WO1999005286A1 - Plant atp-phosphoribosyl transferase and dna coding therefor - Google Patents

Abstract

The present invention provides an isolated DNA molecule encoding the ATP-phosphoribosyl transferase (APRT) enzyme from a plant source or modified forms of the enzyme which are herbicide tolerant. Using the information provided by the present invention, the DNA coding sequence for APRT enzyme(s) from any plant source may be obtained using standard methods. The present invention also embodies the recombinant production of the APRT enzyme, and methods for using recombinantly produced APRT. The present invention is further directed to probes and methods for detecting the presence and form of the APRT gene and quantitating levels of APRT transcripts in an organism. The present invention further embodies expression cassettes and recombinant vectors comprising the said expression cassettes comprising essentially a promoter, but especially a promoter that is active in a plant, operably linked to a DNA molecule encoding the APRT enzyme from a eukaryotic organism according to the invention. The invention is further directed to the preparation of transgenic plants, plant tissue and plant seed which has been stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operably linked to a structural gene encoding an unmodified or modified eukaryotic APRT enzyme.

Description

PLANT ATP-PHOSPHORIBOSYL TRANSFERASE AND DNA CODING THEREFOR

The invention relates generally to a plant enzymatic activity involved in the biosynthesis of L-histidine (L-His). In particular the invention relates to the plant enzyme which catalyzes the first reaction in the histidine biosynthesis, the condensation of adenosine 5'-triphosphate (ATP) and 5-phosphoribosyl 1 -pyrophosphate (PRPP) to form N'- -phosphoribosyl-ATP (PR-ATP); and gene coding therefor. The invention further relates to various utilities including the recombinant production of this enzyme in a heterologous host, incorporation into an assay system for screening chemicals for herbicidal activity, and the development of genetic markers in plants.

One of the enzymes essential to the biosynthesis of L-His in plants is known as ATP- phosphoribosyl transferase (referred to herein as APRT). The APRT enzyme catalyzes the first reaction of the L-His biosynthetic pathway where ATP and PRPP are condensed to form PR-ATP, releasing inorganic pyrophosphate. APRT has been well characterized (Alifano et al., Microbiol.Rev60:44-69 (1996)). The rate of L-His in biosynthesis is totally regulated at the step of APRT by the end product L-His, which inhibits APRT by binding to an allosteric site (Martin, J Biol Chem 238: 257-268 (1963), Fink, Science 146: 525-527 (1964)), and the L-His biosynthesis is also controlled through the regulation of the his operon expression (Johnson and Roth, J Mol Biol 145:735-756 (1981), Hinnebusch, Microbiol.Rev. 52:248-273 (1988)). Genes encoding the APRT enzyme have heretofore not been isolated and characterized from any plant species. However, genes encoding the APRT enzyme have been isolated from a variety of non-plant species including E. coli. S. typhimuriυm, Lactococcus lactis, and Saccharomyces cerevisiae (Alifano et al., Microbiol. Rev 60:44-69 (1996)).

The present invention provides an isolated DNA molecule encoding the ATP- phoshoribosyl transferase (APRT) enzyme from a plant source.

A DNA coding sequence for an APRT enzyme in Arabidopsis thaliana and wheat is provided in SEQ ID NOS: 3, 5, 7 and 9. Using the information provided by the present invention, the DNA coding sequence for APRT enzyme(s) from any plant source may be obtained using standard methods. The present invention also embodies the recombinant production of the APRT enzyme, and methods for using recombinantly produced APRT. In particular, the present invention provides methods of using purified APRT in an assay system to screen for novel inhibitors of APRT activity which may be used as herbicides to control undesirable vegetation in fields where crops are grown, particularly agronomicaliy important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, tobacco, and soybeans.

The present invention embodies an assay system for screening chemicals for herbicidal activity using APRT. Comprised by the present invention is a method for assaying a chemical for the ability to inhibit the activity of a APRT enzyme from a plant comprising

(a) combining said APRT enzyme and phosphoribosyl pyrophosphate (PRPP), ATP and other enzymes of the His biosynthetic enzymes under conditions in which said APRT enzyme is capable of catalyzing the conversion of said phosphoribosyl pyrophosphate (PRPP) to 5-amino-1-ribosyl-4-imidazole carboxamide (AICAR)

(b) combining said chemical, said APRT enzyme, said phosphoribosyl pyrophosphate (PRPP), ATP and said other enzymes of the His biosynthetic enzymes in a second reaction mixture under the same conditions as in said first reaction mixture;

(c) comparing the absorbance of said first and said second reaction mixture at about 550nm, wherein said chemical is capable of inhibiting the activity of said APRT enzyme if the absorbance of said second reaction mixture is significantly less than the absorbance of said first reaction mixture.

Further comprised is a method for assaying a chemical for the ability to inhibit the activity of a APRT enzyme from a plant comprising

(a) combining said APRT enzyme and phosphoribosyl pyrophosphate (PRPP) and ATP under conditions in which said APRT enzyme is capable of catalyzing the conversion of said phosphoribosyl pyrophosphate (PRPP) to PRATP,

(b) combining said chemical, said APRT enzyme, said phosphoribosyl pyrophosphate (PRPP) and ATP in a second reaction mixture under the same conditions as in said first reaction mixture;

(c) comparing the absorbance of said first and said second reaction mixture at about 290 nm, wherein said chemical is capable of inhibiting the activity of said APRT enzyme if the absorbance of said second reaction mixture is significantly less than the absorbance of said first reaction mixture.

The present invention is further directed to probes and methods for detecting the presence and form of the APRT gene and quantitating levels of APRT transcripts in an organism. These methods may be used to diagnose plant disease conditions which are associated with an altered form of the APRT enzyme or altered levels of expression of the APRT enzyme.

In one aspect, the present invention is directed to an isolated DNA molecule which encodes a plant ATP-phosphoribosyl transferase (referred to herein as APRT), the enzyme which catalyzes a step in the biosynthesis of L-His. In particular the invention relates to DNA molecules encoding the plant ATP-phosphoribosyl transferase from dicotyledonous plants, but especially from Arabidopsis plants, such as those given in SEQ ID NO: 7and 9 respectively. Further embodied are DNA molecules encoding the plant ATP-phosphoribosyl transferase from monocotylodonous plants, but especially from wheat (Triticum aestivum) plants, such as those given in SEQ ID NO: 3 and 5 respectively. The invention further relates to an isolated DNA molecule encoding the plant ATP-phosphoribosyl transferase from a dicotyledonous plant, wherein said protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 10. Also comprised is an isolated DNA molecule encoding the plant ATP-phosphoribosyl transferase from a monocotyledonous plant, wherein said protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 6.

Using the information provided by the present invention, the DNA coding sequence for the APRT enzyme may be isolated from the genome of any plant species according to the invention using standard methods. Thus in a further embodiment the present invention provides probes capable of specifically hybridizing to a plant DNA sequence encoding APRT enzyme activity to the respective mRNA and methods for detecting the said DNA sequences in eucaryotic organisms using the probes according to the invention.

The present invention further embodies expression cassetts and recombinant vectors comprising the said expression cassetts comprising essentially a promoter, but especially a promoter that is active in a plant, operably linked to a DNA molecule encoding the APRT enzyme from a eukaryotic organism according to the invention. The expression cassette according to the invention may in addition further comprise a signal sequence operably linked to said DNA molecule, wherein said signal sequence is capable of targeting the protein encoded by said DNA molecule into a suitable cellular compartement, but preferably into the chloroplast or the mitochondria.

In addition, the present invention provides plants, plant tissues and plant seeds with altered APRT activity which are resistant or at least tolerant to inhibition by a herbicide at levels which normally are inhibitory to the naturally occurring APRT activity in the plant. In particular, the invention embodies plants, wherein the altered APRT activity is conferred by over-expression of the wild-type APRT enzyme or by expression of a DNA molecule encoding a herbicide tolerant APRT enzyme. The said herbicide tolerant APRT enzyme may be a modified form of a APRT enzyme that naturally occurs in a eukaryote or a prokaryote; or a modified form of a APRT enzyme that naturally occurs in said plant; or the said herbicide tolerant APRT enzyme may naturally occur in a prokaryote. Plants encompassed by the invention include monocotyledonous and dicotyledonous plants, but especially those which would be potential targets for APRT inhibiting herbicides, particularly agronomicaliy important crops such as maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, tobacco, sunflower, sugar cane, sugar beet, oilseed rape, and soybeans. Further encompassed by the invention is a DNA molecule encoding a modified ATP- phosphoribosyl transferase comprising a plant ATP-phosphoribosyl transferase having at least one amino acid modification, wherein said modified plant ATP-phosphoribosyl transferase is tolerant to a herbicide in amounts which inhibit said a plant ATP- phosphoribosyl transferase.

The invention embodies a DNA molecule according to the invention which is part of a plant genome.

The present invention is further directed to methods for the production of plants, plant tissues, and plant seeds which contain a APRT enzyme resistant to, or tolerant of inhibition by a herbicide at a concentration which inhibits the naturally occurring APRT activity. The said resistance or tolerance may be obtained by expressing in the said transgenic plants either a DNA molecule encoding a modified form of a APRT enzyme that naturally occurs in a eukaryote, or a modified form of a APRT enzyme that naturally occurs in said plant, or a APRT enzyme that naturally occurs in a prokaryot, or a APRT enzyme which is a modified form of a protein which naturally occurs in a prokaryote.

One specific embodiment of the invention is directed to the preparation of transgenic maize plants, maize tissue or maize seed which have been stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operably linked to a structural gene encoding an unmodified prokaryotic APRT enzyme which is resistant to the herbicide.

The invention is further directed to the preparation of transgenic plants, plant tissue and plant seed which has been stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in plants operably linked to a structural gene encoding an unmodified eukaryotic APRT enzyme. This results in over-expression of the unmodified APRT in the plant sufficient to overcome inhibition of the enzyme by the herbicide.

The present invention also embodies the production of plants which express an altered APRT enzyme tolerant of inhibition by a herbicide at a concentration which normally inhibits the activity of wild-type, unaltered APRT. In this embodiment, the plant may be stably transformed with a recombinant DNA molecule comprising a structural gene encoding the resistant APRT, or prepared by direct selection techniques whereby herbicide resistant lines are isolated, characterized and developed.

The present invention is further directed to a method for controlling the growth of undesired vegetation which comprises applying to a population of a plant with altered APRT activity which is resistant to inhibition by a herbicide at levels which normally are inhibitory to the naturally occurring APRT activity in the said plant, an effective amount of a APRT - inhibiting herbicide. Plants to be protected in the described way are especially those which would be potential targets for APRT inhibiting herbicides, particularly agronomicaliy important crops such as, for example, maize and other cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and the like, as well as cotton, sugar cane, sugar beet, oilseed rape, tobacco, sunflower and soybeans. Herbicides that qualify as APRT inhibitors are those selected from the group consisting of aryluracil, diphenylether, oxidiazole, imide, phenyl pyrazole, pyridine derivative, phenopylate and O- phenylpyrrolidino- and piperidinocarbamate analogs of said phenopylate.

The present invention also embodies the recombinant production of the APRT enzyme, and methods for using recombinantly produced APRT. The invention thus further embodies host cells, but especially cells selected from the group consisting of plant cells, animal cells, bacterial cells, yeast cells and insect cells, stably transformed with a recombinant DNA molecule comprising a suitable promoter functional in the respective host cell operably linked to a structural gene encoding an unmodified or modified plant APRT enzyme, wherein said host cell is capable of expressing said DNA molecule.

The present invention further provides methods of using purified APRT to screen for novel herbicides which affect the activity of APRT, and to identify herbicide-resistant APRT mutants.

In a further embodiment of the invention a method is provided for identifying a modified APRT enzyme resistant to a APRT inhibitor present in a population of cells comprising the steps of

(a) culturing said population in the presence of said APRT inhibitor in amounts which inhibit the unmodified form of said APRT enzyme;

(b) selecting those cells from step (a) whose growth is not inhibited; and

(c) isolating and identifying the APRT enzyme present in the cells selected from step (b).

