Background of the invention Riboflavin biosynthesis Riboflavin synthase catalyzes the terminal step of riboflavin biosynthesis, the formation of riboflavin from 6, 7-dimethyl-8-ribityllumazine (Bacher et al., in : Escherichia coli and Salmonella (F. C. Neidhard, ed.), p. 657, American Society for Microbiology, Washington DC, 1996 ; Bacher et a/., Methods Enzymol. 280 : 389, 1997) Despite the importance of this pathway in plants, genes encoding plant riboflavin synthase have not previously been isolated. Thus, there is a need in the art for methods and compositions that provide plant riboflavin synthase genes, plant riboflavin synthase inhibitors useful as herbicides, and herbicide-resistant plant riboflavin synthase variants. The present inventors have isolated the gene encoding plant riboflavin synthase expressed it in E. coli, and demonstrated that bacterially expressed plant riboflavin synthase is enzymatically active and they have found a method for screening for inhibitors of its enzymatic activity.

Brief description of the Drawinqs Fig. 1 is an illustration of the pathway for the biosynthesis of riboflavin.

Fig. 2 is the nucleotide and protein sequence of riboflavin synthase from Arabidopsis thaliana without chloroplast import signal sequence.

Fig. 3 is the nucleotide sequence of the pNC0113 vector.

Summarv of the invention The present invention provides purified isolated nucleic acids encoding plant riboflavin synthase (ribC), in particular derived from Arabidopsis thaliana, as well as sequence-conservative variants and function-conservative variants thereof ; DNA vectors comprising riboflavin synthase-encoding nucleic acid operably linked to a transcription regulatory element ; and cells comprising the riboflavin synthase vectors, including without limitation bacterial, fungal, plant, insect, and mammalian cells. In one embodiment, a bacterial cell expressing high levels of plant riboflavin synthase is provided. Also encompassed are riboflavin synthase polypeptides and enzymatically active fragments derived therefrom.

In another aspect, the invention provides a method for identifying herbicides or riboflavin synthase inhibitors as defined in claim 11.

In the above method, the monitoring step may be achieved, for example, by measuring the concentration of riboflavin. Alternatively, an inhibitor is identified as a compound that inhibits the growth of a test culture, wherein the inhibition may be reversed by the addition of riboflavin to the culture.

In a further aspect, the invention provides methods for identyfing herbicide- resistant riboflavin synthase variants, by producing mutants of riboflavin synthase in vitro or in vivo, notably by (a) providing a population of cells expressing plant riboflavin synthase ; (b) mutagenizing the population of cells ; (c) contacting the mutagenized population of cells with an herbicide, under conditions inbibitory for the growth of non-mutagenized cells ; (d) recovering cells resistant to the inhibitory effects of the herbicide ; and (e) isolating, purifying and optionally sequencing riboflavin synthase-encoding nucleic acid from the recovered cells.

Alternatively, DNA encoding riboflavin synthase is subjected to random or site-directed mutagenesis in vitro, followed by expression in a heterologous cell and screening or selection of cells that exhibit herbicide resistance.

In yet another aspect, the invention encompasses variant riboflavin synthase proteins that are herbicide-resistant. Preferably, an herbicide resistant riboflavin synthase variant protein, when expressed, in a cell that requires riboflavin synthase activity for viability, exhibits (i) catalytic activity alone sufficient to maintain the viability of a cell in which it is expressed ; or catalytic activity in combination with any herbicide resistant riboflavin synthase variant protein also expressed in the cell, which may be the same as or different than the first riboflavin synthase variant protein, sufficient to maintain the viability of a cell in which it is expressed ; and (ii) catalytic activity that is more resistant to the herbicide than is wild type riboflavin synthase.

Also provided are nucleic acids encoding herbicide-resistant variants, DNA vectors comprising the nucleic acids, and cells comprising the variant riboflavin synthase encoding vectors. Genes encoding herbicide-resistant riboflavin synthase variants can be used as genetic markers, such as, for example, in plasmids and methods for the introduction and selection of any other desired gene.

In another aspect, the present invention provides a method for conferring herbicide resistance on a cell or cells, and particularly a plant cell or cells such as, for example, a seed. A riboflavin synthase gene, preferably the Arabidopsis thaliana riboflavin synthase gene, is mutated to alter the ability of an herbicide to inhibit the enzymatic activity of the riboflavin synthase. The mutant gene is cloned into a compatible expression vector, and the gene is transformed into an herbicide- sensitive cell under conditions in which it is expressed at sufficient levels to confer herbicide resistance on the cell.

Also contemplated are methods for weed control, wherein a crop containing an herbicide resistant riboflavin synthase gene according to the present invention is cultivated and treated with a weed-controlling effective amount of the herbicide.

Detailed Description of the Invention The present invention encompasses isolated, purified, nucleic acids that encode plant riboflavin synthase. Riboflavin synthase expression systems in which enzymatically active riboflavin synthase is produced, and screening methods for identifying riboflavin synthase inhibitors.

The present invention also encompasses methods for screening for and producing plant riboflavin synthase variants that are resistant to the inhibitory action of herbicides, DNAs that encode these variants, vectors that include these DNAs, the riboflavin synthase variant proteins, and cells that express these variants.

Additionally provided are methods for producing herbicide resistance in plants by expressing these variants and methods of weed control.

Isolation and Characterization of the Gene Encoding Arabidopsis riboflavin svnthase and of riboflavin svnthase The present inventors have isolated and sequenced the gene encoding Arabidopsis thaliana riboflavin synthase, using the methods outlined below. Briefly, Arabidopsis thaliana cDNA library was screened using a PCR-based method Primers : A forward primer, designated ARARFSF2 (5'-GGAGAAATTAACCATGTTTACTGGAATCGTGGAGGAAATG-3') and a reverse primer, designated ARARFSH (5'-GCGATCTGCAGAGTTACCTTTGGAGAAGC-3') were synthesized based on an Arabidopsis sequence (GenBank ID No : AC006234 from bp 107550-bp 108167) that showed homology to bacterial and yeast riboflavin synthase sequences.

The primers were evaluated in a polymerase chain reaction (PCR) using as template DNA a 1 J aliquot of the Arabidopsis cDNA. For PCR, a 100 1ll reaction contained 1X PCR Buffer, 200 iM of each deoxynucleoside triphosphate, 2 units of Taq DNA Polymerase, and 10 pmoles of each primer. The reaction mixture was heated to 94°C for 2 min and amplified using 25 cycles of : 94°C for 30 sec, 50°C for 30 sec, 72°C for 45 sec. This was followed by incubation at 72°C for 7 min. A fragment of the predicted size of 640 bp was produced. This fragment was cloned into the pNC0113 vector and sequenced and was found to be identical to the Arabidopsis database sequence.

The protein sequence in the Genbank database entry (protein ID AAD20926. 1) comprises only the C-terminal part of the complete riboflavin synthase protein.

