAlaArgAspAlaAle61ySerArgLeuGlnAlaApg61yLeuAlaApgApg61yValLeuAla 5epAspThpPpoArgAleGlyArgCysGlyGlnProAlaThrSerThr61yAlaGlyThrThrTppGly5ep61y TCG6ATACACC6C6C6CG66ACSCT6CG6SCAGCCG & CTACAA6CAC6666GCT6GCAC6ACGT6GGSTTCT6SC 750 AGCCTATGTGGCGCGCGCCCT6CGACGCCCGTCGGCCGAT6TTCGTSCCCCC5ACC5TGCT6CACCCCAA5ACCG ApglleCysApgAlaArgSepAlaAlaPpoLeuApgSerCysAlaArgProSerAlaApgApgPpoThrArgAla, SerVal61yArgAlaProArgGlnProCysGlyAlaValLeuValProAlaProValValHisPpoGluProLeu

ProTyrVaiAlaArgPpoValSerArgAlaAlaProAtt LeuCysPrDPpo61nCy5SerThpPpoAsnGlnCys

ArgAspPheGluLeuProAlaProProApgPpoValArgProValThpGinlle AiaArøLeuArgAlaAlaSlyProAlaProPpoArgProAleArgHisThpAsp SerAlaThr5erSepCysArgProApgProAlaPro5ep61yPro5erHisArg5er ASC6CSACTTCGASCT6CCGSCCCCGCCCCGCCCCSTCCSGCCCGTCACACAGATCT 807 TCGC6CT5AAGCTCGACGSCCG6GGCGGGGCG6GGCAG5CCG6GCAGT6T6TCTAGA AlaArgSerArgAlaAlaPro51yAlaGly61yArg61yAlaArgOP ValSerArg