Genes encoding altered APRT can be used as selectable markers in plant cell transformation methods. The present invention thus further embodies a method of selecting plants, plant tissue or plant cells transformed with a transgene of interest from non-transformed plants, comprising the steps of:

(a) transforming a plant, plant tissue or plant cell with a transgene of interest capable of being expressed by the plant, and a gene encoding an altered APRT resistant to a APRT inhibitor;

(b) transferring the thus-transformed plants or plant cells to a medium comprising the APRT inhibitor; and

(c) selecting the plants or plant cells which survive in the medium. The present invention is further directed to probes and methods for detecting the presence and form of the APRT gene and quantitating levels of APRT transcripts in an organism. These methods may be used to diagnose disease conditions which are associated with an altered form of the APRT enzyme or altered levels of expression of the APRT enzyme.

Plant APRT coding sequences may be isolated according to well known techniques based on their structural sequence homology to the Arabidopsis thaliana and wheat APRT enzymes. In these techniques all or part of the known APRT coding sequence is used as a probe which selectively hybridizes to other APRT coding sequences present in genomic or cDNA libraries from a chosen organism. Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g.. Sambrook et al., Molecular Cloning , eds., Cold Spring Harbor Laboratory Press. (1989)) and amplification by PCR using oligonucleotide primers corresponding to sequence domains conserved among known APRT amino acid sequences (see, e.g. Innis et al., . PCR Protocols, a Guide to Methods and Applications eds., Academic Press (1990)).

The isolated plant APRT sequences may be manipulated according to standard genetic engineering techniques to suit any desired purpose. For example, the entire APRT sequence or portions thereof may be used as probes capable of specifically hybridizing to APRT coding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among APRT coding sequences and are preferably at least 20 nucleotides in length, and most preferably at least 50 nucleotides in length. Such probes may be used to amplify and analyze APRT coding sequences from a chosen organism via the well known process of polymerase chain reaction (PCR). This technique may be used to isolate additional APRT coding sequences from a desired organism or as a diagnostic assay to determine the presence of APRT coding sequences in an organism.

APRT specific hybridization probes may also be used to map the location of the native APRT gene(s) in the genome of a chosen plant using standard techniques based on the selective hybridization of the probe to genomic APRT sequences. These techniques include, but are not limited to, identification of DNA polymorphisms identified or contained within the APRT probe sequence, and use of such polymorphisms to follow segregation of the APRT gene relative to other markers of known map position in a mapping population derived from self fertilization of a hybrid of two polymorphic parental lines (see e.g. Helentjaris et al., Plant Mol. Biol. 5: 109 (1985). Sommer et al. Biotechniques 72:82 (1992); D'Ovidio et al., Plant Mol. Biol. 15: 169 (1990)). While any plant APRT sequence is contemplated to be useful as a probe for mapping APRT genes, preferred probes are those APRT sequences from plants more closely related to the chosen plant, and most preferred probes are those APRT sequences from the chosen plant. Mapping of APRT genes in this manner is contemplated to be particularly useful for breeding purposes. For instance, by knowing the genetic map position of a mutant APRT gene that confers increased production of histidine, flanking DNA markers can be identified from a reference genetic map (see, e.g., Helentjaris, Trends Genet. 3: 217 (1987)). During introgression of this trait into a new breeding line, these markers can then be used to monitor the extent of APRT-linked flanking chromosomal DNA still present in the recurrent parent after each round of back- crossing.

APRT specific hybridization probes may also be used to quantitate levels of APRT mRNA in a plant using standard techniques such as Northern blot analysis. This technique may be used as a diagnostic assay to detect altered levels of APRT expression.

Also comprised by the present invention are DNA molecules which hybridizes to a DNA molecule according to the invention as defined hereinbefore, but preferably to an oligonucleotide probe obtainable from said DNA molecule comprising a contiguous portion of the coding sequence for the said APRT enzyme at least 10 nucleotides in length, under moderately stringent conditions.

Factors that effect the stability of hybrids determine the stringency of the hybridization. One such factor is the melting temperature T_m which can be easily calculated according to the formula provided in DNA PROBES, George H. Keller and Mark M. Manak , Macmillan Publishers Ltd, 1993, Section one: Molecular Hybridization Technology; page 8 ff. The preferred hybridization temperature is in the range of about 25°C below the calculated melting temperature T_m and preferably in the range of about 12-15°C below the calculated melting temperature T_m and in the case of oligonucleotides in the range of about 5-10°C below the melting temperature T_m.

For recombinant production of the enzyme in a host organism, the plant APRT coding sequence may be inserted into an expression cassette designed for the chosen host and introduced into the host where it is recombinantly produced. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the chosen host is within the level of skill of the routineer in the art. The resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into the host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli (see, e.g. Studier and Moffatt, J. Mol. Biol. 189: 113 (1986); Brosius, DNA 8: 759 (1989)), yeast (see, e.g., Schneider and Guarente, Meth. Enzymol. 194: 373 (1991)) and insect cells (see, e.g., Luckow and Summers, Bio/Technol. 6: 47 (1988)). Specific examples include plasmids such as pBluescript (Stratagene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Haven, CT), pTrcHis (Invitrogen, San Diego, CA), and baculovirus expression vectors, e.g., those derived from the genome of Autographica californica nuclear polyhedrosis virus (AcMNPV). A preferred baculovirus/insect system is pVL 1392/Sf21 cells (Invitrogen, San Diego, CA).

Recombinantly produced plant APRT enzyme can be isolated and purified using a variety of standard techniques. The actual techniques which may be used will vary depending upon the host organism used, whether the APRT enzyme is designed for secretion, and other such factors familiar to the skilled artisan (see, e.g. chapter 16 of Ausubel, F. et al., Current Protocols in Molecular Biology, pub. by John Wiley & Sons, Inc. (1994) .

Recombinantly produced plant APRT enzyme is useful for a variety of purposes. For example, it may be used to supply APRT enzyme for an in vitro assay to screen known herbicidal chemicals whose target has not been identified to determine if they inhibit APRT. Such an in vitro assay may also be used as a more general screen to identify chemicals which inhibit APRT activity and which are therefore herbicide candidates. Alternatively, recombinantly produced APRT may be used to elucidate the complex structure of this enzyme. Such information regarding the structure of the APRT enzyme may be used, for example, in the rational design of new inhibitory herbicides.

In one aspect of the invention, the amount of APRT enzyme present in a plant or plant cell is increased by introducing into the plant or plant cell a chimeric gene capable of expressing APRT enzyme in a plant cell. Such a chimeric gene will comprise a promoter capable of regulating gene expression in a plant, operably linked to a DNA sequence which encodes a APRT enzyme, followed by a transcriptional terminator and polyadenylation signal. Coding sequences for APRT enzymes may be genetically engineered for optimal expression in a particular crop plant. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (see, e.g. Perlak et al., Proc. Natl. Acad. Sci. USA 88: 3324 (1991); Koziel er a/., Bio/technol. 11: 194 (1993)).

A DNA sequence coding for a APRT enzyme may be inserted into an expression cassette designed for plants to construct a chimeric gene according to the invention using standard genetic engineering techniques. The choice of specific regulatory sequences such as promoter, signal sequence, 5' and 3' untranslated sequences, and enhancer appropriate for the achieving the desired pattern and level of expression in the chosen plant host is within the level of skill of the routineer in the art. The resultant molecule, containing the individual elements linked in proper reading frame, may be inserted into a vector capable of being transformed into a host plant cell.

Examples of promoters capable of functioning in plants or plant cells (i.e., those capable of driving expression of associated coding sequences such as those coding for biotin biosynthetic enzymes in plant cells) include the cauliflower mosaic virus (CaMV) 19S or 35S promoters and CaMV double promoters; nopaline synthase promoters; pathogenesis-related (PR) protein promoters; small subunit of ribulose bisphosphate carboxylase (ssuRUBISCO) promoters, and the like. Preferred are the rice actin promoter (McElroy et al., Mol. Gen. Genet. 231: 150 (1991 )), maize ubiquitin promoter (EP 0 342 926; Taylor et al., Plant Cell Rep.12: 491 (1993)), and the PR-1 promoter from tobacco, Arabidopsis, or maize (see U.S. Patent Application Serial No. 08/181 ,271 to Ryals et al., incorporated by reference herein in its entirety). Also preferred are the 35S promoter and an enhanced or double 35S promoter such as that described in Kay et al., Science 236: 1299-1302 (1987). The promoters themselves may be modified to manipulate promoter strength to increase expression of the associated coding sequence in accordance with art- recognized procedures. Preferred promoters for use with the present invention will be those which confer high level constitutive expression.

Signal or transit peptides may be fused to the APRT coding sequence in the chimeric DNA constructs of the invention to direct transport of the expressed APRT to the desired site of action. Examples of signal peptides include those natively linked to the plant pathogenesis-related proteins, e.g. PR-1 , PR-2, and the like. See, e.g., Payne et al., Plant Mol. Biol. 11:89-94 (1988). Examples of transit peptides include the chloroplast transit peptides such as those described in Von Heijne et al., Plant Mol. Biol. Rep. 9:104-126 (1991); Mazur et al., Plant Physiol. 85: 1110 (1987); Vorst et al., Gene 65: 59 (1988), and mitochondrial transit peptides such as those described in Boutry et al., Nature 328:340-342 (1987). Also included are sequences that result in localization of the encoded protein to various cellular compartments such as the vacuole. See, for example, Neuhaus et al., Proc. Natl. Acad. Sci. USA 88: 10362-10366 (1991) and Chrispeels, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 21-53 (1991). The relevant disclosures of these publications are incorporated herein by reference in their entirety.

The chimeric DNA construct(s) of the invention may contain multiple copies of a promoter or multiple copies of the coding sequence for a APRT enzyme. In addition, the construct(s) may include coding sequences for markers and coding sequences for other peptides such as signal or transit peptides, each in proper reading frame with the other functional elements in the DNA molecule. The preparation of such constructs are within the ordinary level of skill in the art.

Useful markers include peptides providing herbicide, antibiotic or drug resistance, such as, for example, resistance to hygromycin, kanamycin, G418, gentamycin, lincomycin, methotrexate, glyphosate, phosphinothricin, or the like. These markers can be used to select cells transformed with the chimeric DNA constructs of the invention from untransformed cells. Other useful markers are peptidic enzymes which can be easily detected by a visible reaction, for example a color reaction, for example luciferase, β-glucuronidase, or β-galactosidase.

Chimeric genes designed for plant expression such as those described herein can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant (i.e. monocot or dicot) and/or organelle (i.e. nucleus, chloroplast, mitochondria) targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4.320-334 (1986)), electroporation (Riggs et al, Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986), Agrobacterium mediated transformation (Hinchee et al., Biotechnology 6:915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent 4,945,050; and McCabe et al., Biotechnology 6:923-926 (1988)).see also, Weissinger et al., Annual Rev. Genet. 22:421-477 (1988); Sanford et al., Paniculate Science and Technology 5.27-37 (1987)(onion); Christou et al., Plant Physiol. 87:671-674 (1988)(soybean); McCabe et al., Bio/Technology 6:923-926 (1988)(soybean); Datta et al., Bio/Technology 8:736-740 (1990)(rice); Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305-4309 (1988)(maize); Klein et al., Bio/Technology 6:559-563 (1988)(maize); Klein et al., Plant Physiol. 97:440-444 (1988)(maize); Fromm et al., Bio/Technology 8:833-839 (1990); and Gordon-Kamm et al., Plant Cell 2:603-618 (1990)(maize); Svab et al., Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990)(tobacco chloroplasts); Gordon-Kamm et al, in Transgenic Plants, vol. 2., pp.21-33, pub. by Academic Press (1993)(maize).

Once a chimeric gene encoding a APRT enzyme has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques. Alternatively, the coding sequence for a APRT enzyme may be isolated, genetically engineered for optimal expression and then transformed into the desired variety.

The expression 'progeny' is understood to embrace both, "asexually" and "sexually" generated progeny of transgenic plants. This definition is also meant to include all mutants and variants obtainable by means of known processes, such as for example cell fusion or mutant selection and which still exhibit the characteristic properties of the initial transformed plant, together with all crossing and fusion products of the transformed plant material.

