EP0948603A1 - Sterol glycosyl transferases - Google Patents

Abstract

The invention relates to isolated DNA fragments or recombinant DNA constructs containing at least part of a sterol glycosyl transferase coding sequence. Substances used as sterols are either sterols in the strictest sense (cholesterol, ergosterol, beta-sistosterol, stigmasterol) or made from a skeletal structure of 5-alpha-cholesteran-3-beta-ol or 5-alpha-cholestan-3-alphol-ol (possibly modified by side-chains or double bonds in the ring systems). The invention also relates to DNA sequences, the use thereof to modify the content and/or structure of steryl glycosides and/or synthethic secondary products in transgenic organisms.

Description

Name: Sterol glycosyltransferases

The invention relates to DNA sequences which code for sterol glycosyltransferases, and to their use for changing the content and / or the structure of sterylglycosides and / or their synthetic secondary products in transgenic organisms.

Sterylglycosides and the biosynthetic secondary products Steryloligoglycosidede and acylated Sterylglycoside are natural substances found in plants as well as in some fungi and bacteria. Many physiological effects are described for these substances and their secondary products, e.g. Inhibition of vascular permeability, anti-tumor activity, anti-inflammatory and hemostatic effects (Okuyama, E. and Yamazaki, M. (1983) Yakugaku Zasshi 103: 43 ff; Normura, T .; Watanabe, M .; Inoue, K. and Ohata, K. (1978) Japan J. Pharmacol. 28, Suppl. HOP; Miles, DH; Stagg, DD and Parish, EJ (1979) J. Nat. Prod. 42: 700 ff; King, ML; ing, H . C; Wang, CT. And Su, M. (1979) J. Nat. Prod. 42: 701 ff .; Seki, J .; Okita, A .; Watanabe, M .; Nagagawa, T .; Honda , K .; Tatewaki, N. and Sugiyama, M. (1985) J. Phar. Sci. 74: 1259-1264), which suggest use as therapeutically active substances in humans. So far only ß-sitosterol-ß-D-glucoside isolated from plants is available as a medication for the treatment of prostate hyperplasia (e.g. as Harzol capsules, Hoyer GmbH, Neuss).

A disadvantage of these substances is that they are present in the organisms only in relatively small quantities and must be hiert by complicated processes specially ^¬ and cleaned. Moreover, some organisms that these substances contain pathogenic to humans and can be cultivated only with great effort, what their potential use as drug de- detergents with, emulsifying agents, as a raw material for plastics and for Her ^¬ position of liposomes when large quantities and must be of higher purity, currently appears to be of little use.

The enzymatic synthesis of sterylglycosides in the organisms from sugar nucleotides and sterols with a free OH group is carried out by the sugar-nucleotide-dependent sterol-glycosyltransferases (in short: sterol-glycosyl- transferases) catalyzed. These enzymes can be part of the organisms ^¬ men isolated and purified, but are not available in sufficient quantities and Qulitäten for economic applications.

The activity of these enzymes can be demonstrated using special in vitro enzyme detection systems. In one case, a sterol-glucosyltransferase from oats could also be purified to homogeneity (Warnecke and Heinz, 1994). However, no gene or other nucleic acid that codes for a sterol glycosyltransferase is known.

Furthermore, a number of nucleic acid sequences are known which are similar to the sequences described in this application. In no case was a sterol glycosyltransferase activity of the associated transcription product shown or even discussed. Such nucleic acid sequences may now be used, the content and / or the composition of sterol glycosides and Folgepro ^¬ Dukten to manipulate in certain organisms and to change so economically relevant characteristics of these organisms positive. So crops can be produced with improved tolerance or resistance to Vermin ^¬ approach to environmental influences such as soil salinity, drought, cold and frost. Microorganisms such as baker's and brewing yeast can also be improved with regard to ethanol and temperature tolerance.

In addition to the reaction product sterylglycoside, the enzyme itself can also be economically usable if it is genetically engineered in large quantities. The application for cholesterol quantification may be mentioned here as an example.

Furthermore, the sterol can glycosyltransferases - and this kodie ^¬ leaders DNA sequences - len due to the similarity of solanidine with Stero- - as enzymes, or for the provision of such enzymes, USAGE ^¬ det be used for the synthesis of solanine are responsible in Solanaceen. This could then be used to produce genetically modified solaninar plants or plants. By choosing suitable methods a sol ^¬ che reduction to certain plant parts or stages of development be ^¬ can be limited.

The object of the present invention is to isolate nucleic acid fragments to provide with which transgenic organisms can be produced, which have improved economically relevant properties or with which sterylglycosides and their secondary products can be produced in vivo or in vitro. These substances can a) be produced in larger quantities than in the starting organisms; or b) are produced by organisms that are easier and safer to cultivate than those in which these substances occur naturally; or c) be of novel structure and favorable to use natural ^¬ exhibit properties.

A method has been found to control the synthesis of steryl glycosides and their derivatives. For this purpose, nucleic acid fragments are provided which code for sterol glycosyltransferases in order to generate chimeric genes. These chimeric genes can be used to cell cultures ^¬ to transform plants, animals or microorganisms and change with their Sterylglycosidsynthese as ^¬.

The invention relates

An isolated DNA fragment DNA construct contained ^¬ tend least part of a sterol or sterol glycosyltransferase glycosyltransferase coding sequence in a narrow sense (1) or recombinant;

(2) a protein is the abgeleitetet ^¬ acid sequences from any of the nucleic shown in Figures 1-3 or 11-22.

(3) plasmids, viruses or other vectors, nucleic acids such as finishes in (1) de ^¬ oxide;

(4) genomic clones containing genes or parts of genes encoding a sequence as defined in (1);

(5) a chimeric gene that is capable of changing the content of sterol glycosyl transferase or sterol glycosyl transferase in the narrow sense, in particular sterol glucosyl transferase or sterol glucosyl transferase in the narrow sense, in a transformed cell;

(6) transformed cells, transformed microorganisms, plants or parts of plants which contain a chimeric gene as defined in (5);

(7) a method for producing Sterylclycosiden comprising culturing ^¬ fours defined in (6) transformed organisms;

(8) the steryl clycosides obtainable by the process defined in (7) or their derivatives; (9) a DNA fragment obtainable by one of the following or parts thereof: a.) Use of one of the in Figure 1-3 or 11-13 or 17 gezeig ^¬ th nucleic acid sequences as a hybridization sample; b) using the amino acid sequences shown in FIGS. 4, 5, 14-16, 18, 19, 21 or 22 for the synthesis of peptides or proteins which serve to obtain antisera; or c) i) comparing the nucleotide sequences shown in FIGS. 1-3, 11-13 or 17 or the amino acid sequences shown in FIGS. 4, 5, 14-16, 18, 19, 21 or 22 with each other or with already known ^¬ th nucleotide sequences or derived therefrom, amino acid sequences, ii) deriving and synthesizing appropriate specific oligo nucleotides from similar regions of these sequences, and iii) using these oligonucleotides to using a sequenzab ^¬ dependent protocol, in particular the PCR method, hither ^¬ provide nucleic acids sen for sterol glycosyl or sterol Glycosyltransfera ^¬ in the narrow sense, encode especially sterol glycosyl or sterol glucosyl in the narrow sense or parts thereof;

(10) a chimeric gene which contains a DNA fragment defined in (9) and which is able to determine the content of sterol-glycosyltransferase or sterol-glycosyltransferase in the narrower sense, in particular ^¬ sterol-glucosyltransferase or sterol, in a transformed cell -Glucosyltransferase in the narrower sense, to change;

(11) transformed cells defining a chimeric gene as described in (10) ent, ^¬ hold;

(12) organisms, in particular microorganisms such as bacteria and yeasts, the gene or genes, encoding sterol glycosyl or sterol-Gly ^¬ cosyltransferasen in the narrower sense, particularly sterol Glucosyltransfera ^¬ sen or sterol glycosyl in the narrow sense, by transformation deleted or interrupted with suitable chimeric genes;