Especially the important FTGI-motive is not included in the database entry. The analysis of the nucleotide sequence reveals a possible N-terminal chloroplast import signal sequence of 64 aminoacids (Schatz et a/., Science 271 : 1519, 1996). This import sequence was excluded in the cloned gene to get the mature Arabidopsis protein by expression in a bacterial host. For that purpose a start codon was introduced preceding the FTGI-motive. The chloroplast import sequence may also be included in the cloned gene especially if expressed in a eukaryotic host.

A protein coded by the above gene was expressed in E. coli cells. The cell mass was treated with lysozyme and ultrasound. After centrifugation riboflavin synthase activity was detected in the supernatent. The protein having the riboflavin synthase activity was isolated by anion exchange chromatography. It is surprising that the protein coded by the above nucleic acid sequence could be obtained in a form that is functional for riboflavin synthase activity.

In the screening method the pH may be in the range of 5. 5 to 9, preferably 7 to 8. 5.

The temperature is preferably in the rage of +10 °C from the optimum temperature.

The screening process may be monitored by measuring the formation of riboflavin or the disappearance of 6, 7-dimethyl-8-ribityl-lumazine.

The measurement may be carried out in the reaction mixture or after separation, e. g. by chromotography, notably HPLC. Riboflavin production may be monitored by its absorbance at 445 nm at acidic pH, e. g. pH1, or at 470 nm near neutral pH or by the fluorescence at 516 nm with an excitation at 445 nm.

Nucleic Acids, Vectors, Expression Svstems and Polypeptides In practicing the present invention, many techniques in molecular biology, microbiology, recombinant DNA, and protein biochemistry such as these explained fully in, for example, Sambrook et a/., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ; DNA Cloning : A practical Approach, Volumes 3 and 33, 1985 (D. N.

(Glover ed.) ; Oligonucleotide Synthesis, 1984, (M. L. Gait ed.) ; Transcription and Translation, 1984 (Hames and Higgins eds.) ; A Practical Guide to Molecular Cloning ; the series, Methods in Enzymology (Academic Press, Inc.) ; and Protein Purtification : Principes and Practice, Second Edition (Springer-Verlag, NY.) are used.

The present invention encompasses nucleic acid sequences encoding plant riboflavin synthase, enzymatically active fragments derived therefrom, and related riboflavin synthase derived sequences from other plant species. As used herein, a nucleic acid that is"derived from"an riboflavin synthase sequence refers to a nucleic acid sequence that corresponds to a region of the sequence, sequences that are homologous or complementary to the sequence, and"sequence-conservative variants"and"function-conservative variants". Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.

Function-conservative variants are those in which a given amino acid residue in riboflavin synthase has been changed without altering the overall conformation and function of the riboflavin synthase polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). Fragments of riboflavin synthase that retrain enzymatic activity can be identified according to the methods described herein, e. g, expression in E. coli followed by enzymatic assay of the cell extract. Riboflavin synthase sequences derived from plants other than Arabidopsis thaliana can be isolated by routine experimentation using the methods and compositions provided herein. For example, hybridization of a nucleic acid comprising all or part of the Arabidopsis riboflavin synthase sequence under conditions of intermediate stringency (such as, for example, an aqueous solution of 2X SSC at 65°C) to cDNA or genomic DNA derived from, other plant species can be used to identify riboflavin synthase homologues. cDNA libraries derived from different plant species are commercially available (Clontech, Palo Alto, CA ; Stratagene, La Jolla, CA). Alternatively, PCR-based methods can be used to amplify riboflavin synthase- related sequences from cDNA or genomic DNA derived from other plants.

Expression of the identified sequence in, e. g., E. coli, using methods described in more detail below, is then performed to confirm that the enzymatic activity of the polypeptide encoded by the sequence corresponds to that of riboflavin synthase.

Accordingly, riboflavin synthase sequences derived from dicotyledonous and monocotyledenous plants are within the scope of the invention.

The nucleic acids of the present invention include purine-and pyrimidine- containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single-and double-stranded molecules, i. e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as"protein nucleic acids". (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.

The nucleic acids may be isolated directly from cells. Alternatively, PCR can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates.

Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if dersiable, to introduce appropriate new restriction sites, if desirable, to facilitate incorporation into a given vector for recombinant expression.

The nucleic acids of the present invention may be flanked by natural Arabidopsis regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns. 5'-and 3'-noncoding regions and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modification include methylation,"caps", substitution of one or more of the naturally occuring nucleotides with an analog, and internucleotide modification such as, for example, those with uncharged linkages (e. g., methyl phosphonates, phosphotriesters, phosphoromidates, carbamates, etc.) and with charged linkages (e. g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e. g., nucleases, toxins, antibodies, signal peptides, poly-L-Lysine, etc.), intercalators (e. g., acridine, psoralen, etc.), chelators (e. g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The nucleic acid may be derivarized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.

The invention also provides nucleic acid vectors comprising the disclosed riboflavin synthase sequences or derivatives or fragments thereof. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in an variety of eukaryotic and prokaryotic hosts. Non-limting examples include pNC0113 (American Type Culture Collection PTA-852), pQE plasmids (Quiagen, Hilden, Germany), pUC plasmids, pET plasmids (Novagen, Inc., Madison, USA), or pRSET or pREP (Invitrogen, San Diego, USA), and many appropriate host cells, using methods disclosed or cited herein or otberwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication sytems for cloning or expression, one or more markers for selection in the host, e. g. antibiotic resistance, and one or mose expression cassettes. Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaC12 mediated DNA uptake, tungae infection, microinjection, miroprojectile, or other established methods.

Appropriate host cells include bacteria, archaebacteria, fungi, especially yeast, plant and animal cells, especially mammalian cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosacchromyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lyphoid cell lines. Preferred replications systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, and the like. A large number of transcription initation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologeous proteins in the various hosts.

Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced riboflavin synthase-derived peptides and polypeptides.

Advantageously, vectors may also include a transcription regulatory element (i. e., a promoter) operably linked to the riboflavin synthase (ribC) portion. The promoter may optionally contain operator portions and/or ribosome binding sites.

Non-limiting examples of bacterial promoters compatible with E. coli include : trc promoter, p-tactamase (penicillinase) promoter ; lactose promoter ; tryptophan (trp) promoter ; arabinose BAD operon promoter, lambda-derived Pi promoter and N gene ribosome binding site ; and the hybrid tac promoter derived from sequences of the trp and lac UV5 promoters. Non-limiting examples of yeast promoters include 3- phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase (GALI) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Suitable promoters for mammalian cells include without limitation viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences and poly A addition sequences, and enhancer sequences which increase expression may also be included. Sequences which cause amplification of the gene may also be desirable.

Furthermore, sequences that facilitate secretion of the recombinant product from cells, including, but not limited to, bacteria, yeast, and animal cells, such as secretory signal sequences and/or prohormone pro region sequences, may also be included.