Another object of the invention concerns the proliferation material of transgenic plants.

The proliferation material of transgenic plants is defined relative to the invention as any plant material that may be propagated sexually or asexually in vivo or in vitro. Particularly preferred within the scope of the present invention are protoplasts, cells, calli, tissues, organs, seeds, embryos, pollen, egg cells, zygotes, together with any other propagating material obtained from transgenic plants.

Parts of plants, such as for example flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed by means of the process of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention. It is thus a further object of the present invention to provide plant propagation material for cultivated plants, but especially plant seed that is treated with an seed protectant coating customarily used in seed treatment.

The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed. An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. The invention relates to a bag of seeds comprising seed according to the invention. Comprised by the present invention is a bag of seeds comprising seed of a transgenic plant comprising a DNA encoding a protein having a plant ATP-phosphoribosyl transferase activity together with lable instructions. Further comprised by the present invention is a bag of seeds comprising seed of a transgenic plant comprising a DNA encoding a DNA molecule encoding a modified ATP- phosphoribosyl transferase comprising a plant ATP-phosphoribosyl transferase having at least one amino acid modification, wherein said modified plant ATP-phosphoribosyl transferase is tolerant to a herbicide in amounts which inhibit said plant ATP- phosphoribosyl transferase together with lable instructions.

Such bags containing seed to breed progeny from plants transformed according to the method of the present invention, a method such as that which follows may be used: maize plants produced as described in the examples set forth below are grown in pots in a greenhouse or in soil, as is known in the art, and permitted to flower. Pollen is obtained from the mature tassel and used to pollinate the ears of the same plant, sibling plants, or any desirable maize plant. Similarly, the ear developing on the transformed plant may be pollinated by pollen obtained from the same plant, sibling plants, or any desirable maize plant. Transformed progeny obtained by this method may be distinguished from non- transformed progeny by the presence of the introduced gene(s) and/or accompanying DNA (genotype), or the phenotype conferred. The transformed progeny may similarly be selfed or crossed to other plants, as is normally done with any plant carrying a desirable trait. Similarly, tobacco or other transformed plants produced by this method may be selfed or crossed as is known in the art in order to produce progeny with desired characteristics. Similarly, other transgenic organisms produced by a combination of the methods known in the art and this invention may be bred as is known in the art in order to produce progeny with desired characteristics.

The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tillage of the soil or removal of weeds and infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines which for example increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained which, due to their optimized genetic "equipment" , yield harvested product of better quality than products which were not able to tolerate comparable adverse developmental conditions.

In seeds production germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and well-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof.

In order to treat the seed, the protectant coating may be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, eg treatment directed at the buds or the fruit. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD^Φ), methaiaxyl (Apron^®), and pirimiphos-methyl (Actellic^®). If desired these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit. It is a further aspect of the present invention to provide new agricultural methods such as the methods examplified above which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 : Peptide #1 LTYIFDEETP

SEQ ID NO: 2: Peptide #2 VGDFGGPASAF

SEQ ID NO: 3: DNA molecule encoding the wheat (Triticum aestivum) ATP-phosphoribosyl transferase.

SEQ ID NO 4: Amino acid sequence of wheat (Triticum aestivum) ATP-phosphoribosyl transferase,) encoded by the DNA sequence provided in SEQ ID No:3.

SEQ ID NO: 5: DNA molecule encoding the wheat APRT-phosphoribosyl transferase.

SEQ ID NO: 6: Amino acid sequence of wheat APRT-phosphoribosyl transferase encoded by the DNA sequence provided in SEQ ID NO: 5:.

SEQ ID NO: 7: DNA molecule encoding the Arabidopsis ATP-phosphoribosyl transferase

SEQ ID NO: 8: Amino acid sequence of Arabidopsis ATP-phosphoribosyl transferase encoded by the DNA sequence provided in SEQ ID NO: 7:.

SEQ ID NO: 9 DNA molecule encoding the Arabidopsis ATP-phosphoribosyl transferase

SEQ ID NO: 10: Amino acid sequence of Arabidopsis ATP-phosphoribosyl transferase encoded by the DNA sequence provided in SEQ ID NO: 9:.

SEQ ID NO: 11 : Degenerate oligonucletide 5'-TAYATHTTYGAYGARGARAC-3\ designed as a hybridization probe from the peptide sequence determined from the purified wheat germ APRT set forth in SEQ ID NO: 1

SEQ ID NO: 12: antisense, 5'-GTCTCCTCGTCAAATATGTA-3\ primer designed from the amino acid sequence determined from the purified APRT shown in SEQ ID NO: 1.

SEQ ID NO: 13: Primer: 5'-CGGGATCCATGAAGCGTGACCAGATTCGTCTTG -3' SEQ ID NO: 14: Primer: 5'-GCTCTAGAAGCTTCAGCATATGCATCTTCC-3'.

SEQ ID NO: 15: Double-stranded DNA fragment comprised of oligonucleotide of sequence

5' AAT TAT GAC GTA ACG TAG GAA TTA GCG GCCC GCT CTC GAG T 3'

SEQ ID NO: 16. Double-stranded DNA fragment comprised of oligonucleotide of sequence

5' AATTAC TCG AGA GCG GCC GCG AATTCC TAC GTTACG TCA T 3'

DEPOSITS

The following DNA Sequences comprising SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9 have been deposited with the National Institute of Biosience and Human- Technology (NIBH) Agency of International Trade and Industry 1 -3, Higashi 1-chome Tsukuba-shi, Ibaraki-ken 305, Japan as indicated below:

DNA sequence of SEQ ID NO: 3 encoding SEQ ID NO: 4 as FERM BP-6019 DNA sequence of SEQ ID NO: 5 encoding SEQ ID NO: 6 as FERM BP-6020 DNA sequence of SEQ ID NO: 7 encoding SEQ ID NO: 8 as FERM BP-6017 DNA sequence of SEQ ID NO: 9 encoding SEQ ID NO: 10 as FERM BP-6018.

The invention will be further descirbed by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting.

EXAMPLES

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory manual. Cold Spring Harbor laboratory, Cold Spring

EXAMPLE 1 : Purification of ATP-Phosphoribosyl Transferase (APRT) from Wheat Germ

Preparation of Plant Tissue Extract

Wheat germ purchased from Sigma is used as an enzyme source. Wheat germ is homogenized with acetone in a methanol/dry ice bath using a homogenizer. The acetone- insoluble materials are dried under reduced pressure, and then extracted in ice-cold buffer A (0.1 M potassium phosphate pH 7.5, 0.1 M NaCI, 1 mM L-His, 5 mM EDTA, 30 mM 2- mercaptoethanol and 10% (w/w) polyvinylpyrrolidone). The extract is passed through 4 layers cheese cloth and centrifuged at 10,000 x g for 15 min to remove insoluble materials. The proteins in the supernatant are precipitated with ammonium sulfate (80% saturation) by centrifugation at 10,000 x g for 15 min. The precipitate is redissolved in buffer B (0.1 M Tris- HCI pH 7.5, 30 mM 2-mercaptoethanol) and desalted using a Sephadex G-25 column (PD 10, Pharmacia).

Purification

All purification steps are carried out at 4°C. The acetone powder from wheat germ (1 kg) is extracted for 30 min in 6 L of buffer A . After centrifugation at 20,000 x g for 15 min, the supernatant is fractionated by ammonium sulfate precipitation. The precipitate obtained from the 27-47% saturated fraction is redissolved in 50 mM potassium phosphate buffer pH 7.5. Then, ammonium sulfate is added to give 25% saturation. After centrifugation at 20,000 x g for 15 min, the supernatant solution is applied to a Butyl-Toyopearl 650M column (5 x 51 cm, Tosoh, Tokyo, Japan) in 50 mM phosphate buffer pH 7.5 with 1 M ammonium sulfate, and proteins are eluted by decreasing gradient of ammonium sulfate concentration. Each fraction is desalted using a Sephadex G25 column (Pharmacia, PD10) for activity determination. The combined active fractions is dialyzed against 10 L of 50 mM Tris-HCI pH 7.5, and applied to a DEAE-Toyopearl 650M column (5 x 25 cm, Tosoh, Tokyo, Japan) equilibrated with the same buffer. Proteins are eluted with a gradient of NaCI (0-0.5 M) in the same buffer. The active fractions are concentrated and dialyzed against 10 L of 20 mM potassium phosphate buffer pH 7.5. The dialyzed solution is applied to a Heparin- Sepharose CL-6B column (2.5 x 8 cm, Pharmacia) equilibrated with potassium phosphate pH 7.5, and elution is performed by increasing the buffer concentration from 0.02 M to 0.4 M. The active fractions from the Heparin-Sepharose column are applied to a HiLoad 26/60 Superdex 200 pg column (Pharmacia) equilibrated with 20 mM Tris-HCI (pH 7.5) containing 0.2 M NaCI and proteins are eluted with the same buffer. After concentrating the active fractions, the buffer is changed to 20 mM Tris-HCI pH 7.5 by using a PD10 column (Pharmacia). The enzyme solution is applied to a 1 mL HiTrap Blue column (Pharmacia) and eluted isocratically with 20 mM Tris-HCI (pH 7.5).

The purified APRT protein so obtained from wheat germ is digested with lysyl endopeptidase, and the resulting digest is separated by reverse phase HPLC. The resulting peptides are subjected to automated Edman degradation (Strickler et a;, Anal Biochem. 140: 553-566 (1984)) with an Applied Biosystems (Foster City., CA) 470A protein sequencer, and peptide sequences are determined:

Peptide #1 LTYIFDEETP (SEQ ID NO: 1)

Peptide #2 VGDFGGPASAF (SEQ ID NO: 2)

Enzvme Assav

The standard assay mixture (175 μL) contains 1 1.4 mM Tris-HCI (pH 9.0), 22.8 mM MgCl2, 85.7 mM KCI, 5.7 mM ATP, 0.57 mM phosphoribosyl pyrophosphate (PRPP), and enzyme. The reaction is started by the addition of phosphoribosyl pyrophosphate, and the mixture is incubated at 30°C for 15 min. The reaction is stopped by adding 50μl 1 N HCI. The enzyme assay is performed in the standard reaction mixture supplemented with 10 μl of the extract from a Salmonella typhimurium strain SB3095 (hisG46, pepH102, fla-2055) that is lacking APRT activity (Ames et al., J. Biol. Chem., 2019-2026, 236, 1961 ). This assay mixture contains the plant APRT and the other enzymes of the His biosynthetic enzymes derived from strain SB3095, and 5-amino-1-ribosyl-4-imidazole carboxamide (AICAR) is stoicheometrically released during the L-His biosynthesis as a byproduct. The activity of plant APRT is thus determined by measuring the AICAR production rate. AICAR is determined photometrically with the Bratton-Marshall method as described by Ames et al. (Ames et al., J. Biol. Chem., 2019-2026, 236, 1961). This method is used for the determination of enzyme activity both in crude plant extracts and during purification. 10 μM solution of AICAR gives an absorbance of 0.270 at 550 nm in this assay method (Ames et al., J. Biol. Chem., 2019-2026, 236, 1961). APRT is also spectrophotomerically assayed in the standard reaction mixture without the S. typhimurium extract. In this system, APRT activity is determined by monitoring the PRPP- and ATP-dependent production of PRATP, which can be estimated by following the absorbance increase at 290 nm using an extinction coefficient of 3.6 x 10³ for PRATP (Smith and Ames J. Biol. Chem. 240, 3056-3063, 1965). Purified APRT may be used in assays to discover novel inhibitors of the enzyme, which inhibitors potentially would function as commercially viable herbicides. Typically, the inhibitory effect of a chemical on IGPD is determined in the enzyme assay methods described above. Inhibitor solutions in various concentrations, e.g., 1 mM, 100 mM, 10 mM, and 1 mM are added to the reaction mixture prior to the initiation of the enzyme reaction. If a measure of inhibition greater than IC50 = 10 mM is expected, further assays may be performed using even lower concentrations of inhibitor.