(13) sterol glycosyl or sterol glycosyl in close ^¬ en sense, particularly sterol glycosyl or sterol glucosyl ^¬ transferases in the narrow sense, or portions thereof or fusion proteins with the aforementioned transferases from organisms as in (6) or ( 11) defi ned ^¬, is available; and

(14) Antisera, products from antisera, antibodies or parts thereof, which are directed against proteins as defined in (13). The nucleic acid fragments coding for sterol glucosyltransferases (Fig. 2, 17) could be isolated from Avena sativa and Arabidopsis thalliana. The deduced amino acid sequences of these nucleic acid fragments show surprisingly little resemblance to the familiar Se ^¬ sequences of steroid hormone glucuronosyltransferases. It is therefore surprising that we were able to isolate completely new nucleic acid fragments with our methods. No other nucleic acid fragments that code for sterol glycosyltransferases have been identified. The iso ^¬ profiled eukaryotic nucleic acid fragments are characterized in that they are surprisingly capable provided ^¬ ersequenzen with corresponding STEU, ^¬ mechanisms in both eukaryotes and prokaryotes Orga, and therefore herein difizierung without the typical eukaryotic processing and Mo ^¬, the synthesis effect of enzymatically active sterol-glucosyltransfer ^lawn .

The invention also relates to isolated nucleic acid fragments whose amino acid sequences have ended abgelei ^¬ defined similarities to the deduced amino acid sequences of the sequences shown in Fig. 12 or 13. The invention also relates to all plasmids, viruses and other vectors which contain these isolated nucleic acid fragments or parts thereof.

The amino acid sequences shown in FIGS. 4 and 18 show striking similarities with the derived amino acid sequence of a piece of genomic DNA from S. cerevisiae (see FIG. 9). This is the chromosome XII Cosmid 9470 (Genbank No. gb U17246). The similarity related to the 3 'region of the reverse reading frame of bp 32961-36557 (gene L9470.23). No function is known for this putative gene. Various parts of this gene are provided with suitable control sequences and, after transformation of E. coli with this chimeric gene, were surprisingly able to detect sterol-glucosyltransferase activity in cell homogenates of the transgenic cells.

The invention further relates to the use of Nukleinsäuresequen ^¬ zen of Figs. 1-3, 11-13 and 17, or the sequences derived therefrom Aminosäurese ^¬ for the isolation of genes or cDNAs encoding syltransferasen for other sterol Glyco ^¬. This applies, for example, to the use of the sequences or parts thereof as hybridization samples, the use of antibodies against a polypeptide which is encoded by the nucleic acid fragments or derivatives thereof. In addition, the derivative of oligonucleotides and their use in the PCR method, from the nucleotide or Aminosäu ^¬ resequenzen also by comparison to other sequences affected. The invention relates to all plasmids, viruses and other vectors which contain the nucleic acid fragments from FIGS. 1-3, 11-13, 17 or parts thereof or the yeast gene L9470.23 or parts thereof or nucleic acid fragments or parts thereof which are according to the in last paragraph methods described were iso profiled ^¬ containing and the lawn suitable in transformed cells for expression of sterol Glycosyltransfe ^¬ are. Protection claim is also applicable to all organisms (microorganisms, animals, plants, parts since ^¬ of, cell cultures) containing these chimeric genes and the products or extracts from it if the material composition of the Orga ^¬ mechanisms has been altered by the action of these chimeric genes .

The representation of nucleic acids in the figures is from the 5 'end to the 3' end, that of proteins from the amino terminus to the carboxyter minus. The amino acids are named in the one-letter code. The image ^¬ Ungen serve to illustrate the present invention. They show:

1: DNA partial sequences of an approximately 800 bp long DNA fragment which was obtained from the oat cDNA using the PCR method (see Example 3). A. 5'-terminal sequence walδe. B. 3'-terminal sequence wal9er.

2: DNA sequence of the nucleic acid fragment HaSGT, which was isolated from a cDNA expression gene bank from oat seedlings. It has a length of 2317 base pairs (bp) and contains an open reading frame from positions 1 to 1971. Start and stop codons can be found at positions 148-150 and 1972-1974.

Fig. 3: walδe comparisons of the partial DNA sequences, and 800 bp of the wal9er lan ^¬ gene DNA fragment with the sequence of the oat HaSGT (Fig. 1) (Fig. 2). The comparison was carried out using the CLUSTAL program (Higgins and Sharp, 1988, Gene 73, 237-244). A. Comparison between walδe and Ha ^¬ SGT. B. Comparison between wal9er and HaSGT. The * mark identical bases.

Fig. 4: amino acid sequence HaSGTP in the one-letter code, which from the DNA sequence of the HaSGT nucleic acid fragment was derived and which codes for a sterol glucosyltransferase with a molecular mass of 71 kD.

5: Comparison of the N-terminal amino acid sequence of the purified enzyme (N-TERMINUS) with the amino acid sequence HaSGTP derived from the oat clone HaSGT. The comparison was carried out using the CLUSTAL program (Higgins and Sharp, 1988, Gene 73, 237-244). The * mark identical amino acids. - denote non-existent or unknown amino acids.

6: Thin-layer chromatographic analysis of radioactive products from in vitro enzyme assays, which were carried out with cell-free homogenates from transformed E. coli cells (Example 5.).

The organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which with the eluent chloroform: methanol 85:15 (A) or chloroform: methanol: ammonia (25%) 65: 35: 5 (B). were developed. The Rf values of the radioactive, lipophilic reaction products were determined using a Berthold TLC analyzer and compared with authentic standards ^¬ s, which were detected with α-naphthol-sulfuric acid. Only one product was identified that was identified as sterylglucoside. The Rf value of the steryl glucoside at A in this case deviates from the usual value with this eluent because the eluent was not freshly prepared and the composition had changed due to evaporation. A. The E. coli cells were transformed with the plasmid pBS-ATG (Example 5.). B. The E. coli cells were transformed with the plasmid pBS-HRP (Example 5.).

Fig. 7: Western blot of recombinant sterol glucosyl transferases. Expressing each 40 ug protein from E. coli cells, the different parts of the oat HaSGT was subjected to SDS-polyacrylamide gel electrophoresis un ^¬ terworfen and then transferred to a hydrophobic membrane. The immunostaining was carried out with an antiserum against the sterol-glucosyltransferase purified from oats. Lanes 1 and 2: protein from E. coli cells which were transformed with the plasmid pBS-HRP. Lane 3: protein from E. coli cells transformed with the plasmid pBS-HATG. Lane 4: standard proteins with molecular weights of 31, 45, 66 and 97 kD. The proteins were stained with Ponceaurot, the standard proteins with marked with a pen and discarded.

Fig. 8: Dunnschichtchromatographische analysis of radioactive products of an in vitro enzyme assay which was carried out with cell-free homogenate of transformed with the plasmid pGALHAMl S. cerevisiae cells (In ^¬ game 6). The organic phase was applied to a silica gel 60 plate (Merck, Darmstadt), which was developed with the eluent chloroform: methanol 85:15. The Rf value of the radioactive, lipophilic Reaktionspro ^¬ domestic product was determined using a Berthold TLC analyzer and compared with authentic standards that were detected with α-naphthol-sulfuric acid. Only one product was identified that was identified as steryl glucoside.

9: Amino acid sequence derived from the DNA sequence of the S. cerevisiae gene L9470.23 in the one-letter code. The amino acids with which the second section of the fusion proteins begins, for which the plasmids of cloning 1 to 4 code (example 7), are marked.