AlaValGluLeuGlnApgSlyApgSlyAlaGlyAspProGlyAspCysLeuAsp

ApgSerLysSerSer & lyAla61y61yArgGlyThrArgGlyThrValCy5lle

I DK 175254 B1 I

I 10 I

In DNA Sequence II I

I 1 A & A7CT5GA6CGAC6TCCTG666SCCG & 7CC5 & 7GCT6CCCG6G6AC6ACTTCT7C7CCC I

I 7CTA & ACC7C5CTGCA66ACCCCC6GCCAGGCCACGAC & G6CCCC7GCTGAA6AA6A && 6 I

A A A A * A A A A A I

I 1 BGLII XHOII, 2 DPNI SAU3A. 5 G5U1, 12 AATII ACYI, 13 MAE11 I

I, 17 APYI ECORII, 2B R5RII, 27 AOAII, 35 BBVl, 39 AOAI NCII I

I SMAI, 40 NCII, 52 MBOII, 59 MNLI, I

I E1 7CGGCG6CACCTCCATCTC66C6TT6CGGGT6GTCTC5C6CATCC6CAAG6AACTCG6C6 I

I A5CC6CCST55AG67AGAGCCGCAAC6CCCACCASA5CGC67A6GCST7CCTT6ASCCSC, I

H a · I

I GB HGICl, 70 mi, 97 FNUD3I, 100 SFANI, 101 FOKI, I

I 121 T6CCACTCDG5CTC6CCG7GATCTTC6A5AC6CCGTCCCT6GAAGCGGT66CCGAA7CDG I

I ACG5TGA6GCCGAGCG & CACTA6AA6CTCT6C6GCAGGGACCTTCGCCADC6GCTTAGGC I

A * A A AAA

I 122 BGLl, 140 DPNI 5AU3A, 142 MBOII, 149 ACYI H&AI TTH1111, I

I 158 APYI ECORII, 1B9 CFRI 6DIII. 174 HlNFI, 180 R5AI, I

I 181 TACTCCGCGAACTGAA6GG6ACG7AGTAAAGA & GT6CCCGCCACCCGCTTTCGCA6AACA I

I AT6A66C6CTT6ACTTCCCCT6CATCATTTCTCCACGG6CGST666C6AAAGC6TCTT6T I

Η I

I 18B FNUDII, 201 MAE II. 2Π MNL I, 213 HGICl, 214 5DUI. IN

I 241 CCGAA6GAAGACCACACGT6AGCCCAGAACGACGCCC6G7CGAGATCCGTCCCGCCACCG I

I 6 & CTTCCTTCT6GTGTGCACTCGGG7CTTGCTGC5GGCCAGCTCTAGGCABG6CGST6GC I

A a * A A

I 247 MBOII, 254 AFUII, 255 PMACI, 255 MAEII, 260 HSIJII SDUI I

I, 271 ACYI H&AI, 275 NCII, 283 XHOII. 284 B1NI DPNI SAU3A, I

I 301 CCSCC6ACATB5CG6C6GTCT5C5ACATC6TCAA7CACTACATCSA & ACGAGCACG5TCA I

I 6GCS5CT6TACCSCC6CCA0ACGCTG7A6CA & TTAGTGAT5TAGCTC7GCTCGT5CCAST I

I 303 B6LI. 308 NLAIII, 3Z4 TTH111I, 350 HGIAI 5DUI, 357 HINCI I

I I

I 3G1 ACTTCC & T AC & GA6CC6CAGAC7CC6CAG & AST & GATCSAC6ACCT S6A & C6CCTCCAGS I

I 76AAGGCATGCCTC66CS7CTGASGC6TCCTCACCTA6CT6CT6GACCTCGC6GAS6TCC I

I A I

I 3B7 RSAI, 360 HINFI, 394 BINI, 395 DPNI SAU3A, 404 APYI ECOR I

I II, 405 GSUI, 409 HAEII, 413 MNLI, 414 6SU1. 415 APYI ECORII I

I, 419 AOAII ,. IN

I 421 ACC5CTACCCCT66CTCGTCGCC5AGGT66AG6SCGTCGTCGCCG5CATCGCCTAC6CCG I

I TGGC6ATGG6GACCGAGCA5C5BCTCCACCTCCCGCA5CAGCSGCC5TA6C6GATGC66C I

H A «I

I 430 APYI ECORII, 444 MNLI, 450 MNLI, 453 ACYI, 454 HGAI, 462 I

NAEI, 466 SFANI, 477 NAEI, I

I 4B1 GCCCC7G6AAG6CCC6CAACSCCTAC6ACT56ACCGTCSA57C6AC66T67ACGTCTCCC I

I CGGS5ACC7TCC565C6776C & 6A7GC76ACCTSGCAGC7CAGC76CCACAT6CASA6SG I

I 484 APYI ECORII, 511 AUAII. 519 HINFI, 521 ACCI HINCII SALI, I

I 530 R5AI, 532 MAEII, I

11 DK 175254 B1 DNA Sequence II (continued) 541 ACC6 & CACCAGCGGCTCG6ACTG6GCTCCACCCTCTACACCCACCTGCTGAAGTCCf TGGCC5TGGTCGCCGA6CCT6ACCC6A6GTGG6A6A7GT56ST5GACGACTTCAS5

A A Μ ΑΜΑ AA

544 H5ICI, 549 NSPBII. 563 H6IJII SOUI, 572 MNLI, 578 TAQII, 583 BSPMI, 595 NCOI STY1, 59G NLAIII, G00 MNLI.

B01 AGGCCCAGGSCTTCAAGAGC6T6GTCGCC5TCATCG6ACT6CCCAACGACCCGA6CGTGC TCCG6GTCCCGAAGTTCTCGCACCAGCGGDAGTAGCCTGADG5GTTGCTG6GCT0GCAC6 A Λ APYI EC0R1I 605, 550 AVAI, 661 6CCT6CACGA6GCGCTCG5ATACACCGCGCGC66GAC6CTGCGGGCAGCCGGCTACAA6C CSGACGTGCTCCSCGAGCCTATGTGGCGCGCGCCCT6CGAC6CCC5TC6GCCGATGTTC6 a a a a a a aa aa a Gb9 MnlI, 671 HaeII, Fnu DII B86, E87 BssHII, BB8 Fnu DII, Fnu DII B90, B95 HgaI, 658 BBUI. 705 BB <JI, 708 ΝΑΕ I, 716 ΤΤΗ1ΠΙ I.

721 ACG666GCTGGCACGACGTSGG6TTCTGGCAGC6CGACTTCGAGCTGCCGGCCCCGCCCC TGCCCCC6ACCGTGCT6CACCCCAA6ACC6TCGCGCTGAAGCTCGACGGCCGG6GCG65G

A A A and A A A

732 DRAIII, 736 MAE11, 749 BBUI, 753 FNUDII, 763 ALU], 7B4 B BUI. 767 NAEI,

781 6CCCC6TCCGSCCCGTCACACAGATCT CGGGGCA66CCGG6CA6TGTGTCTAGA

A A A

795 MAEIII, 802 BGLII XHOII, 803 DPNI SAU3A,

Claims (7)

1. Resistance gene sequence against phosphinothricin (PTC) II obtained from the entire DNA for phosphinothricyl II-alanyl-alanine (PTT) resistance selected Streptomyces viri II Dochromogenes DSM 4112 by cloning with BamHI, cloning a . I 4.0 kb fragment and selection with respect to PTT resistance. IN A gene sequence according to claim 1, characterized by the restriction map of FIG. 1. I I 10 A gene sequence according to claims 1 and 2, characterized in I by the DNA sequence I (appendix), from position # 258 to I in # 806. I Use of the gene sequence of claims 1 to 3 I for the production of PTT resistant plants. I I 15 Use of the gene sequence of claims 1-3 as a PTT resistance marker in bacteria. IN Use of the gene sequence according to claims 1-3 as a PTC resistance marker in plant cells. IN Use of the gene sequence according to claims 1-3 to 11 for selective N-acetylation of the L-form of racemic PTC. IN