EXAMPLE 2: Cloning of Wheat cDNAs Encoding APRT Genes

Total RNA is prepared from 7-d-old wheat seedlings by phenol/chloroform extraction followed by lithium chloride precipitation. Poly(A)+ RNA is isolated from the total RNA using a poly(A)+ Quick mRNA isolation kit (Stratagene, LaJolla, CA). A cDNA library is constructed from the poly(A)+ RNA in the bacteriophage vector lambda ZAPII (Stratagene) using the Uni-ZAP XR Gigapack II Gold cloning kit (Stratagene) as described in the manufacturers' instruction. A phage or plasmid cDNA library is plated at a density of approximately 10,000 plaques on a 10 cm Petri dish, and filter lifts of the plaques are made after overnight growth of the plates at 37 °C. A degenerate oligonucletide, 5'- TAYATHTΓΎGAYGARGARAC-3', (SEQ ID NO 11) is designed as a hybridization probe from the peptide sequence determined from the purified wheat germ APRT set forth in SEQ op

ID NO: 1 , and the plaque lifts are probed with the probe labeled with P-ATP by MEGALABEL (TAKARA Shuzo, Kyoto, Japan) Hybridization conditions are 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04 pH 7.0, 1 mM EDTA at 40 °C. After hybridization overnight, the filters are washed with 2X SSC, 1 % SDS. Positively hybridizing plaques are detected by autoradiography. After purification to single plaques, cDNA inserts are isolated, and their sequences determined by the chain termination method using dideoxy terminators labeled with fluorescent dyes (Applied Biosystems, Inc., Foster City, CA). The sequence thus obtained for the wheat APRT cDNA and the protein it encodes are provided in SEQ ID NOS:3 and 4. Peptide #1 (SEQ ID NOS: 1) exactly matches the predicted protein sequence (SEQ ID NO: 4) determined from the wheat APRT cDNA. Additional wheat APRT cDNA clone is isolated from a wheat cDNA library using the cDNA fragment (SEQ ID NO: 3) as a hybridization probe. The DNA is labeled with [^Pj-dCTP by the random priming labeling method and used as a probe to screen 600,000 plaques from the wheat cDNA library. The wheat APRT DNA coding sequence elucidated from the clone thus isolated is provided in SEQ ID NO: 5. The amino acid sequence encoded by this DNA sequence is provided in SEQ ID NO: 6. The isolation of the two DNA sequences coding for individual protein sequences is likely due to the presence of multiple isoforms encoded by different genes in wheat genome. Wheat, with its hexaploid genome, may have even more than two APRT genes. The protein sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 6 are compared in Table I.

TABLE I

Comparison of the Wheat (SEQ ID NOS: 4 and 6) APRT Amino Acid Sequences

Identical residues are denoted by the vertical bar between the two sequences. Alignment is performed using the software, DNASIS version 3.4 (HITACHI software Engineering America, San Bruno, CA). The amino acid sequence (SEQ ID NO: 1 ) determined from the purified wheat APRT protein is underlined.

(SEQ ID NO: 4)1 DAIVDLVSSG TT RENNLKE IDGGIILESQ

MINIMI MINIUM I I! Mill

(SEQ ID NO: 6) 1 HVSFLAGDGA LESYPAMGMA DAIVDLVSSG TTLRENNLKE IEGGVILESQ 31 ATLVASKKSL NRREGVLEIS HEMLERLEAH LTASGKIMVT ANMRGNSAEE l l l l l l l l l l M I N I M I l l l l l l l l l l l l l l l l l l l l M I N I M I !

51 ATLVASKKSL NRREGVLEIS HEMLERLEAH LTASGKIMVT ANMRGNSAEE 81 VAERVLSQTS LCGLQGPTIS PVYCTLDGKV AVDYYAINW VPQKSLYKSI 1111111111 I

101 VAERVLSQTS LCGLQGPTIS PVYCTLDGNV AVDYYAINW VPQKSLYKSI 131 QQLRSIGGSG VLVSKLTYIF DEETPRWRKL LSELGM* I MM

151 QQLRSIGGSG VLVSKLTYIF DEETPR RKL LAELGM*

EXAMPLE 3: Isolation of Additional APRT Genes Based on the Sequence Homology to Known APRT Sequences

Total RNA is prepared from 7-d-old Arabidopsis seedlings by phenol/chloroform extraction followed by lithium chloride precipitation. Poly(A)+ RNA is isolated from the total RNA using a poly(A)+ Quick mRNA isolation kit (Stratagene, LaJolla, CA). A cDNA library is constructed from the poly(A)+ RNA in the bacteriophage vector lambda ZAPII (Stratagene) using the Uni-ZAP XR Gigapack II Gold cloning kit (Stratagene) as described in the manufacturers' instruction. Lambda phage is collected from the Arabidopsis cDNA library, and phage DNA is in a solution containing 0.1 % (w/v) sodium dodecyl sulfate and 20 mM EDTA (pH 8.0), phage DNA is prepared by phenol/chloroform extraction and a part of this is used as a template for PCR with a set of SK primer, 5'-TCTAGAACTAGTGGATC-3' (Stratagene) and an antisense, 5'-GTCTCCTCGTCAAATATGTA-3' (SEQ ID 12), primer designed from the amino acid sequence determined from the purified APRT shown in SEQ ID NO: 1. The PCR is carried out in 50 μl of a reaction mixture consisting of 20 mM of Tris- HCI (pH 8.4) containing 10 pmol of the primers, 200 μM dATP, 200 μM dCTP, 200 μM dTTP, 200 μM dGTP, 2 mM MgCl2 50 mM KCI, 1.25 units/mL Taq DNA polymerase (GIBCO-BRL). The reaction is performed through 35 cycles of 1 min at 94 °C, 1 min at 50 °C and 90 sec at 72 °C using a thermal cycler (Perkin Elmer/Cetus, model 9600). PCR products are separated by agarose gel (1%) electrophoresis. A major band (0.4 kb), which represents the PCR-amplified fragment derived from Arabidopsis APRT, is isolated from the gel and cloned into a pCRII vector using a TA cloning kit (Invitorgen). The DNA insert is used to isolate its corresponding full-length clone from the Arabidopsis cDNA library.

32

Practically, the DNA is labeled with [ P]-dCTP by random priming labeling method and used as a probe to screen 600,000 plaques from the Arabidopsis cDNA library. The APRT DNA coding sequences elucidated from this clone are provided in SEQ ID NOS: 7 and 9. The amino acid sequence encoded by these DNA sequences are provided in SEQ ID NOS: 8 and 10, respectively. TABLE II

Comparison of the Arabidopsis (SEQ ID NOS: 8 and 10) Amino Acid Sequences

Identical residues are denoted by the vertical bar between the two sequences. Alignment is performed using the software, DNASIS version 3.4 (HITACHI software Engineering America, San Bruno, CA).

(SEQ ID NO: 8) 1 MSLLLPTNLQ -QYPS -SSSFPSSTP ILSPPPSTAF SVIVPRRRCL

I I I M l III I I II III I I III

(SEQ ID NO: 10) 1 MPISIPLNAT LQYSSPSSSS SSSSLVPSSP LFSPIPSTTV SLTGIRQRCL 44 RLVTSCVSTV QSSVATNGSS PAPAPAAVW ERDQIRLGLP SKGRMAADAI

I MM I II II I INN! MMIIII I

51 RMVTSCVSNA QKSVLNGATD SVS W GREQIRLGLP SKGRMAADSL

94 DLLKDCQLFV KQVNPRQYVA QIPQLPNTEV WFQRPKDIVR KLLSGDLDLG 1111 ! 111 i I Mill MM 111 ! 111111

96 DLLKDCQLFV KQVNPRQYVA QIPQLPNTEV WFQRPQDIVR KLLSGDLDLG 144 IVGLDTLSEY GQENEDLIIV HEALNFGDCH LSIAIPNYGI FENINSLKEL

Mill I II II ININ! MM Mill

146 IVGLDIVGEF GQGNEDLIIV HEALNFGDCH LSLAIPNYGI FENIKSLKEL 194 AQMPQWSEER PLRLATGFTY LGPKFMKENG IKHWFSTAD GALEAAPAMG III Ml INN II MM Mill III

196 AQMPQWTEER PLRVATGFTY LGPKFMKDNG IKHVTFSTAD GALEAASAMG 244 IADAILDLVS SGITLKENNL KEIEGGWLE SQAALVASRR ALNERKGALN II I M 111 II 11 II

246 IADAILDLVS SGTTLKENNL KEIEGGWLE SQAALVASRR ALTERKGALE 294 TVHEILERLE AHLKADGQFT WANMRGNSA QEVAERVLSQ PSLSGLQGPT f 111111111 MM MM II MM I I

296 TVHEILERLE AHLKPNGQFT WPNMRGTDA EEVAERVKTQ PSLSGLQGPT 344 ISPVYCTQNG KVSVDYYAIV ICVPKKALYD SVKQLRAAGG SGVLVSPLTY I II Mill I II MM II Mill I II

346 ISPVYCKRDG KVTIEYYAIV ICVPKKALYE SVQQLRAVGG SGVLVTPVTY 394 IFDEDTPRWG QLLRNLGI*

II I MM Ml III

396 IFHEETPRWS QLLSNLGL* TABLE III

Comparison of the Wheat (SEQ ID NOS: 4 AND 6) and the

Arabidopsis (SEQ ID NOS: 8 AND 10) amino acid sequences

Identical residues are denoted by the dot above the four sequences. Alignment is performed using the software, DNASIS version 3.4 (HITACHI software Engineering America, San Bruno, CA).

( SEQ ID NO : 8 ) 1 MSLLLPTNLQ -QYPS -SSSFPSSTP ILSPPPSTAF SVIVPRRRCL

( SEQ ID NO : : 10 ) 1 MPISIPLNAT LQYSSPSSSS SSSSLVPSSP LFSPIPSTTV SLTGIRQRCL

( SEQ ID NO : 4 )

( SEQ ID NO : 6 )

44 RLVTSCVSTV QSSVATNGSS PAPAPAAVW ERDQIRLGLP SKGRMAADAI

51 RMVTSCVSNA QKSVLNGATD SVS W GREQIRLGLP SKGRMAADSL

94 DLLKDCQLFV KQVNPRQYVA QIPQLPNTEV WFQRPKDIVR KLLSGDLDLG 96 DLLKDCQLFV KQVNPRQYVA QIPQLPNTEV FQRPQDIVR KLLSGDLDLG

144 IVGLDTLSEY GQENEDLIIV HEALNFGDCH LSIAIPNYGI FENINSLKEL 146 IVGLDIVGEF GQGNEDLIIV HEALNFGDCH LSLAIPNYGI FENIKSLKEL

194 AQMPQWSEER PLRLATGFTY LGPKFMKENG IKHWFSTAD GALEAAPAMG 196 AQMPQWTEER PLRVATGFTY LGPKFMKDNG IKHVTFSTAD GALEAASAMG

HVSFLAGD GALESYPAMG

244 IADAILDLVS SGITLKENNL KEIEGGWLE SQAALVASRR ALNERKGALN

246 IADAILDLVS SGTTLKENNL KEIEGGWLE SQAALVASRR ALTERKGALE

19 MADAIVDLVS SGTTLRENNL KEIDGGIILE SQATLVASKK SLNRREGVLE

1 MADAIVDLVS SGTTLRENNL KEIEGGVILE SQATLVASKK SLNRREGVLE

294 TVHEILERLE AHLKADGQFT WANMRGNSA QEVAERVLSQ PSLSGLQGPT

296 TVHEILERLE AHLKPNGQFT WPNMRGTDA EEVAERVKTQ PSLSGLQGPT

69 ISHEMLERLE AHLTASGKIM VTANMRGNSA EEVAERVLSQ TSLCGLQGPT

51 ISHEMLERLE AHLTASGKIM VTANMRGNSA EEVAERVLSQ TSLCGLQGPT

344 ISPVYCTQNG KVSVDYYAIV ICVPKKALYD SVKQLRAAGG SGVLVSPLTY

346 ISPVYCKRDG KVTIEYYAIV ICVPKKALYE SVQQLRAVGG SGVLVTPVTY

119 ISPVYCTLDG KVAVDYYAIN VWPQKSLYK SIQQLRSIGG SGVLVSKLTY

101 ISPVYCTLDG NVAVDYYAIN VWPQKSLYK SIQQLRSIGG SGVLVSKLTY 394 IFDEDTPR G QLLRNLGI*

396 IFHEETPRWS QLLSNLGL*

169 IFDEETPRWR KLLSELGM*

151 IFDEETPRWR KLLAELGM*

The sequence comparison provided in TABLE III indicates that the wheat DNA sequences (SEQ ID NOS: 3 and 5) lack their N-terminal portion including the n-TERMINAL signal sequence which exhibit general properties of the chloroplast transit sequence found in SEQ ID NOS: 8 and 10. Applicability of this protocol provided in EXAMPLE III for obtaining the complete APRT genes from wheat and other plants has been supported by the observation of specific hybridization of an Arabidopsis APRT coding sequence probe to discrete DNA restriction fragments from the genome of wheat and Zea mays in a standard Southern blot. This result indicates that the degree of homology between the Arabidopsis and Zea mays APRT coding sequences is sufficient for the Arabidopsis sequence to specifically hybridize to the Zea mays sequence present among a pool of DNA representing the entire maize genome. In view of this Southern result, specific hybridization of the Arabidopsis APRT coding sequence to a maize cDNA library according to the protocol above would be expected since such a library represents only a subset of the entire maize genomic DNA (i.e. the coding portion).