Nucleic acids encoding wild-type or variant riboflavin synthase polypeptides may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell, and thereby effect homlogous recombination at the site of endogenous gene or a sequence with substantial identity to the gene. Other recombination-based methods, such as non-homologous recombinations or deletion of endogenous genes by homologous recombination, may also be used.

Riboflavin synthase derived polypeptides according to the present invention, including function-conservative variants of riboflavin synthase, may be isolated from wild-type or mutant Arabidopsis cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalia cells) into which an riboflavin synthase derived protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins. Alternatively, polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.

"Purification"of a riboflavin synthase polypeptide refers to the isolation of the riboflavin synthase polypeptide in a form that allows its enzymatic activity to be measured without interference by other components of the cell in which the polypeptide is expressed. Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the riboflavin synthase protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibobdies produced against riboflavin synthase or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.

The present invention also encompasses derivatives and homologues of riboflavin synthase polypeptides. For some purposes, nucleic acid sequences encoding the peptides may be altered by substitutions, additions, or deletions that provide for functionally equivalent molecules, i. e., function conservative variants. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine, and histidine) ; negatively charged amino acids (aspartate and glutamate) ; polar neutral amino acids ; and non polar amino acids.

The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

Screening Methods to Identifv Riboflavin Svnthase Inhibitors/Herbicides The methods and compositions of the present invention can be used to identify compounds that inhibit the function of riboflavin synthase and thus are useful as herbicides or as lead compounds for the development of useful herbicides. This may be achieved by providing a cell that expresses riboflavin synthase and thereby produces cell cultures expressing riboflavin synthase are incubated in the presence of test compounds to form test cultures, and in the absence of test compounds to form control cultures. Incubation is allowed to proceed for a sufficient time and under appropriate conditions to allow for interference with riboflavin synthase function. At a predetermined time after the start of incubation with a test compound, an assay is performed to monitor riboflavin synthase enzymatic activity. Alternativly riboflavin synthase may be isolated from host cells that expresses riboflavin synthase. The isolated riboflavin synthase protein is incubated in the presence of test compounds to form test assays, and in the absence of test compounds to form control assays.

Incubation is allowed to proceed for a sufficient time and under appropriate conditions to allow for interference with riboflavin synthase function.

Riboflavin synthase activity is monitored in whole cells or alternatively, riboflavin synthase enzymatic activity may be monitored in cell extracts or mixtures containing the isolated riboflavin synthase enzyme using conventional assays such as that described in Example 5-7 below. Additional controls, with respect to both cultur samples and assay samples, are also included, such as, for example, a host cell not expressing riboflavin synthase (e. g., a host cell transformed with an expression plasmid containing the riboflavin synthase gene in a reverse orientation or with no insert). Riboflavin synthase inhibitory compounds are identified as those that reduce riboflavin synthase activity in the test cultures or assays relative to the control cultures or assays.

Host cells that may be used in practicing the present invention include without limitation bacterial, fungal, insect, mammalian, and plant cells. Preferably, bacterial cells are used. Most preferably, the bacterial cell is E. coli or B. subtilis.

Preferably, the methods of the present invention are adapted to a high-throughput screen, allowing a multiplicity of compounds to be tested in a single assay. Such inhibitory compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compount files, and synthetic compound libraries. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co.

(Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milord, CT). A rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondell et al., TibTech 14 : 60, 1996).

Riboflavin synthase inhibitor assays according to the present invention are advantageous in accommodating many different types of solvents and thus allowing the testing of compounds from many sources.

Once a compound has been identified by the methods of the present invention as riboflavin synthase inhibitor, in vivo and in vitro tests may be performed to further characterize the nature and mechanism of the riboflavin synthase inhibitory activity.

For example, the effect of an identified compound on in vitro enzymatic activity of purified or partially purified riboflavin synthase may be determined and enzyme kinetic plots may be used to distinguish, e. g., competitive and non-competitive inhibitors.

Compounds identified as riboflavin synthase inhibitors using the methods of the present invention may be modified to enhance potency, efficacy, uptake, stability, and suitability for use in commercial herbicide applications, etc. These modifications are achieved and tested using methods well-known in the art.

Isolation of Herbicide-Resistant Riboflavin Svnthase Variants The present invention encompasses the isolation of riboflavin synthase variants that are resistant to the action of riboflavin synthase inhibitors/herbicides. The riboflavin synthase variants may be naturally occurring or may be obtained by random or site- directed mutagenesis.

In one embodiment, a population of cells or organisms expressing riboflavin synthase is mutagenized using procedures well-known in the art, after which the cells or organisms are subjected to a screening or selection procedure to identify those that are resistant to the toxic effects of an riboflavin synthase inhibitor. The variant riboflavin synthase gene is then isolated from the resistant cell or organism using, e. g., PCR techniques.

In another embodiment, an isolated riboflavin synthase gene is subjected to random or site-directed mutagenesis in vitro, after which mutagenized versions of the gene are reintroduced into an appropriat cell such as, e. g., E. coli, and the cells are subjected to a selection or screening procedure as above.

The variant riboflavin synthase genes are expressed in an appropriate host cell, and the enzymatic properties of variant riboflavin synthase polypeptides are compared to the wild-type riboflavin synthase. Preferably, a given mutation results in an riboflavin synthase variant polypeptide that retains in vitro enzymatic activity, while exhibiting catalytic activity that is relatively more resistant to the selected herbicide (s) than is wild-type riboflavin synthase. Preferably, when expressed in a cell that requires riboflavin synthase activity for viability, the variant exhibits (i) catalytic activity alone sufficient to maintain the viability of a cell in which it is expressed ; or catalytic activity in combination with any herbicide resistant riboflavin synthase variant protein also expressed in the cell, which may be the same as or different than the first riboflavin synthase variant protein, sufficient to maintain the viability of a cell in which it is expressed ; and (ii) catalytic activity that is more resisant to the herbicide than is wild type riboflavin synthase.

Therefore, any one specific riboflavin synthase variant protein need not have the total catalytic activity necessary to maintain the viability of the cell, but must have some catalytic activity in an amount, alone or in combination with the catalytic activity of additional copies of the same riboflavin synthase variant and/or the catalytic activity of other riboflavin synthase variant protein (s), sufficient to maintain the viability of a cell that requires riboflavin synthase activity for viability. For example, catalytic activity may be increased to minimum acceptable levels by introducing multiple copies of a variant encoding gene into the cell or by introducing the gene which further includes a relatively strong promoter to enhance the production of the variant.

More resistant means that the catalytic activity of the variant is diminished by the herbicide (s), if at all, to a lesser degree than wild-type riboflavin synthase catalytic activity is diminished by the herbicide (s). Preferred more resistant variant riboflavin synthase retains sufficient catalytic activity to maintain the viability of a cell, plant, or organism wherein at the same concentration of the same herbicide (s), wild-type riboflavin synthase would not retain sufficient catalytic activity to maintain the viability of the cell, plant or organism.