10: Thin-layer chromatographic analysis of radioactive products from in vitro enzyme assays, which were carried out with cell-free homogenates from transformed S. cerevisiae cells (see Example 7). The organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which were developed with the eluent chloroform: methanol 85:15. The Rf values determined to the radioactive, lipophilic reaction products WUR ^¬ with a Berthold TLC analyzer and compared with authentic standards that were detected with α-naphthol-sulfuric acid. Only one product could be detected that was identified as steryl glucoside. A. The S. cerevisiae cells were transformed with the cloning 2 ^¬ the Plas mid. B. The S. cerevisiae cells were transformed with the plamide of cloning 4 (Example 5.).

11: DNA sequence of the DNA fragment Aper, which was isolated from Arabidopsis thalliana using the PCR method (Example 8).

Fig. 12: DNA sequence of the DNA fragment Kpcr, which was from the Solanum tuberosum with the PCR method (Example 8.).

Fig. 13: DNA partial sequence of the DNA fragment Cpcr, which with the PCR method was isolated from Candida albicans (Example 8.).

14: A. Amino acid sequence ApcrP in the one-letter code, which was derived from the DNA sequence of the DNA fragment Aper. B. Comparison of the amino acid sequence ApcrP with the oat sequence HaSGTP. The comparison WUR ^¬ de using the program CLUSTAL (Higgins and Sharp, 1988, Gene 73, 237- 244) was performed. The * mark identical amino acids.

15: A. Amino acid sequence KpcrP in the one-letter code, which was derived from the DNA sequence of the DNA fragment Kpcr. B. Comparison of the amino acid sequence KpcrP with the oat sequence HaSGTP. The comparison WUR ^¬ de using the program CLUSTAL (Higgins and Sharp, 1988, Gene 73, 237- 244) was performed. The * mark identical amino acids.

16: A. Amino acid sequence CpcrP in the one-letter code, which was derived from the partial DNA sequence of the DNA fragment Cpcr. B. Comparison of the amino acid sequence CpcrP with the oat sequence HaSGTP. The comparison was carried out using the CLUSTAL program (Higgins and Sharp, 1988, Gene 73, 237-244). The * mark identical amino acids.

17: DNA sequence of the nucleic acid fragment AtSGT, which was isolated from a cDNA expression gene bank from oat seedlings (Example 9). It has a length of 2353 base pairs (bp) and an open entält Le ^¬ seraster from position 1 to 2023. start or stop codon found at positions 113-115 and 2023-2025.

18: Amino acid sequence AtSGTP in the one-letter code, which was derived from the DNA sequence of the nucleic acid fragment AtSGT.

Fig. 19: Comparison of the amino acid sequences HaSGTP and AtSGTP. The Ver ^¬ was immediately performed using the program CLUSTAL (Higgins and Sharp, 1988, Gene 73, 237-244). The * mark identical amino acids.

Fig. 20: Dunnschichtchromatographische analysis of radioactive products ei ^¬ nes in vitro enzyme assays, the E. transformed with cell-free homogenate of the ^¬ Plas mid pBS-AtSGT was performed coli cells (Example 10). The organic phase was that corresponds on silica gel 60 plates (Merck, intestinal ^¬ city) coated with an eluent Chloroforr methanol 85:15 was wrapped. The Rf value of the radioactive, lipophilic reaction product was determined with a Berthold TLC analyzer and compared with authentic standards, which were detected with α-naphthol-sulfuric acid. Only one product was identified that was identified as sterylglucoside.

Fig. 21: Partial amino acid sequence of the A HaSGTP Letter B ^¬ ben code.

Fig. 22: Partial amino acid sequence of the A AtSGTP Letter B ^¬ ben code.

Fig. 23: Partial amino acid sequence in the one-letter code, which was derived from the S. cerevisiae gene L9470.23.

The invention is illustrated below using examples:

1. Purification of UDP-glucose: sterol glucosyl transferase, antiserum, N-terminal sequencing:

The purification of the enzyme, the production of an antiserum to the Pro ^¬ tein and Western blot analysis was corner by known methods warning ^¬, DC and Heinz, E. (1994) Plant Physiol. 105: 1067-1073. An analysis of partial sequences of the amino acid sequence of the protein was then carried out. The protein, which had been purified to homogeneity, was subjected to SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon P, Millipore, Eschborn). The protein was stained with Coomassie Brilliant Blue R 250 (Biorad, Munich) and the band corresponding to a molecular mass of 56 kD was cut out of the membrane. The protein was then sequenced directly N- terminal or proteolytically cut to internal Bruchstüc ^¬ ke to obtain. The protein was according to Bauw, G .; van den Bulcke, M .; van Damme, J .; Puype, M .; digested with trypsin 194-196 and the proteolytic breakdown ^¬ pieces with a high-performance liquid chromatography system (130A, Applied Biosystems,: Van Montagu, M. and Vandekerckhove, J. (1988) J. Chem Prot. 7. Weiterstadt) separated on a "reverse phase" column (Vydac C4, 300 Angstrom pore diameter, 5 μm particle size). The peptides were eluted with a linear gradient (0-80% B, solution A: water with 0.1% trifluoroacetic acid, solution B: 70 acetonitrile with 0.09% trifluoroacetic acid) at a flow rate of 0.2 ml / min. The elution pattern of the peptides corresponded to the pattern which typically corresponds to a trypsin self-digest. Even after repeating the experiment several times, no peptide could be assigned to the purified protein due to the retention time. Most peptides were then sequenced. However, the sequences all corresponded to the amino acid sequence of the trypsin. This Versu ^¬ che showed that the purified very hydrophobic membrane protein the Try-resists psinverdau well and that let the hydrophobic Peptidbruchstuecke apparently hardly solve even from the membrane. However, the trials continued with an alternative strategy. After renewed digestion attempts, the eluted peptides were subjected to rechromatography (using a Nucleosil C8 column, 120 × 1.6 mm, gradient as above). Surprisingly, it turned out that a translucent bar ^¬ homogeneous peptide Trypsinselbstverdaus ent a minor component ^¬ held, not corresponded to the amino acid sequence of trypsin. In the one-letter code, this sequence is: MTETTIIQALEMTGQ. The Proteinse ^¬ quenzierungen were carried out by the standard Edman degradation on an automatic Sequenziergerdt (473A, Applied Biosystems, Weiterstadt).

Were determined nosäuren the N-terminal amino acid sequence to a length of 15 Ami ^¬. In the one-letter code, this is: DVGGEDGYGDVT-VEE. - In addition, the sequence of a peptide fragment was determined over a length of 14 amino acids. In the one-letter code, this is: MTETIIQALEMTGQ.

2. Creation of an oat cDNA bank:

A cDNA expression ^{gene bank} from oats was created in order to isolate complete clones of the sterol-glucosyltransferase.

First, RNA was old of 4 days and in the dark cultivated oat germ ^¬ lingen (Avena sativa, variety Alfred) isolated. For this purpose, the seedlings were ground in liquid nitrogen. The powder was taken up in a buffer with guanidine isothiocyanate and filtered. The RNA was sedimented by a cesium chloride solution in the ultracentrifuge. The sediment was ^removed in aqua dest. recorded and the RNA precipitated with 2 parts of ethanol and 0.05 parts of acetic acid and sedimented. The sediment was in Aqua dest. added. MRNA was isolated from the oat RNA. This was done with Dynabeads Oligo (dT) from Dynal GmbH (Hamburg) according to the instructions for use. Using the ZAP-cDNA Synthesis Kit (Stratagene, Heidelberg) cDNA was synthesized from the isolated RNA according to Her ^¬ manufacturers specifications synthesized and a cDNA library created.