EXAMPLE 5: Heterologous Expression in Insect Cells

The APRT protein is expressed using the baculovirus expression vector system according to the method described previously (Summers and Smith, 1987), using a baculovirus transfer vector pVL1392 (Invitrogen, San Diego, CA), Spodoptera furugiperda 21 (Sf21) cells (Invitrogen) and an infectious BaculoGold Baculovirus DNA (Pharmingen, San Diego, CA).

Sf21 cells are maintained at 27 ⁹C as a monolayer culture in a Grace's medium supplemented with 0.33% TC yeastolate, 0.33% lactoalbumin, 10% fetal bovine serum, and 50 μg/ml of gentamycin sulfate. The expressed APRT protein is purified from the infected Sf21 cells. Briefly, the infected cells are sonicated in buffer A containing 0.1 M potassium phosphate (pH 7.5), 0.1 M NaCI, 1 mM L-His, 5 mM EDTA, 30 mM 2-mercaptoethanol and 10% (w/w) polyvinylpyrrolidone) and centrifuged at 10,000 x g for 15min. The recombinantly produced APRT is purified by the method provided by EXAMPLE 1. EXAMPLE 6: Heterologous Expression in E.coli

The coding region without putative chloroplast transit sequence is amplified by PCR under a condition: 95 °C for 5 min, 30 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72°C, using a set of primers: 5'-CGGGATCCATGAAGCGTGACCAGATTCGTCTTG -3' (SEQ ID 13) and 5'-GCTCTAGAAGCTTCAGCATATGCATCTTCC-3' (SEQ ID NO: 14). The DNA fragment thus amplified is subcloned into a PCRII vector using a TA cloning kit (Invitrogen), and then inserted into an expression plasmid pMAL-C2 vector (New England Biolabs, Inc. MA, USA). The pMAL-C2 carrying the insert of the APRT DNA is used for the transformation of an E. coli strain JM109. The APRT proteinis recombinantly produced in the E. coli cells as a fusion protein with a maltose binding protein, which is purified through a one-step amylose resin affinity column chromatography as described by the manufacturer.

EXAMPLE 7: Selecting for plant APRT genes resistant to APRT-inhibitory herbicides in the E. coli expression system.

Inhibition of plant APRT enzymes in a bacterial system is useful for large-scale screening for herbicide-resistant mutations in the plant genes. Transforming APRT plasmids into an available E. coli APRT defective mutant, (e.g. strain JC411 , which carries the hisG1 mutation (Delorme et al., J. Bacteriol. 174: 6571 -6579 (1992)) and histidine prototrophic colonies are selected on M9-0.2% (w/v) glucose minimal plates supplemented with 100 mg/ml ampicillin and amino acids except for histidine (Sambrook J, Fritsch EF, Maniatis T, ed. eds. Molecular cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor, Cold Spring Harbor Press, NY (1989)). Positive histidine prototrophic colonies are purified by restreaking on the M9 plates described above. The E. coli harboring plant APRT gene is cultivated in a liquid media supplemented with 100 mg ampicilin. Initial dose response experiments are done on plates containing 100 mg/ml ampicillin and APRT inhibitory herbicide. High frequency "resistant" colonies are identified even at high concentrations of herbicide, and retransformation/herbicide sensitivity assay is performed to confirm that this resistance is not plasmid-borne.

The plant APRT plasmids are mutagenized in a variety of ways, using published procedures for chemical (e.g. sodium bisulfite (Shortle et al., Methods Enzymol. 700:457-468 (1983); methoxylamine (Kadonaga et al., Nucleic Acids Res. 73:1733-1745 (1985); oligonucleotide-directed saturation mutagenesis (Hutchinson et al., Proc. Natl. Acad. Sci. USA, 83:710-714 (1986); or various polymerase misincorporation strategies (see, e.g. Shortle et al., Proc. Natl. Acad. Sci. USA, 79:1588-1592 (1982); Shiraishi et al., Gene 64.313-319 (1988); and Leung et al., Technique 7:11-15 (1989)). The expected up-promoter mutants from whole-plasmid mutagenesis are eliminated by recloning the coding sequence into a wild-type vector and retesting. Given that higher expression is likely to lead to better growth in the absence of herbicide, a visual screen for coding sequence mutants is also possible.

Any plant APRT gene expressing herbicide resistance in the bacterial system may be engineered for optimal expression and transformed into plants using standard techniques as described herein. The resulting plants may then be treated with herbicide to confirm and quantitate the level of resistance conferred by the introduced APRT gene.

EXAMPLE 8: Production of herbicide-tolerant plants by overexpression of plant APRT genes

To express the APRT protein in transgenic plants, the appropriate full length APRT cDNA is inserted into the plant expression vector pCGN1761 ENX, which is derived from pCGN1761 as follows. pCGN1761 was digested at its unique EcoRI site, and ligated to a double-stranded DNA fragment comprised of two oligonucleotides of sequence 5' AAT TAT GAC GTA ACG TAG GAA TTA GCG GCCC GCT CTC GAG T 3' (SEQ ID NO: 15) and 5' AAT TAC TCG AGA GCG GCC GCG AAT TCC TAC GTT ACG TCA T 3' (SEQ ID NO: 16). The resulting plasmid, pCGN1761 ENX, contained unique EcoRI, Notl, and Xhol sites that lie between a duplicated 35S promoter from cauliflower mosaic virus (Kay et al., Science 236:1299-1302 (1987)) and the 3' untranslated sequences of the tml gene of Agrobacterium tumefaciens. This plasmid is digested and ligated to a fragment resulting from restriction enzyme digestion of one of the plasmids bearing a APRT cDNA, such that it carries the complete APRT cDNA. From this plasmid is excised an Xbal fragment comprising the Arabidopsis APRT cDNA flanked by a duplicated 35S promoter and the 3' untranslated sequences of the tml gene of A. tumefaciens. This Xbal fragment is inserted into the binary vector pCIB200 at its unique Xbal site, which lies between T-DNA border sequences. The resulting plasmid, designated pCIB200APRT, is transformed into A. tumefaciens strain CIB542. See,e.g. Uknes et al., Plant Cell 5:159-169 (1993). Leaf disks of Nicotiana tabacum cv. Xanthi-nc are infected with A. tumefaciens CIB542 harboring pCIB200IGPD as described by Horsch et al, Science 227: 1229 (1985). Kanamycin-resistant shoots from 15 independent leaf disks are transferred to rooting medium, then transplanted to soil and the resulting plants grown to maturity in the greenhouse. Seed from these plants are collected and germinated on MS agar medium containing kanamycin. Multiple individual kanamycin resistant seedlings from each independent primary transformant are grown to maturity in the greenhouse, and their seed collected. These seeds are germinated on MS agar medium containing kanamycin. Plant lines that give rise to exclusively kanamycin resistant seedlings are homozygous for the inserted gene and are subjected to further analysis. Leaf disks of each of the 15 independent transgenic lines are excised with a paper punch and placed onto MS agar containing various increasing concentrations of a APRT inhibitory herbicide.

After three weeks, two sets of 10 disks from each line were weighed, and the results recorded. Transgenic lines more resistant to the inhibitor than wild type, non-transformed plants are selected for further analysis.

RNA is extracted from leaves of each of these lines. Total RNA from each independent homozygous line, and from non-transgenic control plants, is separated by agarose gel electrophoresis in the presence of formaldehyde (Ausubel et al., Current Protocols in Molecular Biology. Wiley & Sons, New York (1987)). The gel is blotted to nylon membrane (Ausubel et al., supra.) and hybridized with the radiolabeled Arabidopsis APRT cDNA. Hybridization and washing conditions are as described by Church and Gilbert, Proc. Natl. Acad. Sci. USA 87:1991-1995 (1984). The filter is autoradiographed, and intense RNA bands corresponding to the APRT transgene are detected in all herbicide-tolerant transgenic plant lines.

To further evaluate resistance of the APRT-overexpressing line, plants are grown in the greenhouse and treated with various concentrations of a APRT-inhibiting herbicide. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: NOVARTIS AG

(B) STREET: Schwarzwaldallee 215

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : 4002

(G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991

(ii) TITLE OF INVENTION: Plant ATP Phosphoribosyl Transferase and DNA Coding Therefor

(iii) NUMBER OF SEQUENCES: 16

(iv) CHtFUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) C MPUTER: IBM PC cαrrpatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) rTynPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Leu Thr Tyr lie Phe Asp Glu Glu Thr Pro 1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYTrOTHETTCAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Val Gly Asp Phe Gly Gly Pro Ala Ser Ala Phe 1 5 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CTARACTERISTICS:

(A) LENGTH: 911 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:!..498

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAC GCT ATC GTG GAT CTT GTG ACT ACT GGA ACG ACT TTG CGT GAG AAT 48 Asp Ala He Val Asp Leu Val Ser Ser Gly Thr Thr Leu Arg Glu Asn 1 5 10 15

AAT TTG AAG GAA ATT GAT GGT GGA ATC ATT TTG GAA AGC CAG GCA ACA 96 Asn Leu Lys Glu He Asp Gly Gly He He Leu Glu Ser Gin Ala Thr 20 25 30

CTT GTA GCA ACT AAG AAA TOT CTG AAC AGG CGT GAA GGT GTG TTA GAG 144 Leu Val Ala Ser Lys Lys Ser Leu Asn Arg Arg Glu Gly Val Leu Glu 35 40 45

ATT TCA CAT GAA ATG CTT GAA AGA TTA GAG GCT CAC CTC ACA GCG TCT 192 He Ser His Glu Met Leu Glu Arg Leu Glu Ala His Leu Thr Ala Ser 50 55 60

GGC AAG ATA ATG GTA ACA GCA AAT ATG AGG GGC AAT ACT GCA GAA GAA 240 Gly Lys He Met Val Thr Ala Asn Met Arg Gly Asn Ser Ala Glu Glu 65 70 75 80

GTG GCA GAG AGA CTT CTC AGC CAA ACA TCA TTA TCT GGA TTA CAG GGC 288 Val Ala Glu Arg Val Leu Ser Gin Thr Ser Leu Cys Gly Leu Gin Gly 85 90 95

CCA ACT ATA ACT CCA GTG TAT TGC ACA CTT GAT GGC AAG CTT GCC GTG 336 Pro Thr He Ser Pro Val Tyr Cys Thr Leu Asp Gly Lys Val Ala Val 100 105 110

GAC TAC TAT GCT ATT AAT CTT GTA GTT CCC CAA AAG TCG CTT TAC AAG 384 Asp Tyr Tyr Ala He Asn Val Val Val Pro Gin Lys Ser Leu Tyr Lys 115 120 125