Preferably, the catalytic activity in the absence of herbicide (s) is at least about 5 % and, most preferably, is more than about 20 % of the catalytic activity of the wild- type riboflavin synthase in the absence of herbicide (s).

Herbicide-resistant riboflavin synthase variants can be used as genetic markers in any cell that is normally sensitive to the inhibitory effects of the herbicide formation.

In one embodiment, DNA encoding an herbicide-resistant riboflavin synthase variant is incorporated into a plasmid under the control of a suitable promoter. Any desired gene can then be incorporated into the plasmid, and the final recombinant plasmid introduced into an herbicide-sensitive cell. Cells that have been transformed with the plasmid are then selected or screened by incubation in the presence of a concentration of herbicide sufficient to inhibit growth and/or riboflavin synthase activity.

Chemical-resistant Plants and Plants Containinc Variant Riboflavin Svnthase Genes The present invention encompasses transgenic cells, including, but not limited to seeds, organisms, and plants into which genes encoding herbicide-resistant riboflavin synthase variants have been introduced. Non-timiting examples of suitable recipient plants are listed in Table 1 below : TABLE 1 RECIPIENT PLANTS COMMONNAME FAMILY LATIN NAME Maize Gramineae Zea mays Maize ; Dent Gramineae Zea mays dentiformis Maize, Flint Gramineae Zea mays vulgaris Maize, Pop Gramineae Zea mays microsperma Maize, Soft Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea mays amyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize, Waxy Gramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum spelta Wheat, Durum Pooideae Triticum durum Wheat, English Pooideae Triticum turgidum Wheat, Large Spelt Pooideae Triticum spelta Wheat, Polish Pooideae Triticum polonium Wheat, Poulard Pooideae Triticum turgidum Wheat, singlegrained Pooideae Triticum momococcum Wheat, Small Spelt Pooideae Triticum monoccoccum Wheat, Soft Pooideae Triticum aestivem Rice Gramineae Oryza sativa Rice, American Wild Gramineae Zizania aquatica Rice, Australian Gramineae Oryza australiensis Rice, Indian Gramineae Zizania aquatica Rice, red Gramineae Oryza glaberrima Rice, Tuscarora Gramineae Ziazana aquatica Rice, West African Gramineae Oryza glaberrima Barley Pooideae Hordeum vulgare Barley, Abyssinian Pooideae Hordeum irregulare intermediate, also Irregular Barley, Ancestral Tworow Pooideae Hordeum spontaneum Barley, Beardless Pooideae Hordeum trifurcatum Barley, Egyptian Pooideae Hordeum trifurcatum Barley, fourrowed Pooideae Hordeum vulgare polystichon Barley, sixrowed Pooideae Hordeum vulgare hexastichon Barley, Tworrowed Pooideae Hordeum distichon Cotton, Abroma Dicotyledoneae Abroma augusta Cotton, American Upland Malvaceae gossypium Hirsutum Cotton, Asiatic Tree also Malvaceae gossypium arboreaum indien Tree Cotton, Brazilian, also, Malvaceae Gossypium barbadense Kidney, and, brasiliense Pernambuco Cotton, Levant Malvaceae Gossypium herbaceum Cotton Long Sil, also Malvaceae Gossypium barbadense Long Staple, Sea Island Cotton Mexican, also Malvaveae Gossypium hirsutum Short Staple Soybean, Soya Leguminosae Glycine max Sugar beet Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plant Arenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato, Cherry Solanaceae Lycopersicon esculentum cerasiforme Tomato, Common Solanaceae Lycopersicon esculentum commune Lycopersicon Tomato, Currant Solanaceae pimpinellifolium Tomato, Husk Solanaceae Physalis ixocarpa Tomato, Hyenas Solanaceae Solanum incanum Tomato, Pear Solanaceae Lycopersicon esculentum pyriforme Tomato, Tree Solancaeae Cyphomandra betacea Potato Solanaceae Solanum tuberosum Potato, Spanish, Sweet Convolvulaceae Ipormoca batatas poatao Rye, Common Pooideae Secale cereale Rye, Mountain Pooideae Secale montanum Pepper, Bell Solanaceae Capsicum annuum grossum Pepper, Bird, also Solanaceae Capsicum annuum Cayenne, Guinea minimum Pepper, Bonnet Solanaceae Capsicum sinense Pepper, Bullnose, also Solanaceae Capsicum annuum Sweet grossum Pepper, Cherry Solanaceae Capiscum annuum cerasiforme Pepper, Cluster, also Red Solanaceae Capsicum annuum Cluster fasciculatum Pepper, Cone Solanaceae Capsicum annuum conoides Pepper, Goat, also Spur Solanaceae Capsicum frutescens Pepper, Long Solanaceae Capsicum frutescens longum Pepper, Ornamental Red, Solanaceae Capsicum annuum also Wrinkled abbreviatum Pepper, Tabasco Red Solanaceae Capsicum annuum conoides Lettuce, Garden Compositae Lactuca sativa Lettuce, Asparagus, also Compositae Lactuca sativa Celery asparagina Lettuce, Blue Compositae Lactuca perennis Lettuce, Blue, also Compositae Lactuca pulchella Chicory Lettuce, Cabbage, also Compositae Lactuca satica capitata Head Lettuce, Cos, also Compositae Lactuca sativa longifolia Longleaf, Romain Lettuce, Crinkle, also Compositae Lactuca sativa crispa Curled, Cutting, Leaf Celery Umbelliferae Apium graveolens dulce Celery, Blanching, also Umbelliferae Apium graveolens dulce Garden Celery, Root, also Umbelliferae Apium graveolens Turniproote rapaceum Eggplant, Garden Solanaceae Solanum melongena Sorghum Sorghum All crop specie Alfalfa Leguminosae Medicago sativium Carrot Umbelliferae Daucus carota sativa Bean, Climbing Leguminosea Phaseolus vulgaris vulgaris Bean, Sprouts Leguminosae Phaseolus aureus Bean, Brazilian Broad Leugminoseae Canavalia ensiformis Bean, Broad Leugminosae Vicia faba Bean, Common, also Leguminosae Phaseolus vulgaris French, White, Kidney Bean, Egyptian Leguminosae Dolichos lablab Bean, Long, also Leguminosae Vigna sesquipedalis Yardlong Bean, Winged Leguminosae Psophocarpus teragonolobus Oat, also Common, Side, Avena Sativa Tree Oat, Black, also Bristle, Avena Strigosa Lopsided Oat, Bristle Avena Pea, also Garden, Green, Leguminosae Pisum, sativum sativum Shelling Pea, Blackeyed Leguminosae Vigna sinensis Pea, Edible Podded Leguminosae Pisum sativum axipluum Pea, Grey Leguminosae Pisum sativum speciosum Pea, Winged Leguminosae Tetragonolobus purpureus Pea, Wrinkled Leguminosae Pisum sativum meduilare Sunflower Compositae Helianthus annuus Squash, Autumn, Winter Dicotyledoneae Cucurbita maxima Squash, Bush, also Dicotyledoneae Cucurbita pepo melopepo Summer Squash, Turban Dicotyledoneae Cucurbita maxima turbaniformis Cucumber Dicotyledoneae Cucumis sativus Cucumber, African, also Momordica charantia Bitter Cucumber, Squirting, also Ecbalium elaterium Wild Cucumber, Wild Cucumis anguria Poplar, California Woody-Plant Populus trichocarpa Popular, European Black Populus nigra Poplar, Gary Populus cancescens Poplar, Lombardy Populus italica Poplar, Silverleaf, also Populus alba White Poplar, Wester Balsam Populus trichocarpa Tobacco Solanaceae Nicotiana Arabidopsis Thaliana Cruciferae Aradbidoposis thaliana Turfgrass Lolium Turfgrass Agrostis Other families of turfgrass Clover Leguminosae Expression of the variant polypeptides in transgenic plants confers a high level of resistance to herbicides allowing the use of these herbicides during cultivation of the transgenic plants.