3. Isolation of partial DNA sequences of sterol glucosyltransferase from oats using the PCR method.

From the sequences of the N-terminal amino acid sequencing (see

1.) Oligonucleotide primers were derived:

DW1 = 5'-GGITAYGGIGAYGTNACIGTIGARGA-3 '(forward sprayer)

DW2 = 5'-GAYGTIGGIGGIGARGAYGGNTA-3 '(forward sprayer)

served as a reverse primer

XXS4T = 5'-GATCTAGACTCGAGGTCGACTTTTTTTTTTTTTT-3 '

Abbreviations: Y = C and T - D = G and A and T - I = inosine - N = A and G and C and T - R = G and A - K = G and T - S = G and C - H = A and T and C - B = G and T and C - V = G and A and C - X = C and I - W = A and T - M = A and C

The polymerase chain reaction (PCR) method was carried out as follows:

Reaction mix: 46 μl aqua dest .; 5 ul Boehringer (Mannheim) 10 x PCR buffer; 1 µl each of 10 mM dATP, dGTP, dCTP, dTDP; 1 μl each 100 μM DW1 (or DW2), XXS4T; 0.25 ul Boehringer Taq polymerase; 0.5 μl cDNA from oat seedlings (see 2., concentration not determined). Reaction conditions: 94 ° C, 3 min; 30x (94 "C, 40 s; 53 ° C, 1 min; 72 ° C, 3 min); 72 C °, 10 min.

These PCR reactions with specific primers (DW1 and DW2) and a non-specific primer (XXS4T) mounted on all of the clones of the cDNA library am ^¬ det, the polyA tail containing a so-called, were unsuccessful. This means that there could no amplified DNA fragment was cloned and sequenced, the sequence portions contained which set the introduced corresponded ^¬ primers. The PCR reaction was carried out in numerous modifications (different temperature program, so-called nested PCR with the primers DW1 and DW2), but remained unsuccessful.

As a result, attempts were made to sequence peptide fragments of the purified protein (see FIG. 1) in order to be able to carry out PCR reactions with two specific primers.

The following oligonucleotide primer was derived from the sequences of the peptide amino acid sequencing (see 1.):

Whale = 5'-GCYTGDATDATIGTYTCIGTC-3 '(reverse primer)

The polymerase chain reaction (PCR) method was carried out as follows:

Reaction mix: 46 μl aqua dest .; 5 ul Boehringer (Mannheim) 10 x PCR buffer; 1 µl each of 10 mM dATP, dGTP, dCTP, dTDP; 1 μl each 100 μM DWI, whale; 0.25 ul Boehringer Taq polymerase; 0.5 μl cDNA from oat germ (see 2nd, concentration not determined).

Reaction conditions: 94 ° C, 3 min; 30x (94 ° C, 40 s; 53 ° C, 1 min; 72 ° C, 3 min); 72 ° C, 10 min.

A successful PCR reaction could only be carried out using the specific reverse primer whale: agarose gel electrophoresis with 15 μl of the reaction mixture gave a DNA band e of approx. 800 bp length.

This piece of DNA was cloned using the Sure Clone ligation kit (Pharmacia, Freiburg) in a plasmid vector and the 5 'and 3' end part ^¬ sequenced. These sequences (walδe and wal9er) are shown in Fig. 1.

4. Isolation of complete clones

The cloned piece of DNA (see 3) was labeled and used to screen a cDNA library (see 2) used transferase to complete clones of the sterol glucosyl ^¬ isolate.

The DNA piece was labeled with the PCR DIG Probe Synthesis Kit (Boehringer, Mannheim) according to the manufacturer's instructions, non-radioactive, DIG = one di goxigenin-containing system for labeling nucleic acids from Boehringer (Mannheim). The labeled sample was then used to search the oat cDNA library. The method is described in the Boehringer DIG System User4s Guide for Filter Hybridization (Plaque Hybridization, Colorimetric Detection with NBT and BCIP). There were 250,000 infektionsfä ^¬ hige phage particles scanned (hybridization temperature 69 ° C). It found WUR ^¬ the 50 positive clones, 13 of which a second and third screening were subjected. These 13 positive clones were converted from the phage form into the plasmid form (in vivo excision according to the Stratagene Protocol ZAP-cDNA synthesis kit, Heidelberg).

An approximately 2300 bp long clone (hereinafter referred HaSGT) completeness, ^¬ sequenced dig and double-stranded. This sequence is shown in FIG. 2: The partial sequences (walδe and wal9er) of the cloned PCR fragment are over 95% identical to the clone HaSGT (FIG. 3). This clone is 2317 bp long and shows an open reading frame from bp 1 to bp 1971. A start ^¬ codon (ATG) for the translation starts at bp 148. If the open reading ^¬ mimic in an amino acid sequence translated (HaSGTP, Fig. 4 ), the amino acid sequence shows complete identity with the amino acid sequence of the peptide fragment of the purified protein and almost complete Identitä ^¬ th to the N-terminal amino acid sequence of the purified protein (14 amino acids of 15 are identical, Fig. 5). This agreement clearly shows that the cloned cDNA corresponds to the purified protein. The Un ^¬ terschied at an amino acid presumably due to allelic differentiation ^¬ zen. Since the first amino acid of the N-terminal amino acid sequence agreed of ger ^¬ protein (D) of the amino acid 133 corresponds to the open reading frame of clone HaSGT, it can be expected that the clone for a pre coded ^¬ protein, the cut in vivo a mature protein (mature ripe protein). The plasmid which contains the 2317 bp long oat clone in the vector pBluescript I SK (inserted between the EcoRI and the Xhol interface) is called pBS-HaSGT in the following.

5. Functional expression of parts of the clone HaSGT in E. coli.

To prove that the cloned DNA sequence (see 4.) codes for a sterol glucosyltransferase, parts of the clone HaSGT were functionally expressed in E. coli.

-It were performed two cloning into appropriate vectors for expression by ^¬: a) This cloning generated a plasmid (pBS-HATG), which encodes a fusion protein ^¬, the first amino acids from the pBluescript lacZ OPE ron and the polylinker derived (normal printed, see below) and the following amino acids which after initiating methionine, the sequence in an amino acid ^¬ translated nucleotide sequence of the HaSGT corresponding (under ^¬ painted, see below).

The plasmid pBS-HaSGT was cut with the restriction enzymes Eael and Eagl and the linearized part, which contains the vector sequences, ligated together with itself. The resulting plasmid codes for a fusion protein, the beginning of which looks like this: MTMITPSSELTLTKGNKSWSSTAVAADADEPTGG ...

b) This cloning generated a plasmid (pBS-HRP), which encodes a Fusionspro ^¬ tein, the first amino acids from the pBluescript lacZ operon and the polylinker derived (normal printed, see below) and the second part of the putative mature protein from oats equivalent (unterstri ^¬ chen, see below).

A PCR test is carried out for this cloning, in which the DNA of the

Plasmid pBS-HaSGT was used as the matrix DNA. The following were

Primer used:

DW 15 = GATGAGGAAATTCACTAGTTG

DW 20 = GATGGATCCACTTGATGTTGGAGG

A PCR fragment of approximately 500 bp in length was nigt gerei ^¬ on an agarose gel, cut with the restriction enzymes BamHI and NdeI and still ^¬ again gel purified, from which a fragment was isolated from approximately 450 bp in length.

The plasmid pBS-HaSGT was cut with the restriction enzymes BamHI and Ndel and a fragment of approximately 4300 bp in length was eluted from an agarose gel. This fragment was ligated with the cut PCR fragment and used to transform E. coli. From the transformier ^¬ th cells plasmid DNA was isolated and partially sequenced. The plasmid DNA codes for the following fusion protein: MTMITPSSELTLTKGNKSWSSTAVAALELVDLDVGGEDGY ...

In the E. coli cells transformed with the plasmids pBS-HATG and pBS-HRP, it was checked whether the corresponding fusion protein was expressed by an in vitro enzyme assay for the detection of sterol-glucosyl transferase activity was carried out with cell homogenates.

The cells of 2 ml overnight culture (2 ml LB-ampicillin, 37 ° C, 14 h) WUR ^¬ sedimented and resuspended in 1 ml lysis buffer (50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT; 1 mg / ml lysozyme (from Hühnerei, Boehringer, Mannheim); 200 μM Pefabloc (Merck, Darmstadt); 0.1% Triton X100 After 5 minutes incubation at 20 ° C, the suspensions were placed on ice and the cells were treated by 3x 3 seconds treatment broken up with the ultrasonic wand.