TCT ATT CAA CAA CTG AGA TCT ATT GGT GGC AGC GGC GTC TTG GTG TCG 432 Ser He Gin Gin Leu Arg Ser He Gly Gly Ser Gly Val Leu Val Ser 130 135 140

AAA CTG ACC TAC ATA TTT GAC GAG GAG ACT CCT AGG TGG CGC AAG CTT 480 Lys Leu Thr Tyr He Phe Asp Glu Glu Thr Pro Arg Trp Arg Lys Leu 145 150 155 160

CTG TCG GAG CTG GGA ATG TGAGGCTTCC TGCAAGGTCG GCCATGCTCT 528

Leu Ser Glu Leu Gly Met 165

CTCCTCTAGA ATGGACCGTC TCAGTGTGAG CATCTGAACT TATGCTCGCT CTAGTGTCTA 588

CATCATACAC AGAAGCGACC CGAGTTTGAA AAAAACGTAT GCAAAGTGAA CCCCCATGTG 648

GACTGCCTGA GGAGATTTCG ATATCTAGCG CXX3GATATTG TGCTCAGCTA AOJΓATTCCC 708

TGGACAGGCA GTGGGTGGTT TATCCCTTTT ATGTACCCAC CAGTTGATAG AGTTGGGGCT 768

CCGGGATGTT Q3GTCTATCT TGTTTGTGTG TACCGGATCT TGCACCTTTT GTTGGCACGG 828

CCCTGAATAA TTGGGGCTAC CTGATATGAT GATAATAATG AATCCCATTG TGGTTTGGTC 888

TAAAAAAAAA AAAAAAAAAA AAA 911

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 166 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Asp Ala He Val Asp Leu Val Ser Ser Gly Thr Thr Leu Arg Glu Asn 1 5 10 15

Asn Leu Lys Glu He Asp Gly Gly He He Leu Glu Ser Gin Ala Thr 20 25 30

Leu Val Ala Ser Lys Lys Ser Leu Asn Arg Arg Glu Gly Val Leu Glu 35 40 45

He Ser His Glu Met Leu Glu Arg Leu Glu Ala His Leu Thr Ala Ser 50 55 60

Gly Lys He Met Val Thr Ala Asn Met Arg Gly Asn Ser Ala Glu Glu 65 70 75 80

Val Ala Glu Arg Val Leu Ser Gin Thr Ser Leu Cys Gly Leu Gin Gly 85 90 95

Pro Thr He Ser Pro Val Tyr Cys Thr Leu Asp Gly Lys Val Ala Val 100 105 110

Asp Tyr Tyr Ala He Asn Val Val Val Pro Gin Lys Ser Leu Tyr Lys 115 120 125

Ser He Gin Gin Leu Arg Ser He Gly Gly Ser Gly Val Leu Val Ser 130 135 140

Lys Leu Thr Tyr He Phe Asp Glu Glu Thr Pro Arg Trp Arg Lys Leu 145 150 155 160

Leu Ser Glu Leu Gly Met 165

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 973 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..558

(D) OTHER INFORMATION: /codon_start=

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CAT CTT TCA TTT TTA GCT GGT GAT GCT GCT CTC GAG TCA TAT CCT GCT 48 His Val Ser Phe Leu Ala Gly Asp Gly Ala Leu Glu Ser Tyr Pro Ala 1 5 10 15

ATG GCT ATG GCT GAC GCT ATT GTG GAT CTT GTG ACT ACT GGA ACG ACT 96 Met Gly Met Ala Asp Ala He Val Asp Leu Val Ser Ser Gly Thr Thr 20 25 30

TTG CGT GAG AAT AAT TTG AAG GAA ATT GAA GGT GGA GTA ATT TTG GAA 144 Leu Arg Glu Asn Asn Leu Lys Glu He Glu Gly Gly Val He Leu Glu 35 40 45

AGC CAG GCA ACA CTT GTA GCA ACT AAG AAA TCT CTG AAC AGA CGT GAG 192 Ser Gin Ala Thr Leu Val Ala Ser Lys Lys Ser Leu Asn Arg Arg Glu 50 55 60

GCT CTG TTA GAG ATT TCA CAT GAA ATG CTT GAG AGA TTG GAG GCT CAC 240 Gly Val Leu Glu He Ser His Glu Met Leu Glu Arg Leu Glu Ala His 65 70 75 80

CTC ACA GCG TCT GGC AAG ATA ATG GTA ACA GCA AAT ATG AGG GGC AAT 288 Leu Thr Ala Ser Gly Lys He Met Val Thr Ala Asn Met Arg Gly Asn 85 90 95

ACT GCA GAA GAA GTG GCA GAG AGA GTT CTC AGC CAA ACA TCA TTA TCT 336 Ser Ala Glu Glu Val Ala Glu Arg Val Leu Ser Gin Thr Ser Leu Cys 100 105 110

GGA TTA CAG GGC CCA ACT ATA AGC CCA GTG TAT TGC ACG CTT GAT GGC 384 Gly Leu Gin Gly Pro Thr He Ser Pro Val Tyr Cys Thr Leu Asp Gly 115 120 125

AAT GTT GCT GTG GAC TAC TAT GCT ATT AAT GTT GTA GTT CCC CAA AAG 432 Asn Val Ala Val Asp Tyr Tyr Ala He Asn Val Val Val Pro Gin Lys 130 135 140

TCG CTT TAC AAG TCT ATT CAA CAA CTG AGA TCT ATT GGT GGC AGC GGA 480 Ser Leu Tyr Lys Ser He Gin Gin Leu Arg Ser He Gly Gly Ser Gly 145 150 155 160

CTC TTG GTG TCG AAA CTG ACC TAC ATA TTT GAC GAG GAG ACT CCT AGG 528 Val Leu Val Ser Lys Leu Thr Tyr He Phe Asp Glu Glu Thr Pro Arg 165 170 175

TGG CGC AAA CTT CTC GCG GAG CTG GGA ATG TGAGACCTTG CTGCAAGGTC 578 Trp Arg Lys Leu Leu Ala Glu Leu Gly Met 180 185

GGCCATGCCC TCCGCTAGAA TGGACCGTCT CAGTGAGCAT CTGAACTTAT GCT03CTGTA 638

GTGTCTACAT CATACACAGA AGCGACCCGA GTTTGAAAAA AAAACCCCTA TGCAAAGTGA 698

GCTCCCATGT GGATTGCCTG ACGAGATTTC GATATCTAGC GCCGGATATT GTGCTCAGCT 758

AACCTATTCC CCTGGACAGG CAGTGGTTGG TTTATCCCTT TTATCTACCA α_ACTCGATA 818

CGGTTGGGGG TCCGGGATGT TGGGTGTATG TTGTTTGTCT CTGCCGGATC TTGCACCTTT 878 TGTTGGTGCG GCCCTGAATA ATTGGGGCTA CTTGATATGA TGACAATAAT GAATCCCATT 938 GTGσrTTGGT CTCAAAAAAA AAAAAAAAAA AAAAA 973

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 186 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

His Val Ser Phe Leu Ala Gly Asp Gly Ala Leu Glu Ser Tyr Pro Ala 1 5 10 15

Met Gly Met Ala Asp Ala He Val Asp Leu Val Ser Ser Gly Thr Thr 20 25 30

Leu Arg Glu Asn Asn Leu Lys Glu He Glu Gly Gly Val He Leu Glu 35 40 45

Ser Gin Ala Thr Leu Val Ala Ser Lys Lys Ser Leu Asn Arg Arg Glu 50 55 60

Gly Val Leu Glu He Ser His Glu Met Leu Glu Arg Leu Glu Ala His 65 70 75 80

Leu Thr Ala Ser Gly Lys He Met Val Thr Ala Asn Met Arg Gly Asn 85 90 95

Ser Ala Glu Glu Val Ala Glu Arg Val Leu Ser Gin Thr Ser Leu Cys 100 105 110

Gly Leu Gin Gly Pro Thr He Ser Pro Val Tyr Cys Thr Leu Asp Gly 115 120 125

Asn Val Ala Val Asp Tyr Tyr Ala He Asn Val Val Val Pro Gin Lys 130 135 140

Ser Leu Tyr Lys Ser He Gin Gin Leu Arg Ser He Gly Gly Ser Gly 145 150 155 160

Val Leu Val Ser Lys Leu Thr Tyr He Phe Asp Glu Glu Thr Pro Arg 165 170 -> 175

Trp Arg Lys Leu Leu Ala Glu Leu Gly Met 180 185

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE (CHARACTERISTICS: (A) LENGTH: 1564 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 85..1317

(D) OTHER INFORMATION: /codon_start= 25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TGAAAGTGTG TTGTGTCTTA TTCGTTATAT AATTCCTTCT CCGGTCTAAA AGAAACCCTT 60

TCACTCTCGA AAAGCCACAC AACA ATG TCT CTC CTT CTC CCT ACG AAT TTA 111

Met Ser Leu Leu Leu Pro Thr Asn Leu 1 5

CAA CAA TAC CCT TCT TCT ICC ICC TTC CCA TCT TCA ACA CCT ATC CTA 159 Gin Gin Tyr Pro Ser Ser Ser Ser Phe Pro Ser Ser Thr Pro He Leu 10 15 20 25

TCT CCG CCT CCT TCC AGC GCT TTC TCC CTC ATC CTA CCT CGT CGG AGA 207 Ser Pro Pro Pro Ser Thr Ala Phe Ser Val He Val Pro Arg Arg Arg 30 35 40

TCT CTC AGA TTG GTT ACT TCT TCT CTC TCC ACC CTT CAA AGC TCC GTC 255 Cys Leu Arg Leu Val Thr Ser Cys Val Ser Thr Val Gin Ser Ser Val 45 50 55

GCA ACA AAC GCT TCC TCT CCA GCT CCT GCT CCG GCC GCT GTT GTC GTT 303 Ala Thr Asn Gly Ser Ser Pro Ala Pro Ala Pro Ala Ala Val Val Val 60 65 70

GAG CCT GAC CAG ATT CGT CTT GGT CTT CCT ACT AAA GGA CGT ATG GCT 351 Glu Arg Asp Gin He Arg Leu Gly Leu Pro Ser Lys Gly Arg Met Ala 75 80 85

GCT GAT GCA ATC GAT CTT CTC AAG GAC TCT CAA CTG TTT GTT AAA CAA 399 Ala Asp Ala He Asp Leu Leu Lys Asp Cys Gin Leu Phe Val Lys Gin 90 95 100 105

GTC AAT CCT AGG CAA TAT CTT GCA CAG ATT CCC CAG TTA CCA AAC ACT 447 Val Asn Pro Arg Gin Tyr Val Ala Gin He Pro Gin Leu Pro Asn Thr 110 115 120

GAA GTC TGG TTT CAA CGG CCA AAA GAT ATT GTC AGA AAG TTA CTC TCA 495 Glu Val Trp Phe Gin Arg Pro Lys Asp He Val Arg Lys Leu Leu Ser 125 130 135

GGA GAT TTG GAT CTA GGT ATC GTT GCT CTT GAC ACA CTT ACT GAA TAT 543 Gly Asp Leu Asp Leu Gly He Val Gly Leu Asp Thr Leu Ser Glu Tyr 140 145 150

GCT CAG GAA AAT GAA GAT CTT ATC ATT GTC CAT GAA GCT CTC AAC TTT 591 Gly Gin Glu Asn Glu Asp Leu He He Val His Glu Ala Leu Asn Phe 155 160 165

GGA GAC TCT CAC CTG TCT ATT GCG ATT CCA AAC TAT GGG ATA TTT GAG 639 Gly Asp Cys His Leu Ser He Ala He Pro Asn Tyr Gly He Phe Glu 170 175 180 185

AAT ATA AAT TCT CTG AAG GAG CTA GCG CAA ATG CCC CAA TGG ACT GAA 687 Asn He Asn Ser Leu Lys Glu Leu Ala Gin Met Pro Gin Trp Ser Glu 190 195 200