Methods for the introduction of foreign genes into plants are known in the art.

Non-limiting examples of such methods include Agrobacterium infection, particle bombardment, polyethylene glycol (PEG) treatment of protoplasts, electroperation of protoplasts, microinjection, macroinjection, tiller injection, pollen tube pathway, dry seed inhibition, laser perforation, and electrophoresis. These methods are described in, for example, B. Jenes et al., and S. W, Ritchie et al. In Transgenic Plants, Vol. 1, Engineering and Utilization, ed. S.-D. Kung, R. Wu, Academic Press, Inc., Harcourt Brace Jovanovich 1993 ; and L. Mannonen et al., Critical Reviews in Biotechnology, 14 : 287-310, 1994.

In a preferred embodiment, the DNA encoding a variant riboflavin synthase is cloned into a DNA vector containing an antibiotic resistance marker gene, and the recombinant riboflavin synthase DNA-containing plasmid is introduced into Agrobacterium tumefaciens containing a Ti plasmid. This"binary vector system"is described in, for example, U. S. Patent No. 4, 490, 838, and in An et al.. Plant Mol.

Biol. Manual A3 : 1-19 (1988). The transformed Agrobacterium is then co-cultivated with leaf disks from the recipient plant to allow infection and transformation of plant cells. Transformed plant cells are then cultivated in regeneration medium, which promotes the formation of shoots, first in the presence of the appropriate antibiotic to select for transformed cells, then in the presence of herbicide. In plant cells successfully transformed with DNA encoding herbicide-resistant riboflavin synthase, shoot formation occurs even in the presence of levels of herbicide that inhibit shoot formation from non-transformed cells. After confirming the presence of variant riboflavin synthase DNA using, for example, polymerase chain reaction (PCR) analysis, transformed plants are tested for their ability to withstand herbicide spraying and for their capabilities for seed germination and root initiation and proliferation in the presence of herbicide.

The methods and compositions of the present invention can be used for the production of herbicide-resistant riboflavin synthase variants, which can be incorporated into plants to confer selective herbicide resistance on the plants.

Intermediate variants of riboflavin synthase (for example, variants that exhibit sub- optimal specific activity but high herbicide resistance, or the converse) are useful as templates for the design of second-generation riboflavin synthase variants that retain adequate specific activity and high resistance.

Herbicide resistant riboflavin synthase genes can be transformed into crop species in single or multiple copies to confer herbicide resistance. Generic engineering of crop species with reduced sensitivity to herbicides can : (1) Increase the spectrum and flexibility of application of specific effective and enviromentally benign herbicides ; (2) Enhance the commercial value of these herbicides ; (3) Reduce weed pressure in crop fields by effective use of herbicides on herbicide resistant crop species and a corresponding increase in harvest yields ; (4) Increase sales of seed for herbicid resistant plants ; (5) Increase resistance to crop damage from carry-over of herbicides applied in previous planting ; (6) Decrease susceptiblity to changes in herbicide characteristics due to adverse climate conditions ; and (7) Increase tolerance to unevenly or mis-applied herbicides.

For example, transgenic riboflavin synthase variant protein containing plants can be cultivated. The crop can be treated with a weed controlling effective amount of the herbicide to which the riboflavin synthase variant transgenic plant is resistant, resulting in weed control in the crop without detrimentally affecting the cultivated crop.

Description of the Preferred Embodiments The follow examples are intended to illustrate the present invention without limitation.

Example 1 Construction of the expression vector pNCOt 13 Destruction of the Ncol recognition site of pQE30 : 2. 0 pg of the vector pQE30 (Qiagen, Hilden, Germany) ist digested with 30 U Ncol (New England Biolabs, Schalbach, Germany (NEB)) in total volume of 60 Ll containing 6 ut of NEB4 buffer.

The reaction mix is incubated for 3 h at 37 °C. After adding 33 LM of each dNTP (NEB) and 5 U Klenow fragment of polymerase I from E. coli (NEB) the reaction mix is incubated for additional 30 min at 25 °C. The vector DNA is purified using the PCR purification kit from Qiagen as described in example 3.

20 ng of vector DNA is religated with 1 U of T4-Ligase from Gibco-BRL (Eggenstein, Germany), 2 pl of T4-Ligase buffer (Gibco-BRL) in a total volume of 10 ut yielding the plasmid pQE_noNco. The ligation mixture is incubated over night at 4 °C. With 2 ut of the ligation mixture electrocompetent E. coli XL1-Blue (Bullock et al., 1987, commercial source : Stratagene, LaJolla, CA, USA) cells are transformed.

Preparation of electrocompetent cells : 1 liter of LB medium is inoculated 1 : 100 with fresh overnight culture of E. coli XL1-Blue. The cells are grown at 37 °C with shaking at 220 rpm to an optical density of 0. 5 at 600 nm. The cells are chilled on ice for 20 min and centrifuged for 15 min at 4, 000 rpm at 4 °C. The supernatant is removed and the pellet is resuspended in 1 liter of ice-cold sterile 10 % (v/v) glycerol. The cells are centrifuged two times as described before resuspending the cells in 0. 5 liter and in 20 ml of ice-cold sterile 10 % (v/v) glycerol, respectively. The cells are centrifuged an additional time and the pellet is resuspended in a volume of 2 mi of ice-cold 10 % (v/v) glycerol. This suspension is frozen in aliquots of 80 pi and stored in liquid nitrogen.