The reaction solution of the in vitro enzyme assay had a volume of 60 μl and was composed as follows (1/17/1996):

100 mM Tris / HCl pH 8.0 (at 30 ° C); 1mM DTT; 0.2% Triton X100; 1 mM Cho ^¬ lesterol, 5 ul E. coli homogenate (1-2 mg protein / ml), 100,000 dpm UDP- [U- ¹⁴ C] glucose (144 uM).

The reaction was stopped after 20 min (at 30 ° C) by mixing with 0.5 ml of water and 1.6 ml of ethyl acetate. After phase separation by brief centrifugation, the upper organic phase was removed and the radioactivity contained therein determined with a scintillation counter: E. coli homogenate with pBS-HaATG: 620 disintegration per minute (radio ^¬ active disintegrations per minute) (dpm) E. coli homogenate with pBS-HRP: 3100 dpm E. coli homogenate, untransformed: 0 dpm

The radioactivity present in the organic phase of incubated parallel samples was subjected to thin-layer chromatography analysis:

The organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which were developed with the eluent chloroform: methanol 85:15. The Rf values determined to the radioactive, lipophilic reaction products WUR ^¬ with a Berthold TLC analyzer and compared with authentic standards that were detected with α-naphthol-sulfuric acid. Only one product could be detected that was identified as steryl glucoside (see FIG. 6). It was thus possible to demonstrate that the transformed E. coli cells express a protein which shows sterol-glucosyltransferase activity. Non-transformed control cells show no sterol-glucosyltransferae activity.

Expression of the plant peptide sequences was also determined by Western blot analyzes demonstrated:

40 μg protein of the E. coli homogenates was precipitated with 8% trichloroacetic acid and then subjected to SDS-polyacrylamide gel electrophoresis (10%) (with Biorad Mini Protean II apparatus, Munich). The proteins were electroblotted onto a nitrocellulose membrane and immunostained (anti-sterol-glucosyltransferase antiserum 1: 1000, staining with hydrogen peroxide and 4-chloro-naphthol). The Western blot membrane is shown in Fig. 7. In E. coli with pBS-HRP, a band of approx. 59 kD is clearly stained. In E. coli with pBS-HaATG, a 74 kD band is the most intensely colored. These proteins han ^¬ it delt by the encoded proteins on the plasmids.

6. Functional expression of part of the clone HaSGT in S. cerevisiae.

For this purpose, a vector was produced which is suitable for the expression of the plant cDNA in Saccharomyces cerevisiae.

- Amplification of the CYC1 terminator Zaret, J.K. and Sherman, F. (1982) Cell 28: 563-573 with the PCR method using the primers 5'-GATATCTAGAGGCCGCAAATTAAAGCCTTC-3 'and

5'-CCCGGGATCCGAGGGCCGCATCATGTAATT-3 'and cloning into the vector pRS316 Sikorski, R. S. and Hieter, P. (1989) Genetics 122: 19-27. The resulting plasmid was named pRS316t. Cloning of the GAL1 promoter (0.5 kb Spel / Xbal fragment) from the pYES vector (Invitrogen) into the vector Bluescript KS (Stratagen, Heidelberg). PGALl resulted from the cloning.

Cloning of the GAL1 promoter (0.5 kb Xbal / PvuII fragment) from pGALl into the vector Bluescript KS (HincII / Xbal). The resulting plasmid was called pGAL2.

Cloning of the fragment via Xhol / Sacl in the pYES2.0 vector (Invitro ^¬ gen, Leek, Holland). This resulted in pGAL3. Cloning of the fragment from the pGAL3 via Kpnl / Xhol in the pRS316t.

From the single copy yeast pGAL4 originated with follow ^¬ Features: Single copy plasmid, URA marker, GAL1 promoter, CYC1 terminator MCS.

Part of the oat clone HaSGT was extracted from plasmid pBS- with Sall / Kpnl HaSGT cut out and cloned into the pSP72 Vector (Promega, Heidelberg, Sall / Kpnl). The SalI / Kpnl fragment of the resulting plasmid pSPHAMl comprises the corresponding portion of the HaSGT and was cloned into the vector pGAL4 (XhoI / BamHI). The resulting plasmid was used pGALHAMl and to transform the Saccharomyces cerevisiae strain UTL-7A (MATa, ura3-52, trpl, leu2-3 / 112).

The sterol-glucosyltransferase activity was an in vitro enzyme assay performed with cell-free homo ^¬ Genat of the yeast cells to detect the expressed plant sequence. The yeast cells were grown on the following medium (aerobically shaken for 72 h at 29 ° C):

6.7 g / 1 Difco Yeast Nitrogen Base without Amino Acids; 10 mg / 1 tryptophan; 60 mg / 1 leucine; 1% galactose.

The cells of a 30 ml culture are sedimented and taken up in 1 ml of lysis buffer:

50 M Tris / HCl pH 7.5; 15% glycerol; 0.1% Triton X100; 200 µM Pefabloc (Merck, Darmstadt); 1mM DTT; 0.5 mg / ml Lyticase (Sigma, Deisenhofen). After an incubation of 25 min at 20 ° C., the cells were broken up by ultrasound treatment ( ³ × 10 s).

The reaction solution of the in vitro enzyme assay had a volume of 150 μl and was composed as follows (March 10, 1996):

100 mM Tris / HCl pH 8.0 (at 30 ° C); 1mM DTT; 0.2% Triton X100; 1 mM Cho ^¬ lesterol, 20 ul yeast homogenate, 350,000 dpm UDP- [U- ¹⁴ C] glucose (4.2 uM). The reaction was stopped after 45 min (at 30 ° C) by mixing with 0.5 ml of water and 1.6 ml of ethyl acetate. After phase separation by brief centrifugation, the upper organic phase was removed and the radioactivity contained therein was determined with a scintillation counter: yeast homogenate with pGAL4: 0 dpm yeast homogenate with pGALHAMl: 13000 dpm

The ^{radioactivity} present in the organic phase was subjected to thin-layer chromatography analysis from parallel ^samples : the organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which were developed with the eluent chloroform: methanol 85:15. The Rf values determined to the radioactive, lipophilic reaction products WUR ^¬ with a Berthold TLC analyzer and compared with authentic standards that were detected with α-naphthol-sulfuric acid. Only one product could be identified that was cosid was identified (see Fig. 8). It could thus be demonstrated that the transformed yeast cells express a protein which shows sterol-glucosyltransferase activity. Control cells show no sterol-glucosyltransferae activity.

7. Functional expression of genomic DNA sequences from Saccharomyces cerevisiae in E. coli.

The deduced from the cloned by us oats sequence Aminosäurese ^acid sequence showed striking similarities with the deduced amino acid sequence of a genomic piece of DNA from S. cerevisiae (see Fig. 9) This is the chromosome XII cosmid 9470 (Genbank no. GB U17246) . The similarity related to the 34 area of the reverse reading frame of bp 32961-36557 (gene L9470.23). No function was previously known for this putative gene.