GAG AGA CCC TTA CGC TTA GCT ACT GGC TTC ACT TAT CTC GGC CCC AAA 735 Glu Arg Pro Leu Arg Leu Ala Thr Gly Phe Thr Tyr Leu Gly Pro Lys 205 210 215

TTT ATG AAA GAA AAT GGC ATA AAG CAT GTG GTG TTT TCA ACT GCA GAC 783 Phe Met Lys Glu Asn Gly He Lys His Val Val Phe Ser Thr Ala Asp 220 225 230

GGA GCA CTG GAG GCA GCT CCA GCG ATG GGG ATA GCT GAT GCC ATT TTG 831 Gly Ala Leu Glu Ala Ala Pro Ala Met Gly He Ala Asp Ala He Leu 235 240 245

GAT CTT GTG ACT ACT GGT ATA ACA CTC AAA GAG AAC AAC TTG AAA GAA 879 Asp Leu Val Ser Ser Gly He Thr Leu Lys Glu Asn Asn Leu Lys Glu 250 255 260 265

ATT GAA GGA GGT GTT GTG CTG GAA AGC CAG GCG GCA CTT GTG GCA ACT 927 He Glu Gly Gly Val Val Leu Glu Ser Gin Ala Ala Leu Val Ala Ser 270 275 280

AGA AGA GCA TTA AAC GAG AGA AAA GGG GCA CTA AAC ACA CTA CAC GAG 975 Arg Arg Ala Leu Asn Glu Arg Lys Gly Ala Leu Asn Thr Val His Glu 285 290 295

ATT CTT GAG AGA TTG GAG GCC CAT CTA AAG GCG GAT GGC CAA TTC ACT 1023 He Leu Glu Arg Leu Glu Ala His Leu Lys Ala Asp Gly Gin Phe Thr 300 305 310

GTT GTT GCA AAC ATG AGA GGA AAT ACT GCT CAG GAA GTG GCT GAG CGT 1071 Val Val Ala Asn Met Arg Gly Asn Ser Ala Gin Glu Val Ala Glu Arg 315 320 325

GTG CTG AGC CAA CCA TCA TTG TCA GGA TTG CAG GGA CCG ACA ATA AGC 1119 Val Leu Ser Gin Pro Ser Leu Ser Gly Leu Gin Gly Pro Thr He Ser 330 335 340 345

CCA GTG TAC TCT ACA CAA AAT GGA AAA CTA TCG GTT GAC TAC TAT GCC 1167 Pro Val Tyr Cys Thr Gin Asn Gly Lys Val Ser Val Asp Tyr Tyr Ala 350 355 360

ATC GTG ATT TCT GTA CCA AAA AAG GCC CTA TAC GAC TCT GTG AAG CAA 1215 He Val He Cys Val Pro Lys Lys Ala Leu Tyr Asp Ser Val Lys Gin 365 370 375

CTT AGA GCG GCC GGA GGC ACT GGG CTA TTA GTT TCA CCT TTG ACC TAC 1263 Leu Arg Ala Ala Gly Gly Ser Gly Val Leu Val Ser Pro Leu Thr Tyr 380 385 390

ATT TTT GAT GAG GAT ACT CCA AGA TGG GCT CAG CTC CTG AGA AAC CTC 1311 He Phe Asp Glu Asp Thr Pro Arg Trp Gly Gin Leu Leu Arg Asn Leu 395 400 405

GGG ATT TAAATGCTTT GGTAAGGAAG ATGCATATGC TGAACAAGTT TTAAGAAGAC 1367

Gly He

410

TCTTTGGTGT AAATCTCTGA GTTTTTTTGG TACCTTTGCG TGTCTCTTTT TTTTTTTTCT 1427

TTCCCTTTIC TGCTGCAGTC GTCCTCCTCC CTGCTCTTCT AATTTTTACA TCTCTTTTCT 1487

CTATGGATCC TATTCGTTGT TGTTAATCCC GTTGGAACAA TAAAAACAAG CC1T1T1T1Α 1547

ATAAAAAAAA AAAAAAA 1564

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHAT^CTERISTICS:

(A) LENGTH: 411 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Ser Leu Leu Leu Pro Thr Asn Leu Gin Gin Tyr Pro Ser Ser Ser 1 5 10 15

Ser Phe Pro Ser Ser Thr Pro He Leu Ser Pro Pro Pro Ser Thr Ala 20 25 30

Phe Ser Val He Val Pro Arg Arg Arg Cys Leu Arg Leu Val Thr Ser 35 40 45

Cys Val Ser Thr Val Gin Ser Ser Val Ala Thr Asn Gly Ser Ser Pro 50 55 60

Ala Pro Ala Pro Ala Ala Val Val Val Glu Arg Asp Gin He Arg Leu 65 70 75 80

Gly Leu Pro Ser Lys Gly Arg Met Ala Ala Asp Ala He Asp Leu Leu 85 90 95

Lys Asp Cys Gin Leu Phe Val Lys Gin Val Asn Pro Arg Gin Tyr Val 100 105 110

Ala Gin He Pro Gin Leu Pro Asn Thr Glu Val Trp Phe Gin Arg Pro 115 120 125

Lys Asp He Val Arg Lys Leu Leu Ser Gly Asp Leu Asp Leu Gly He 130 135 140

Val Gly Leu Asp Thr Leu Ser Glu Tyr Gly Gin Glu Asn Glu Asp Leu 145 150 155 160

He He Val His Glu Ala Leu Asn Phe Gly Asp Cys His Leu Ser He 165 170 175

Ala He Pro Asn Tyr Gly He Phe Glu Asn He Asn Ser Leu Lys Glu 180 185 190

Leu Ala Gin Met Pro Gin Trp Ser Glu Glu Arg Pro Leu Arg Leu Ala 195 200 205

Thr Gly Phe Thr Tyr Leu Gly Pro Lys Phe Met Lys Glu Asn Gly He 210 215 220

Lys His Val Val Phe Ser Thr Ala Asp Gly Ala Leu Glu Ala Ala Pro 225 230 235 240

Ala Met Gly He Ala Asp Ala He Leu Asp Leu Val Ser Ser Gly He 245 250 255

Thr Leu Lys Glu Asn Asn Leu Lys Glu He Glu Gly Gly Val Val Leu 260 265 270

Glu Ser Gin Ala Ala Leu Val Ala Ser Arg Arg Ala Leu Asn Glu Arg 275 280 285

Lys Gly Ala Leu Asn Thr Val His Glu He Leu Glu Arg Leu Glu Ala 290 295 300

His Leu Lys Ala Asp Gly Gin Phe Thr Val Val Ala Asn Met Arg Gly 305 310 315 320

Asn Ser Ala Gin Glu Val Ala Glu Arg Val Leu Ser Gin Pro Ser Leu 325 330 335

Ser Gly Leu Gin Gly Pro Thr He Ser Pro Val Tyr Cys Thr Gin Asn 340 345 350

Gly Lys Val Ser Val Asp Tyr Tyr Ala He Val He Cys Val Pro Lys 355 360 365

Lys Ala Leu Tyr Asp Ser Val Lys Gin Leu Arg Ala Ala Gly Gly Ser 370 375 380

Gly Val Leu Val Ser Pro Leu Thr Tyr He Phe Asp Glu Asp Thr Pro 385 390 395 400

Arg Trp Gly Gin Leu Leu Arg Asn Leu Gly He 405 410

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHAPΛCTERISTICS:

(A) LENGTH: 1496 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(B) CLONE: Arabidopsis ATP-PRT2

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 16..1254

(D) OTHER INFORMATION: /codon_start= 16

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CGAGGGAGAG CAACC ATG CCA ATC TCA ATT CCC CTT AAC GCC ACT CTA CAA 51 Met Pro He Ser He Pro Leu Asn Ala Thr Leu Gin 1 5 10

TAC TCG TCT CCT TCT TCT TCT TCT TCT TCT TCT TCT CTC GTA CCT TCT 99 Tyr Ser Ser Pro Ser Ser Ser Ser Ser Ser Ser Ser Leu Val Pro Ser 15 20 25

TCC CCT CTT TTC TCT CCG ATT CCT TCA ACC ACT GTT TCC CTT ACC GGA 147 Ser Pro Leu Phe Ser Pro He Pro Ser Thr Thr Val Ser Leu Thr Gly 30 35 40

ATT CGC CAG CGA TGC CTC AGG ATG GTC ACT TCT TGC GTC TCC AAC GCT 195 He Arg Gin Arg Cys Leu Arg Met Val Thr Ser Cys Val Ser Asn Ala 45 50 55 60

CAG AAA TCC GTC CTG AAT GCT GCC ACC GAT TCC GTC TCT GTC GTC GGG 243 Gin Lys Ser Val Leu Asn Gly Ala Thr Asp Ser Val Ser Val Val Gly 65 70 75

CGG GAG CAG ATT CGT CTT GGT CTT CCT AGC AAA GGA CGC ATG GCC GCC 291 Arg Glu Gin He Arg Leu Gly Leu Pro Ser Lys Gly Arg Met Ala Ala 80 85 90

GAT TCG CTG GAT CTT CTC AAG GAT TGC CAA TTG TTT GTC AAA CAA GTC 339 Asp Ser Leu Asp Leu Leu Lys Asp Cys Gin Leu Phe Val Lys Gin Val 95 100 105

AAT CCA CGA CAA TAT GTT GCT CAA ATT CCT CAG TTA CCA AAT ACT GAA 387 Asn Pro Arg Gin Tyr Val Ala Gin He Pro Gin Leu Pro Asn Thr Glu 110 115 120

GTT TGG TTT CAA CGA CCA CAA GAC ATT GTC AGA AAG TTG CTA TCA GGA 435 Val Trp Phe Gin Arg Pro Gin Asp He Val Arg Lys Leu Leu Ser Gly 125 130 135 140

GAT CTG GAT CTA GGC ATC GTT GCT CTT GAC ATA GTT GCT GAA TTT GCT 483 Asp Leu Asp Leu Gly He Val Gly Leu Asp He Val Gly Glu Phe Gly 145 150 155

CAG GGA AAT GAA GAT CTT ATC ATT GTC CAC GAA GCT CTC AAC TTT GGA 531 Gin Gly Asn Glu Asp Leu He He Val His Glu Ala Leu Asn Phe Gly 160 165 170

GAT TCT CAT CTC TCC CTT GCC ATA CCC AAT TAT GGA ATA TTT GAG AAT 579 Asp Cys His Leu Ser Leu Ala He Pro Asn Tyr Gly He Phe Glu Asn 175 180 185

ATA AAA TCT CTG AAG GAG CTA GCA CAA ATG CCT CAA TGG ACC GAG GAA 627 He Lys Ser Leu Lys Glu Leu Ala Gin Met Pro Gin Trp Thr Glu Glu 190 195 200

AGA CCT TTA CGA GTT GCT ACA GGA TTC ACT TAT CTA GGC CCC AAA TTT 675 Arg Pro Leu Arg Val Ala Thr Gly Phe Thr Tyr Leu Gly Pro Lys Phe 205 210 215 220

ATG AAA GAC AAC GGT ATA AAG CAT GTG ACT TTC TCA ACT GCA GAT GGA 723 Met Lys Asp Asn Gly He Lys His Val Thr Phe Ser Thr Ala Asp Gly 225 230 235

GCC CTA GAG GCA GCG TCT GCG ATG GGG ATA GCT GAT GCC ATT TTA GAC 771 Ala Leu Glu Ala Ala Ser Ala Met Gly He Ala Asp Ala He Leu Asp 240 245 250

CTT GTG AGC ACT GGG ACA ACC CTT AAA GAG AAC AAC TTA AAA GAA ATT 819 Leu Val Ser Ser Gly Thr Thr Leu Lys Glu Asn Asn Leu Lys Glu He 255 260 265

GAA GGA GGG GTT GTG CTG GAA ACT CAG GCT GCA CTA GTG GCG ACT AGA 867 Glu Gly Gly Val Val Leu Glu Ser Gin Ala Ala Leu Val Ala Ser Arg 270 275 280