Electro-transformation using the Gene Pulser apparatus from Biorad (Munich, Germany) : The electrocompetent cells are thawed on ice. 40 ul of the cell suspension are mixed with 2 NI of ligation mixture and transferred into a prechilled, sterile 0. 2 cm cuvette (Biorad). The suspension is shaked to the bottom and the cuvette is placed into the prechilled chamber slide. The chamber slide is pushed into the chamber and the cells are pulsed at 2. 50 kV, 25 uF and Pulse Controller setting 200 Q. The cuvette is removed from the chamber and the cells are suspended in 1 mi of SOC medium (2 % (w/v) casein hydrolysate, 0. 5 % (w/v) yeast extract, 10 mM NaCI, 2. 5 mM KCI, 10 mM MgCI2, 10 mM MgS04 and 20 mM glucose). The suspension is shaked for 1 h at 37 °C and 100 NI of the suspension is plated on LB plates containing 150 mg/l ampicillin for maintenance of the plasmid pQEnoNco.

Plasmid isolation, pQE_noNco : Cells of Escherichia coli XL1-Blue harboring the vector pQE_noNco, are grown overnight in Luria Bertani (LB) medium containing 180 mg/l of ampicillin for maintenance of the plasmid in the host cells. 7 ml of the culture are centrifuged for 20 min at 5, 000 rpm. The cell pellet is used for isolation of the plasmid pQEnoNco with the mini plasmid isolation kit from Qiagen (Hilden, Germany). The pellet is resuspended in 0. 3 ml of 10 mM EDTA in 50 mM Tris hydrochloride, pH 8. 0. 30 ug RNase are added. 0. 3 ml of 1 % (w/v) SDS in 200 mM sodium hydroxide are added and incubated for 5 min at room temperature. 0. 3 ml of chilled 3. 0 M sodium acetate, pH 5. 5 are added and incubated for 10 min on ice. The mixture is centrifuged for 15 min at 14, 000 rpm in a minifuge. The supernatant is applied onto a Quiagen-tip 20, which is previously equilibrated with 1 ml of 750 mM NaCI, 15 % (v/v) ethanol and 0. 15 % (v/v) Triton X-100 in 50 mM MOPS, pH 7. 0. The Quiagen-tip is washed four times with 1 ml of of 1000 mM NaCI and 15 % (v/v) ethanol in 50 mM MOPS, pH 7. 0. The DNA is eluted with 0. 8 ml of 1250 mM NaCI and 15 % (v/v) ethanol in 50 mM Tris hydrochloride, pH 8. 5. The DNA is precipated with 0. 56 ml of isopropanol, centrifuged 30 min at 14, 000 rpm and washed with 1 ml of ice-cold 70 % (v/v) ethanol. After drying in a vacuum centrifuge for 5 min, the DNA is dissolved in 50 ut of redistilled H20. The solution contained 8. 3 pg of DNA.

Construction of pNC0113 : 2. 0 pg of the vector pQEnoNco is digested with 30 U EcoRl and 30 U Sall (NEB) in a total volume of 60 pI containing 6 ut of EcoRl-buffer (NEB). The reaction mix is incubated for 3 h at 37 °C. The vector DNA is purified using the PCR purification kit from Qiagen. By this procedure fragments smaller than 100bp will not be isolated.

25 pmol of the oligonucleotides 5'-CACACAGAATTCATTAAAGAGGAGAAATTAA CCATGGGAGGATCCGTCGACCTGCAGCC-3'and 5'-GGCTGCAGGTCGACGGA TCCTCCCATGGTTAATTTCTCCTCTTTAATGAATTCTGTGTG-3'are dissolved in 6 pI EcoRl-buffer (NEB) and 54 pI H2O. The solution is heated to 96°C for 2 min and cooled down to 10°C within 12 h in order to hybridisate the linker. The reaction mix is supplied with 30 U EcoRl and 30 U Sall (NEB) and incubated for 3h at 37°C. The reaction mix is heated to 65°C for 30 min in order to inactivate the enzymes and cooled down to 10°C within 12 h for hybridisation. The reaction mix is yielding approximately 730 ng of the linker.

20 ng of vector DNA and 300 pg of the linker are ligated together with 1 U of T4- Ligase from Gibco-BRL (Eggenstein, Germany), 2 pi of T4-Ligase buffer (Gibco- BRL) in a total volume of 10 ut yielding the plasmid pNC0113. The ligation mixture is incubated over night at 4 °C. With 2 pi of the ligation mixture electrocompetent E. coli XL1-Blue cells are transformed. The E. coli strain XL1-Blue harbouring plasmid pNC 0113 has been deposited on October 14, 1999 and received the patent deposit designation PTA-852 at ATCC.

Example 2 Isolation of cDNA from A. thaliana 1 g of 2 weeks old Arabidopsis thaliana var. Columbia plants (stems and leafs) are frozen and homogenisated in liquid nitrogen. 8 ml of a sterile solution of 600 g/t guanidine thiocyanate, 5 9/l sodium-N-lauroylsarcosine, 50 mM trisodiumcitrate and 5 ml/I 2-mercaptoethanol are added. This mixture is added carefully to 3 mi of a solution (autoclaved) of 959 gui CsCl and 37, 2 g/i EDTA and centrifugated at 33000 rpm at 18°C for 24 h. The supernatant is dicarded and the pellet is airdried for 10 min. The dried pellet is dissolved in 360 ti H20 (bidestillated, sterile). The solution is centrifugated at 14000 rpm for 10 min. The supernatant is mixed with 40, ul 3 M sodium acetate and 1 ml ethanol. The RNA is precipitated over night at-20°C, centrifugated at 14000 rpm at 4°C for 15 min. and washed twice with 500 ml 75% ethanol. The pellet is airdried and dissolved in 200 pI H20 (bidestillated, sterile).

500 µg RNA are obtained.

A mixture containing 2. 75 ig RNA, 50 nmol dNTP's, 1 jig random hexameric primer, 1 p9 T15-primer and 20 % first strand 5x buffer (Promega) in a total volume of 50 1ll is incubated for 5 min. at 95 °C, cooled on ice and 500 U M-MLV reverse transkriptase (Promega) are added. The mixture is incubated for 1 h at 42 °C. After incubation at 92 °C for 5 min., RNase A (20 U) and RNase H (2 U) are added and the mixture is incubated for 30 min. at 37 °C.

The resulting cDNA (1, ul of this mixture) is used for the amplification of ribC by PCR as described in example 3.

Example 3 Construction of an expression clone for riboflavin synthase (ribC) from A. thaliana Cells of Escherichia coli XL1-Blue (Bullock et al., 1987) harboring the expression vector pNCO113 are grown overnight in Luria Bertani (LB) medium containing 180 mg/l of ampicillin for maintenance of the plasmid in the host cells. 7 ml of the culture are centrifuged for 20 min at 5, 000 rpm. The cell pellet is used for isolation of the plasmid pNCO113 with the mini plasmid isolation kit from Qiagen (Hilden, Germany).