Parts of this open reading frame were functionally expressed by us in E. coli:

For this purpose, a 6359 bp fragment was isolated from a Cosmid 9470 DNA preparation by cutting with the enzymes Ndel and Spei (Cosmid bp 31384-37744). This sequence einhielt the desired reading frame and was constructed by cloning into the vector pBluescript II KS (with EcoRV ge ^¬ cut) are used for further subcloning. This plasmid was called pBS-HSC. Four subclonings were carried out which should lead to the expression of parts of the open reading frame of different lengths. These clonings are tabulated below:

Cloning 2 3 4

Cutting pBS-HSC with Eco47III PstI EcoRI Sspl Smal BamHI

Approx. Length of the isolated fragment in bp 3900 5000 3δ00 2500 expression vector pUC19 pUCδ pBSIIKS pUC19 Cutting the expression vector with Smal PstI EcoRI Smal

BamHI

All of these clonings lead to plasmids which code for fusion proteins which, in the first section, consist of the lacZ operon and parts of the poly- the left of the vectors are derived and in the second section consist of polypeptides that correspond to parts of the gene L9470.23. Figure 9 shows the derived protein sequence of the open reading frame (gene L9470.23). In this figure, the amino acids are selected, with which the second from ^¬ cut the fusion proteins of different clones begins. The plasmids of cloning 1-4 were used to transform E. coli. To our surprise, we were able to detect cell-free homogenates of these cells with an in vitro enzyme assay for sterol-glucosyltransferase activity. For this purpose, the cells were sedimented from 15 ml overnight culture (15 ml LB ampicillin, 37 ° C., 14 h) and taken up in 1.5 ml lysis buffer (50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT; 1 mg / ml Lysozyme (from egg, Boehringer, Mannheim). 200 uM Pefabloc (Merck, intestinal ^¬ city) After a 5 minute incubation at 20 ° C, the suspension was placed on ice and the cells by 3x 3 seconds of treatment with the Ultra ^¬ sound rod broken.

The reaction solution of the in vitro enzyme assay had a volume of 100 μl and was composed as follows (May 22, 1996):

50 mM Tris / HCl pH 8.0 (at 30 ° C); 1mM DTT; 1 mM MgCl ^ 10 ul 2 mM Ergo ^¬ sterol in ethanol; 45 ul E. coli homogenate; 150,000 dpm UDP- [U- ¹⁴ C] glucose (2.2 µM).

The reaction was stopped after 45 min (at 30 ° C) by mixing with 0.5 ml of water and 1.6 ml of ethyl acetate. After phase separation by brief centrifugation, the upper organic phase was removed and the radioactivity contained therein was determined using a scintillation counter:

E. coli homogenate with clone 7500 dpm E. coli homogenate with clone 2 10700 dpm E. coli homogenate with clone 3 35000 dpm E. coli homogenate with clone 4 32700 dpm

E. coli homogenate, untransformed: 2000 dpm

The radioactivity present in the organic phase of parallel samples from clones 2 and 4 was subjected to a thin-layer chromatographic analysis:

The organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which were developed with the eluent chloroform: methanol 85:15. The Rf values determined to the radioactive, lipophilic reaction products WUR ^¬ with a Berthold TLC analyzer and compared with authentic standards that were detected with α-naphthol-sulfuric acid. Only one product was identified in each case that was identified as steryl glucoside (see FIG. 10). This allowed us to demonstrate to our surprise that the transformed E. coli cells Prote ^¬ in expressing the sterol glucosyltransferase activity shows. Although the organi ^¬ rule phases of assay with untransformed control cells also contain some radioactivity; however, this is not a labeled steryl glucoside. The deduced amino acid sequence of the gene 9470.23 is mentioned in Fol ^¬ constricting ScSGTP (see Fig. 9).

8. PCR ~ experiments with Arabidopsis, Candida and potato.

Oligonucleotide primers which were used for PCR experiments could be derived from similar amino acid sequence regions between HaSGTP (see 4.) and ScSGTP (see 7.): DW3 = GSIWCIVSIGGIGAYGTHYWICC WA3 = GTIGTICCISHICCISCRTGRTG WA6 = GTISKIGTCCAIGGCATIG

Abbreviations see 4.

The polymerase chain reaction method was carried out as follows:

Reaction mix: 40 μl distilled water; 5 ul Boehringer (Mannheim) 10 x PCR buffer; 1 µl each of 10 M dATP, dGTP, dCTP, dTDP; 1 μl each 100 μM oligonucleotide primer,

0.25 ul Boehringer Taq polymerase; 0.5 µl matrix DNA.

Reaction conditions: 94 ° C, 3 min; 30x (94 ° C, 45 s; 53 ° C, 1 min; 72 ° C, 2 min); 72 °, 10 min. a.) Primers DW3 and Wa6, cDNA was used as the matrix DNA

Arabidopsis mRNA was synthesized. b.) Primers DW3 and Wa6, a phage mixture of a

Lamda ZAP cDNA bank (Stratagene, Heidelberg) of potato with approx. 10

Plaque-forming units used per ml. c.) Primers DW3 and Wa3, genomic DNA from Candida albicans (approx. 50 ng / μl) was used as the matrix DNA.

Result: Agarose gel electrophoresis with 15 μl of the reaction batches gave DNA bands of approximately 340 bp in length (Arabidopsis, potato) and approximately 940 bp in length (Candida albicans). These DNA pieces were analyzed using the pGEM-T Vector System (Promega, Heidel- berg) cloned into a plasmid vector and sequenced partially or completely se ^¬. These sequences are shown in Figures 11-13 (Arabidopsis = Aper; Potato = Kpcr; Candida = Cpcr). The abgelei ^¬ ended amino acid sequences from these sequences (ApcrP, KpcrP and CpcrP) were acid sequences with Ami ^¬ of the oat HaSGTP or the yeast gene L9470.23 (Sc ^¬ SGTP) compared (see Figure 14-16.) It is to our surprise

the potato sequence KpcrP is 86% identical to the corresponding part of the oat sequence HaSGTP,

- The Arabidopsis sequence ApcrP is 90% identical to the corresponding part of the HaSGTP and oat sequence

the candidate sequence CpcrP is 64% identical to the corresponding part of the S. cerevisiae sequence ScSGTP.

9. Isolation of complete Arabidopsis clones

The Arabidopsis PCR clone Aper was using a method as described in the fourth ^¬ be issued for the isolation of complete clones from a lambda Zap Arabidopsis cDNA library (obtained from the Stock Center of the Max Planck Institute for breeding research ^¬, Cologne) used. It was a 2300 bp long clone (the consequences ^¬ AtSGT called) completely sequenced and double-stranded (Fig. 17). This clone is 2353 bp long and shows an open reading frame from bp 1 to bp 2023. A start codon (ATG) for translation begins at bp 113. If the open reading frame is translated into an amino acid sequence (AtSGTP, FIG. 18), it shows the amino acid sequence is very similar to the oat sequence HaSGTP (see Fig. 19).

10. Functional expression of the clone AtSGT in E. coli.

To prove that the clone AtSGT codes for a sterol glucosyltransferase, it was expressed in E. coli. The cloning produced an Plas ^¬ mid (pBS-AtSGT) encoding for a fusion protein whose first amino ^¬ acids from the pBluescript lacZ operon and the polylinker derived (nor ^¬ printed times, see below) and the following amino acids to those of the open Correspond to the reading frame of the clone AtSGT (underlined, see below).

The beginning of the fusion protein looks like this: MTMITPSSELTLTKGNKSWSSTAVAAALELVDPPGCRNSEFGTPLILSFTFWD. ". In the E. coli cells transformed with the plasmid pBS-AtSGT, it was checked whether the corresponding fusion protein was expressed by carrying out an in vitro enzyme assay for the detection of sterol-glucosyltransferase activity with cell homogenates.

The cells from 1.5 ml overnight culture (1.5 ml LB-ampicillin, 37 ° C., 14 h) were sedimented and taken up in 1 ml lysis buffer (50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT ; 1 mg / ml lysozyme (from Hühnerei, Boehringer, Mannheim); 200 μM Pefabloc (Merck, Darmstadt); 0.1% Triton X100. After δminute incubation at 20 ° C the suspension was placed on ice and the cells were passed through 3x 3 Seconds of treatment with the ultrasound wand.