The pellet is resuspended in 0. 3 ml of 10 mM EDTA in 50 mM Tris hydrochloride, pH 8. 0. 30 ug RNase are added. 0. 3 mi of 1 % (w/v) SDS in 200 mM sodium hydroxide are added and incubated for 5 min at room temperature. 0. 3 ml of chilled 3. 0 M sodium acetate, pH 5. 5 are added and incubated for 10 min on ice. The mixture is centrifuged for 15 min at 14, 000 rpm in a centrifuge. The supernatant is applied onto a Qiagen-tip 20, which is previously equilibrated with 1 ml of 750 mM NaCI, 15 % (v/v) ethanol and 0. 15 % (v/v) Triton X-100 in 50 mM MOPS, pH 7. 0. The Qiagen-tip is washed four times with 1 ml of of 1000 mM NaCI and 15 % (v/v) ethanol in 50 mM MOPS, pH 7. 0. The DNA is eluted with 0. 8 mi of 1250 mM NaCI and 15 % (v/v) ethanol in 50 mM Tris hydrochloride, pH 8. 5. The DNA is precipated with 0. 56 ml of isopropanol, centrifuged 30 min at 14, 000 rpm and washed with 1 mi of ice-cold 70 % (v/v) ethanol. After drying in a vacuum centrifuge for 5 min, the DNA is dissolved in 50 pl of sterile H20. The solution contained 8. 3 pg of pNC0113-DNA.

The A. thaliana sequence (accession no. gb AC006234) from basepair (bp) position 107550 to 108164 is amplifie by PCR using cDNA (prepared as described in example 2) as template. The reaction mixture contained 1. 5 mM MgCI2, 50 mM KCI, 10 mM Tris-hydrochloride, pH 8. 8, 0. 1 % (w/w) Triton X-100, 10 pmol of primer 5'-GGAGAAATTAACCATGTTTACTGGAATCGTGGAGGAAATG-3'10 pmol of primer 5'-GCGATCTGCAGAGTTACCTTTGGAGAAGC-3', 1 pl of A. thaliana cDNA, 2U of Taq DNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in a total volume of 100 J.

The mixture is denaturated for 3 min at 95 °C. Then 25 PCR cycles for 30 sec at 94 °C, 30 sec at 50 °C and 45 sec at 72 °C follow. After further incubation for 7 min at 72 °C, the mixture is cooled to 4 °C.

The PCR product is used as template for a second PCR reaction. The reaction mixture contained 1. 5 mM MgC12, 50 mM KCI, 10 mM Tris-hydrochloride, pH 8. 8, 0. 1 % (w/w) Triton X-100, 10 pmol of primer 5'- ACACAGAATTCATTAAAGAGGAGAAATTAACCATG-3', 10 pmol of primer 5'-GCGATCTGCAGAGTTACCTTTGGAGAAGC-3', 1 Nl of the first PCR amplification, 2 U of Taq DNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in a total volume of 100 pI.

The mixture is denaturated for 3 min at 95 °C. Then 25 PCR cycles for 45 sec at 94 °C, 45 sec at 50 °C and 60 sec at 72 °C follow. After further incubation for 20 min at 72 °C, the mixture is cooled to 4 °C.

The PCR amplificate is purified with a PCR purification kit from Qiagen. 500 ut of buffer PB (Qiagen) are added to 98 ut of PCR reaction mixture and applied to a Qiaquick column and centrifuged for 1 min at 14, 000 rpm. The flow through is discarded. 0. 75 ml of buffer PE (Qiagen) are loaded on the column and centrifuged as before. The flow through is discarded and the column is centrifuged for an additional 1 min at 14, 000 rpm. The column is placed in a clean 1. 5 ml eppendorf tube. 50 NI of H20 (redistilled, sterile) are added to the column and it is centrifuged for 1 min at 14, 000 rpm. The flow through contained 2 pg PCR product.

2. 0 pg of the vector pNC0113 and 1. 5 pg of the purified PCR product are digested in order to produce DNA fragments with overlapping ends. Each restriction mixture contained 7 pl of EcoRl-buffer from New England Biolabs (Schalbach, Germany) (NEB), 30 U of EcoRl (NEB), 30 U of Pstl (NEB) and the DNA in a total volume of 70 pi and is incubated for 3 h at 37 °C. Digested vector DNA and PCR product are purified using the PCR purification kit from Qiagen.

20 ng of vector DNA and 16 ng of PCR product are ligated together with 1 U of T4- Ligase from Gibco-BRL (Eggenstein, Germany), 2 pl of T4-Ligase buffer (Gibco) in a total volume of 10 pi yielding the plasmid pNCOararibC. The ligation mixture is incubated over night at 4 °C. With 1 pl of the ligation mixture electrocompetent E. coli XL1-Blue cells are transformed as described in example 1.

Example 4 Preparation of recombinant riboflavin synthase from A. thaliana 0. 5 liter of Luria Bertani (LB) medium containing 90 mg of ampicillin are inoculated with 10 ml of an overnight culture of E. coli strain XL1-Blue harboring plasmid pNCOararibC. The culture is grown in a shaking culture at 37 °C. At an optical density (600 nm) of 0. 7, the culture is induced with 2 mM isopropyl-p-D- thiogalactopyranoside The culture is grown for further 5 h. The cells are harvested by centrifugation for 20 min at 5, 000 rpm and 4 °C. The cells are washed with 25 mM Tris hydrochloride pH 8. 2, centrifuged as above and frozen at-20 °C for storage.

The cells are thawed in 10 ml of 25 mM tris hydrochloride pH 8. 2 containing 0. 02 % sodium azide (buffer A) in the presence of 1 mM phenylmethylsulfonylfluorid, 4 mg/ml lysozyme and 10 ug/ml DNasel. The mixture is incubated at 37 °C for 0. 5 h, cooled on ice and sonified 6 x 10 sec with a Branson Sonifier 250 (Branson SONIC Power Company, Danbury, USA) set to 70 % duty cycle output, control value of 4.

The suspension is centrifuged at 15, 000 rpm at 4°C for 30 min. The supernatant is applied on a column of Sepharose QFF (size 30 cm3, Amersham Pharmacia Biotech, Freiburg, Germany) previously equilibrated with 200 ml buffer A. The column is washed with 50 ml buffer A. Riboflavin synthase is eluted from the column with a gradient from 0-1 M sodium chloride in 300 ml of buffer A. The enzyme is identified by SDS-PAGE showing a band at 22 kDa. Fractions showing this protein band are collecte and dialyse against buffer A overnight.