The reaction solution of the in vitro enzyme assay had a volume of 50 μl and was composed as follows (11.3.1996):

100 M Tris / HCl pH 8.0 (at 30 ° C); 1mM DTT; 0.2% Triton X100; 1 mM Cho ^¬ lesterol; 7.5 ul E. coli homogenate, 100,000 dpm UDP- [U- ¹⁴ C] glucose (2.8 uM). The reaction was stopped after 20 min (at 30 ° C) by mixing with 0.5 ml of water and 1.6 ml of ethyl acetate. After phase separation by brief centrifugation, the upper organic phase was removed and the radioactivity contained therein was determined using a scintillation counter: E. coli homogenate with pBS-AtSGT: 1300 dpm

E. coli homogenate, untransformed: 100 dpm (blank value)

The radioactivity present in the organic phase of incubated parallel samples was subjected to thin-layer chromatography analysis:

The organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which were developed with the eluent ChloroforπuMethanol 85:15. The Rf values determined to the radioactive, lipophilic reaction products WUR ^¬ with a Berthold TLC analyzer and compared with authentic standards that were detected with α-naphthol-sulfuric acid. Only one product could be detected that was identified as steryl glucoside (see Fig. 20). This enabled proved who the that the transformed E. coli cells ming a protein expri, shows the sterol glucosyltransferase activity ^¬. Non-transformed control cells show no sterol-glucosyltransferae activity. All molecular biological work steps, which are not described in detail in the examples, were, unless otherwise stated, according to work instructions from Sambroock, J .; Fritsch, EF and Maniatis, T. (1989): Molecular cloning. A Laboratory Manual. Second edition. Cold Spring Laboratory Press, Cold Spring Laboratory.

Definitions:

-Stereols are substances that have the following structural features: They consist of a 5α-cholestan-3-ß-ol or 5α-cholestan-3-α-ol backbone. This basic structure can be modified on the side chain and / or by various double bonds in the ring systems.

-STEROLS IN THE NARROW SENSE are cholesterol, ergosterol, ß-sitosterol, stigmasterol.

-STERYLGLYCOSIDES are sterols or sterols in the narrower sense, which are linked to a sugar molecule or to the C3 atom via the oxygen atom. These sugars can e.g. Glucose, galactose, mannose, xylose, arabinose, other sugars or sugar derivatives in furanosidic or pyridosidic form and in an α or β linkage. Compounds containing glucuronic acid are excluded from this definition.

-FOLGEPRODUKTE sterylglycosides are te to a secondary production ^¬, which can be synthesized cosiden in organisms, or in in vitro systems enzymatically from Sterylgly- (eg Steryldiglycoside, -triglycoside, oligoglycosides or acylated steryl glycosides). On the other hand, these are punch Sub ^¬, which can be represented by methods of organic chemistry from sterylglycosides.

-STEROL-GLYCOSYL TRANSFERASES are enzymes that transfer a sugar molecule, especially from activated sugars or activated sugar derivatives, especially from sugar nucleotides or sugar derivative nucleotides, to the 0H group on the C3 atom of sterols or sterols in their own sense. The transfer of glucuronic acid sen from this definition out Schlos ^¬. STEROL glucosyltransferases are enzymes that carry a glucose molecule, particularly of activated glucose, especially from uridine diphosphate to the OH group on the C3 atom of sterols or sterols in their own sense about ^¬.

STEROL glycosyltransferases in the narrower sense are enzymes, the gene is a sugar molecule, especially from activated sugars or activated sugar derivatives, especially from sugar nucleotides or Zuckerderivatnukleoti- to the OH group on the C3 atom of sterols in the narrower sense übertra ^¬. The transfer of Glucuronic acid is excluded from this definition.

STEROL glucosyltransferases in the narrower sense are enzymes that phosphate is a glucose molecule, particularly of activated glucose, especially from Uridindi ^¬ on the OH group on the C3 atom of sterols in the narrower sense transmitted.

In this sense, SUGAR are hexoses or pentoses in furanosidic or pyranosidic form.

SUGAR DERIVATIVES are sugars which have been modified in their structure by oxidation or reduction or by the addition or removal of functional groups. As an example may be mentioned here N-Ace ^{'tylglucosamin} and Desoxyri ^¬ bose.

SUGAR NUCLEOTIDES in the sense used here are substances in which one of the organic bases thymine, adenine, guanine, uracil or cytosine is linked to a ribose or deoxyribose and this sugar is in turn linked to another sugar molecule via two phosphoric acid residues.

-PLANT PARTS are parts of a plant such as Leaves, roots, seeds or fruits.

-VECTORS are nucleic acid fragments that are capable of replication under certain conditions and for the incorporation of foreign nucleic acid fragments for the purpose of multiplying this fragment or the expression of this fragment (for example for the production of a pro- teins) can be used. Typical examples are plasmids and phages.

CHIMARY GENE is a nucleic acid fragment that is composed of different parts and does not naturally occur in this form. It comprises a numeral for a sequence encoding a polypeptide sequence and geeig ^¬ control sequences that allow expression. The coding Se ^acid sequence may in this case the control sequences with respect to "sense" - or be "anti-sense" orientation.

Spread the insulation is to obtain certain things from a mixture ver ^¬ VARIOUS things. Things can thereby substances (eg proteins, Nu ^¬ small acid fragments, mRNA, DNA, cDNA, cDNA clones, genes) lines, cell components (eg membranes), cells (eg, bacterial cells, plant cells, protoplasts), Zel- or organisms and their descendants his.

bibliography

1. Bauw, G .; van den Bulcke, M .; van Damme, J .; Puype, M .; van Montagu, M. and Vandekerckhove, J. (19δ8) J. Prot. Chem. 7: 194-196

2. King, M. L .; Ling, H. C; Wang, CT. and Su, M. (1979) J. Nat. Prod. 42: 701 ff.

3. Miles, D. H .; Stagg, D. D. and Parish, E.J. (1979) J. Nat. Prod. 42: 700 ff

4. Normura, T .; Watanabe, M .; Inoue, K. and Ohata, K. (1978) Japan J. Pharacol. 28, Suppl. HOP

5. Okuyama, E. and Yamazaki, M. (1983) Yakugaku Zasshi 103: 43 ff.

6. Seki, J .; Okita, A .; Watanabe, M .; Nakagawa, T .; Honda, K .; Tatewaki, N. and Sugiyama, M. (1985) J. Pharm. Sei. 74: 1259-1264

7. Sikorski, R. S. and Hieter, P. (1989) Genetics 122: 19-27

8. Warnecke, D.C and Heinz, E. (1994) Plant Physiol. 105: 1067-1073

9. Zaret, J.K. and Sherman, F. (1982) Cell 28: 563-573

10. Sambroock, J .; Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning. A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Habor.

Claims

PATENT CLAIMS

Contains 1. An isolated DNA fragment or recombinant DNA construct, the syltransferase least part of a sterol or sterol glycosyltransferase Glyco ^¬ in the narrower sense coding sequence.

2. An isolated DNA fragment or recombinant DNA construct according to claim 1, which contains at least part of a sterol-glucosyltransferase or a sterol-glucosyltransferase coding sequence in the narrower sense.

3. An isolated DNA fragment or recombinant DNA construct in the narrower sense, encoded according to claim 1 or 2, the at least syltransferase an enzymatically active sterol or sterol ^¬ Glyco-glycosyltransferase in the narrower sense, insbeson ^¬ particular sterol glycosyl or sterol glycosyl , wherein the encoded protein after expression is enzymatically active, directly or after processing or modification in vivo or in vitro under th geeigne ^¬ conditions.

4. Isolated DNA fragment or recombinant DNA construct according to one or more of claims 1-3, wherein the nucleic acid from a) plants or b) plants, fungi and bacteria or c) eukaryotes or d) multicellular eukaryotes or e) fungi or f) bacteria can be isolated.

5. Isolated DNA fragment or recombinant DNA construct ge a one or more of claims 1-4, which has the following properties comprising: a) exists for the use deduced amino acid sequence at least 14 aufein ^¬ other amino acids that are identical with those listed in Figure 4 or 18 sequences, and b) the deduced amino acid sequence comprises the Aminosäurese ^acid sequence LHA..