Example 5 Screening of riboflavin synthase activity Assay mixtures containing 100 mM Tris hydrochloride pH 8. 0, 0. 6 mM 6, 7-dimethyl- 8-ribityl-lumazine (prepared according to Bacher, Methods Enzymol. 122 : 192, 1986) and 0. 1 mg enzyme sample in a total volume of 0. 5 mi are incubated for 30 min at 30 °C. The assay is stopped by adding 0. 5 ml 15 % trichloroacetic acid. The mixture is centrifuged for 10 min at 14000 rpm. The absorbance of the supernatant at 445 nm is detected. The absorbance at 445 nm of an analogous prepared assay mixture without enzyme is detected. The absorbance difference (dE) between the assay with and without enzyme is determined. The concentration of produced riboflavin (c) is calculated with the absorbance difference (dE). The extinction coefficient at 445 nm is 11500 cm-'M-1. One unit of enzyme activity catalyzes the formation of 1 nmol riboflavin per h.

Example 6 Screening of riboflavin synthase activity Assay mixtures containing 100 mM Tris hydrochloride pH 8. 0, 0. 6 mM 6, 7-dimethyl- 8-ribityl-lumazine (prepared according to Bacher, Methods Enzymol. 122 : 192, 1986) and 0. 1 mg enzyme sample in a total volume of 0. 5 mi are incubated for 30 min at 30 °C. The assay is stopped by adding 0. 5 mi 15 % trichloroacetic acid. The mixture is centrifuged for 10 min at 14Q00 rpm. The riboflavin concentration of the supernatant is determined by HPLC with a reverse phase Nucleosil RP18 column.

The eluent contains 100 mM ammonium formate in 40 % methanol. The effluent is monitored fluorometrically (excitation 445 nm ; emission 516 nm). Riboflavin from Sigma (Deisenhofen, Germany) is used as standard. The riboflavin concentration of an analogous prepared assay mixture without enzyme sample is determined as control.

Example 7 Screening of riboflavin synthase activity Assay mixtures containing 100 mM Tris hydrochloride pH 8. 0, 0. 6 mM 6, 7-dimethyl- 8-ribityl-lumazine (prepared according to Bacher, Methods Enzymol. 122 : 192, 1986) and 0. 1 mg enzyme sample in a total volume of 1 ml are incubated at 30 °C. After 30 min the absorbance of the assay mixture at 470 nm is detected. The absorbance at 470 nm of an analogous prepared assay mixtures without enzyme sample, which is incubated for the same time at 30 °C, is detected. The absorbance difference (dE) between the assay with and without enzyme sample is determined. The concentration of produced riboflavin (c) is calculated with the absorbance difference (dE). The extinction coefficient at 470 nm is 9100 cm-'M-'. One unit of enzyme activity catalyzes the formation of 1 nmol riboflavin per h.

ATCC 10801 University Blvd a Manassas, VA 20110-2209 * Telephone : 703-365-2700 * FAX : 703- BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7. 3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10. 2 To : (Name and Address of Depositor or Attorney) Institut Fuer Organische Chemie Und Biochemie Attn : Stefan Herz Lehrstuhl III (Prof. Bacher) Technische Universitaet Muenchen Lichtenbergstr. 4, 85747 Garching Germany Deposited on Behalf of : Prof. A. Bacher, Inst. Fur Org. Chemie V. Biochemie, TV-Miinchen Identification Reference by Depositor : Patent Deposit Designation Escherichia coli strain XLI-Blue harbouring plasmid pNC0113 PTA-852 The deposit was accompanied by :-a scientific description X a proposed taxonomic description indicated above.

The deposit was received October 14, 1999 by this International Depository Authority and has been accepted.

AT YOUR REQUEST : X We will not inform you of requests for the strain.

X The strain is available to the, scientific public upon request as of October 14, 1999.

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same.

The strain will be maintained for a period of at least 30 years from date of deposit, or five years after the most recent request for a sample, whichever is longer. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested November 2. 1999. On that date, the culture was viable.

International Depository Authority : American Type Culture Collection, Rockville, Md. 20852 USA Signature of person having authority to re resent ATCC : < t (, gaz Date : November 2. 1999 Barbara M. Hailey, Administrator, Patent Depository ATCC 10801 Univcrsity Blvd Mannssas, VA 20110-2209 Telophone : 703-365-27no * FAX : 703- BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7. 3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10. 2 To : (Name and Address of Depositor or Attorney) Institut Fuer Organische Chemie Und Biochemie Attn : Stefan Herz Lehrstuhl III (Prof. Bacher) Technische Universitaet Muenchen Lichtenbergstr. 4, 85747 Garching Germany Deposited on Behalf of : Prof. A. Bacher, Inst. Fiir Org. Chemie V. Biochemie, TV-Miinchen Identification Reference by Depositor : Patent Deposit Designation Escherichia coli strain XL 1-Blue harbouring plasmid pNC0113 PTA-852 The deposit was accompanied by : _ a scientific description X a proposed taxonomic description indicated above.

The deposit was received October 14. 1999 by this International Depository Authority and has been accepted.

AT YOUR REQUEST : X We will not inform you of requests for the strain.

X The strain is available to the, scientific public upon request as of October 14. 1999.

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same.

The strain will be maintained for a period of at least 30 years from date of deposit, or five years after the most recent request for a sample, whichever is longer. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested November 2. 1999. On that date, the culture was viable.

International Depository Authority : American Type Culture Collection, Rockville, Md. 20852 USA Signature of person having authority to represent ATCC : Date _ Date : November 2. 1999 Barbara M. Hailey, Administrator, Patent Depository ATCC 10801 Universily Blvd Manssas, VA 20110-2209 Telephone : 703-365-2700 * FAX : 703- BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT ISSUED PURSUANT TO RULE 7. 3 AND VIABILITY STATEMENT ISSUED PURSUANT TO RULE 10. 2 To : (Name and Address of Depositor or Attorney) Institut Fuer Organische Chemie Und Biochemie Attn : Stefan Herz Lehrstuhl III (Prof. Bacher) Technische Universitaet Muenchen Lichtenbergstr. 4, 85747 Garching Germany Deposited on Behalf of : Prof. A. Bacher, Inst. Fur Org. Chemie V. Biochemie, TV-Miinchen Identification Reference by Depositor : Patent Deposit Designation Escherichia coli strain XLI-Blue harbouring plasmid pNC0113 PTA-852 The deposit was accompanied by : _ a scientific description X a proposed taxonomic description indicated above.

The deposit was received October 14. 1999 by this International Depository Authority and has been accepted.

AT YOUR REQUEST : X We will not inform you of requests for the strain.

X The strain is available to the scientific public upon request as of October 14. 1999.

If the culture should die or be destroyed during the effective term of the deposit, it shall be your responsibility to replace it with living culture of the same.

The strain will be maintained for a period of at least 30 years from date of deposit, or five years after the most recent request for a sample, whichever is longer. The United States and many other countries are signatory to the Budapest Treaty.

The viability of the culture cited above was tested November 2. 1999. On that date, the culture was viable.

International Depository Authority : American Type Culture Collection, Rockville, Md. 20852 USA Signature of person having authority to represent ATCC : Vii. QAA"L Date : November 2. 1999 Barbara M. Hailey, Administrator, Patent Depository