6. Isolated DNA fragment or recombinant DNA construct according to one or more of claims 1-4, which can be isolated from plants and whose derived amino acid sequence comprises the amino acid sequence HHGGxxxxxxxxxxxxPxxxxxxxxxQ, where x represents any amino acids.

7. Isolated DNA fragment or recombinant DNA construct according to one or more of claims 1-4, which a) can be isolated from plants, b) has a length of at least 400 bp, and c) whose derived amino acid sequence is at least 8 consecutive ^¬ Includes amino acids that are identical to the sequences shown in Fig. 4 or lδ.

δ. An isolated DNA fragment or recombinant DNA construct according to one or more of claims 1-4, which derived Aminosäurese ^acid sequence a) over a length of at least 350 amino acids, at least 75%, in particular 90%, especially 100%, identical to the sequences in Fig. 21 or 22) b over a length of at least 150 amino acids, at least 70% identical to the sequence in Fig. 16, or c) over a length of 100 amino acids, at least 93% identical ^¬ table with of the sequence in Fig. 15.

9. protein is the abgeleitetet acid sequences from any of the nucleic ^¬ in Fig. 1-3 or 11-22 shown.

10. Plasmids, viruses or other vectors, characterized in that they contain nucleic acids according to one or more of claims 1-δ.

11. Genomic clones, characterized in that they contain genes or parts of genes which code for a sequence or parts thereof according to one or more of claims 1-δ.

12. Chimeric gene which is able to change the content of sterol glycosyltransferase or sterol glycosyltransferase in the narrow sense, in particular sterol glucosyltransferase or sterol glucosyltransferase in the narrow sense, in a transformed cell.

13. A chimeric gene according to claim 12, which comprises a nucleic acid fragment, the deduced amino acid sequence of which corresponds at least to 60% of the sequence in FIG. 23 or parts thereof and which is linked in an effective form to suitable control sequences.

14. Chimeric gene according to claim 12, which comprises a DNA fragment defined in one or more of claims 1-8.

15. A chimeric gene products of one or more of claims 12-14, since ^¬ characterized by that it is capable in a transformed cell h ^¬ le a) the synthesis of sterol glycosides or biosynthetic sequence ^¬ such as acylated steryl glycosides or Steryloligoglycoside, for For ) ^¬ b solve the content of sterol glycosides or secondary products to countries changed ^¬, or produce c) new sterylglycosides or derived products.

16. A chimeric gene according to claim 15, characterized in that it is initiating the synthesis of Sterylglycosi ^¬ in a transformed cell is capable of changing the content of sterol glycosides or produce new sterylglycosides.

17. Chimeric gene according to one or more of claims 12-16, since ^¬ characterized in that the construction is in "sense" and / or "antise- sic" orientation.

18. Transf ied cells, transformed microorganisms, plants or Plant parts containing a chimeric gene according to one or more of claims 12-18.

19. Transformed cells, transformed microorganisms, plants or parts of plants according to claim 18, characterized in that they contain sterol-glycosyltransferases or sterol-glycosyltransferases in the narrower sense, in particular sterol-glucosyltransferases or sterol-glucosyltransferases in the narrower sense, which do not naturally or in these occur in a different form or in a different concentration.

20. Transformed cells, transformed microorganisms, plants or parts of plants according to claim 18, characterized in that they have sterol-glycosyltransferase activities or sterol-glycosyltransferase activities in the narrower sense, in particular sterol-glucosyltransferase activities or sterol-glucosyltransferase activities in the narrower sense , show that naturally do not occur in ^these or at a different level or with a different specificity.

21. Transfomierte cells, transformed microorganisms, plants or plant parts according to claim 18, characterized in that they contain steryl glycosides or derivatives that are naturally ren in these non-or in any other quantity or in any other form, in particular having resistors ^¬ sterol or sugar component or other link, occur.

22 plants or plant parts according to one or more of the Ansprü ^¬ che 18-21, characterized in that it tolerant or resistant ge ^¬ genüber one or more of the harmful environmental effects

- dryness,

- high salt concentration in the substrate,

- cold or frost,

- Fungus infestation is a non-transformed control plant.

23. Plants or plant parts according to claim 22, characterized characterized ^¬ marked in that they are more tolerant or resistant to cold or freezing as a non-transformed control plants.

24. Transformed microorganisms according to one or more of the Proverbs 18-22, especially bacteria or yeasts, characterized in that they are more resistant or tolerant to one or more of the environmental influences

- high salt or ethanol concentration in the medium,

- cold or frost,

- High temperatures are as non-transformed microorganisms.

25. A method for producing sterylglycosides or secondary products comprising culturing transformed cells, transformed microorganisms, plants or parts of plants according to one or more of claims 18-24.

26. The method according to claim 25, characterized in that homogenization sate or extracts of the transfomierten cells, micro Orga ^¬ transformed mechanisms, plants or parts of plants are cultivated in vitro.

27. Sterylglycosides or secondary products obtainable by the process according to claim 25 or 26.

28. DNA fragment, obtainable by one of the following or parts thereof: a.) Use of one of the gezeig ^¬ th nucleic acid sequences as a hybridization probe in Figures 1-3 or 11-13 or 17; b) using the amino acid sequences shown in FIGS. 4, 5, 14-16, 18, 19, 21 or 22 for the synthesis of peptides or proteins which serve to obtain antisera; or c) i) comparing the nucleotide sequences shown in FIGS. 1-3, 11-13 or 17 or the amino acid sequences shown in FIGS. 4, 5, 14-16, 18, 19, 21 or 22 with each other or with already known ^¬ th nucleotide sequences or derived therefrom, amino acid sequences, ii) deriving and synthesizing appropriate specific oligo nucleotides from similar regions of these sequences, and iii) using these oligonucleotides to using a sequenzab ^¬ dependent protocol, in particular the PCR method, nucleic acids near, ^¬ ask for sterol glycosyl or sterol Glycosyltransfera ^¬ sen in the narrow sense, especially sterol glycosyl or sterol glucosyl in the narrow sense or parts encode it.

29. Chimeric gene which contains a DNA fragment according to claim 28 and which is capable, in a transformed cell, of the content of sterol-glycosyltransferase or sterol-glycosyltransferase in the narrower sense, in particular sterol-glucosyltransferase or sterol-glucosyltransferase in the narrow sense , to change.

30. Transformed cells containing a chimeric gene according to claim 29.

31. Transformed cells according to claim 30, characterized in that they contain sterol glycosyltransferases or sterol glycosyltransferases in the narrower sense, in particular sterol glucosyltransferases or sterol glucosyltransferases in the narrower sense, which naturally do not exist in these or in another form or in another Concentration.

32. organisms, in particular microorganisms such as bacteria and yeasts, the gene or genes that glucosyltransferases sterol or sterol glycosyl-Gly ^¬ cosyltransferasen in the narrower sense, particularly sterol or sterol glycosyl in the narrower sense encode, by transformation with appropriate chimeric Genes deleted or interrupted.

33. Organisms according to claim 32, which have a change in the content of sterylglycosides and / or secondary products.

34. Organisms according to claim 32 or 33, wherein the chimeric gene comprises a DNA fragment according to one or more of claims 1-9 or 28 um ^¬ .

35. sterol glycosyl or sterol glycosyl in the narrower sense, particularly sterol glycosyl or sterol Gluco ^¬ syltransferasen in the narrow sense, or portions thereof or fusion proteins with the aforementioned transferases from organisms according to one or more of claims 18-25, or 30-32 are available.

36. Antisera or products from antisera which are directed against a protein according to claim 35 or parts thereof and by immunization of Animals are available.

37. Antibodies or parts thereof which are against a protein according to claim

35 or parts thereof are directed rather than classical Immunisie ^¬ tion of animals is.