AGG GCA TTG ACT GAG AGA AAA GGA GCA CTA GAA ACA GTG CAT GAG ATT 915 Arg Ala Leu Thr Glu Arg Lys Gly Ala Leu Glu Thr Val His Glu He 285 290 295 300

CTC GAG AGA TTA GAG GCT CAT TTG AAG CCA AAT GGT CAA TTC ACT GTG 963 Leu Glu Arg Leu Glu Ala His Leu Lys Pro Asn Gly Gin Phe Thr Val 305 310 315

GTT CCA AAC ATG AGA GGA ACG GAT GCT GAA GAA GTG GCT GAA CCT GTG 1011 Val Pro Asn Met Arg Gly Thr Asp Ala Glu Glu Val Ala Glu Arg Val 320 325 330

AAA ACC CAA CCA TCA CTA TCA GGA TTG CAG GGA CCA ACA ATA ACT CCA 1059 Lys Thr Gin Pro Ser Leu Ser Gly Leu Gin Gly Pro Thr He Ser Pro 335 340 345

GTT TAT TCT AAA CGA GAT GGA AAA CTA ACA ATT GAG TAC TAT GCC ATT 1107 Val Tyr Cys Lys Arg Asp Gly Lys Val Thr He Glu Tyr Tyr Ala He 350 355 360 GTG ATA TCT CTA CCA AAA AAA GCG CTT TAT GAG TCT GTG CAG CAA CTG 1155 Val He Cys Val Pro Lys Lys Ala Leu Tyr Glu Ser Val Gin Gin Leu 365 370 375 380

AGA GCG GTG GGA GGA ACT GGG CTA CTA GTT ACT CCT CTA ACA TAC ATT 1203 Arg Ala Val Gly Gly Ser Gly Val Leu Val Thr Pro Val Thr Tyr He 385 390 395

TTT CAT GAG GAG ACT CCC AGA TGG ACT CAG CTT CTG ACT AAC CTC GGC 1251 Phe His Glu Glu Thr Pro Arg Trp Ser Gin Leu Leu Ser Asn Leu Gly 400 405 410

CTT TGATGCTGCT TCTCAGACAT ACTTTGGTTT AGTCAGATGC ATTCGTTGGA 1304

Leu

GGAACTCTTT TGGTCAAACA CTACAATGTC TTTCTTTTTT GGGTTTTATC TTTGTGGATC 1364

TTATAGTAGA AAAAACAGAG GTTGTCTCTT TTAAGCCTTT TAAGTCCATC TATGCCATTT 1424

CAGTTTTTCA GGAGTCAACA CTTTTAAAAA ( AATTTTAT CAAAAGCATT CGTAAAAAAA 1484

AAAAAAAAAA AA 1496

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE ⁽CHARACTERISTICS:

(A) LENGTH: 413 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Pro He Ser He Pro Leu Asn Ala Thr Leu Gin Tyr Ser Ser Pro 1 5 10 15

Ser Ser Ser Ser Ser Ser Ser Ser Leu Val Pro Ser Ser Pro Leu Phe 20 25 30

Ser Pro He Pro Ser Thr Thr Val Ser Leu Thr Gly He Arg Gin Arg 35 40 45

Cys Leu Arg Met Val Thr Ser Cys Val Ser Asn Ala Gin Lys Ser Val 50 55 60

Leu Asn Gly Ala Thr Asp Ser Val Ser Val Val Gly Arg Glu Gin He 65 70 75 80

Arg Leu Gly Leu Pro Ser Lys Gly Arg Met Ala Ala Asp Ser Leu Asp 85 90 95

Leu Leu Lys Asp Cys Gin Leu Phe Val Lys Gin Val Asn Pro Arg Gin 100 105 110

Tyr Val Ala Gin He Pro Gin Leu Pro Asn Thr Glu Val Trp Phe Gin 115 120 125

Arg Pro Gin Asp He Val Arg Lys Leu Leu Ser Gly Asp Leu Asp Leu 130 135 140

Gly He Val Gly Leu Asp He Val Gly Glu Phe Gly Gin Gly Asn Glu 145 150 155 160

Asp Leu He He Val His Glu Ala Leu Asn Phe Gly Asp Cys His Leu 165 170 175

Ser Leu Ala He Pro Asn Tyr Gly He Phe Glu Asn He Lys Ser Leu 180 185 190

Lys Glu Leu Ala Gin Met Pro Gin Trp Thr Glu Glu Arg Pro Leu Arg 195 200 205

Val Ala Thr Gly Phe Thr Tyr Leu Gly Pro Lys Phe Met Lys Asp Asn 210 215 220

Gly He Lys His Val Thr Phe Ser Thr Ala Asp Gly Ala Leu Glu Ala 225 230 235 240

Ala Ser Ala Met Gly He Ala Asp Ala He Leu Asp Leu Val Ser Ser 245 250 255

Gly Thr Thr Leu Lys Glu Asn Asn Leu Lys Glu He Glu Gly Gly Val 260 265 270

Val Leu Glu Ser Gin Ala Ala Leu Val Ala Ser Arg Arg Ala Leu Thr 275 280 285

Glu Arg Lys Gly Ala Leu Glu Thr Val His Glu He Leu Glu Arg Leu 290 295 300

Glu Ala His Leu Lys Pro Asn Gly Gin Phe Thr Val Val Pro Asn Met 305 310 315 320

Arg Gly Thr Asp Ala Glu Glu Val Ala Glu Arg Val Lys Thr Gin Pro 325 330 335

Ser Leu Ser Gly Leu Gin Gly Pro Thr He Ser Pro Val Tyr Cys Lys 340 345 350

Arg Asp Gly Lys Val Thr He Glu Tyr Tyr Ala He Val He Cys Val 355 360 365

Pro Lys Lys Ala Leu Tyr Glu Ser Val Gin Gin Leu Arg Ala Val Gly 370 375 380

Gly Ser Gly Val Leu Val Thr Pro Val Thr Tyr He Phe His Glu Glu 385 390 395 400

Thr Pro Arg Trp Ser Gin Leu Leu Ser Asn Leu Gly Leu 405 410 (2) INFORMATICN FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TAYATHTTYG AYGARGARAC 20

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYT^THETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CπCTCCTCCT CAAATATGTA 20

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii ) HYTrOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CGGGATCCAT GAAGCGTGAC CAGATTCGTC TTG 33

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GCTCTAGAAG CTTCAGCATA TGCATCTTCC 30

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

AATTATGACG TAACCTAGGA ATTAGCGGCC CGCTCTCGAG T 41

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid

(C) STT^S EDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYrrXDTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AATTACTCGA GAGCGGCCGC GAATTCCTAC GTTACGTCAT 40

Claims

We claim:

1. An isolated DNA molecule encoding a protein having a plant ATP-phosphoribosyl transferase activity.

2. The isolated DNA molecule of claim 1 , wherein said protein comprises the amino acid sequence selected from the group consisting of SEQ ID Nos. 4, 6, 8, and 10.

3. A DNA molecule encoding a modified ATP-phosphoribosyl transferase comprising a plant ATP-phosphoribosyl transferase having at least one amino acid modification, wherein said modified plant ATP-phosphoribosyl transferase is tolerant to a herbicide in amounts which inhibit said plant ATP-phosphoribosyl transferase.

4. The DNA molecule of claim 3, wherein said eukaryotic plant ATP-phosphoribosyl transferase comprises the amino acid sequence selected from the group consisting of SEQ ID Nos. 4, 6, 8 and 10.

5. The DNA molecule according to any one of claims 3 to 4 which is part of a plant genome, whereby said DNA molecule is heterologous with respect to the plant genome.

6. A chimeric gene comprising a promoter operably linked to a heterologous DNA molecule encoding a protein from a plant having ATP-phosphoribosyl transferase activity.

7. A chimeric gene comprising a promoter which is active in a plant operably linked to the DNA molecule of claim 3.

8. A chimeric gene according to claims 6 or 7, additionally comprising a signal sequence operably linked to said DNA molecule, wherein said signal sequence is capable of targeting the protein encoded by said DNA molecule into the chloroplast or into the mitochondria.

9. The chimeric gene according to any one of claims 6 to 8 which is part of a plant genome.

10. A recombinant vector comprising the chimeric gene of any one of claims 6 to 9, wherein said vector is capable of being stably transformed into a host cell, preferably a plant cell.

11. A host cell stably transformed with a vector according to claim 10 or with a chimeric gene according to anyone of claims 6 to 9, wherein said host cell is capable of expressing said DNA molecule.

12. A plant or plant cell including the progeny thereof comprising a DNA molecule of any one of claims 1 to 5 or a chimeric gene according to anyone of claims 6 to 9 or a recombinant vector according to claim 10, wherein said DNA molecule is expressed in said plant.

13. A plant or plant cell including the progeny thereof comprising a DNA molecule of any one of claims 3 to 5 or a chimeric gene comprising said DNA molecule, wherein said DNA molecule is expressed in said plant and confers upon said plant and plant cell, respectivly, tolerance to a herbicide in amounts which inhibit naturally occurring plant ATP-phosphoribosyl transferase activity.

14. A plant and its progeny including parts thereof having altered plant ATP- phosphoribosyl transferase activity, wherein said altered plant ATP-phosphoribosyl transferase activity confers upon said plant and its progeny tolerance to a herbicide in amounts which inhibit naturally occurring plant ATP-phosphoribosyl transferase activity.

15. The plant of any one of claims 12 to 14, wherein said plant is selected from the group consisting of soybean, cotton, tobacco, sunflower, sugar beet, soybean and oilseed rape.

16. The plant of any one of claims 12 to 14, wherein said plant is selected from the group consisting of maize, wheat, sorghum, rye, oats, forage and turf grass, barley, sorghum, millet, sugar cane, and rice.

17. The plant of any one of claims 12 to 16, wherein said altered plant ATP- phosphoribosyl transferase activity is conferred by over-expression of a plant ATP- phosphoribosyl transferase which naturally occurs in said plant.

18. The plant of any one of claims 12 to 16, wherein said altered plant ATP- phosphoribosyl transferase activity is conferred by expression of a DNA molecule encoding a herbicide tolerant plant ATP-phosphoribosyl transferase.

19. The seed of a plant according to any one of claims 12 to 18.

20. Propagating material of a plant according to any one of claims 12 to 19 treated with a protectant coating.

21. A method for controlling the growth of undesired vegetation which comprises applying to a population of the plant of any one of claims 12 1 to 18 an effective amount of a ATP-phosphoribosyl transferase-inhibiting herbicide.

22. A method of identifying a modified ATP-phosphoribosyl transferase resistant to a ATP-phosphoribosyl transferase inhibitor present in a population of cells comprising the steps of

(a) culturing said population in the presence of said protox inhibitor in amounts which inhibit the unmodified form of said ATP-phosphoribosyl transferase;

(b) selecting those cells from step (a) whose growth is not inhibited; and

(c) isolating and identifying the ATP-phosphoribosyl transferase present in the cells selected from step (b).

23. A method of selecting plants, plant tissue or plant cells transformed with a transgene of interest from non-transformed plants, comprising the steps of:

(a) transforming a plant, plant tissue or plant cell with a transgene of interest capable of being expressed by the plant, and a gene encoding an altered ATP- phosphoribosyl transferase resistant to a protox inhibitor;

(b) transferring the thus-transformed plants or plant cells to a medium comprising the ATP-phosphoribosyl transferase inhibitor; and

(c) selecting the plants or plant cells which survive in the medium.

24. A probe capable of specifically hybridizing to a plant APRT gene or mRNA, wherein said probe comprises a contiguous portion of the coding sequence for a APRT from a eukaryote at least 10 nucleotides in length.

25. The probe of claim 24 wherein said coding sequence is selected from the group consisting of SEQ ID Nos. 3, 5, 7 and 9.

26. A method of producing a host cell, preferably a plant cell, comprising an isolated DNA molecule encoding a protein from a plant having APRT activity comprising transforming the said host cell with a recombinant vector molecule according to claim 10.

27. A method of producing transgenic progeny of a transgenic parent plant comprising an isolated DNA molecule encoding a protein from a plant having APRT activity comprising transforming the said parent plant with a recombinant vector molecule according to claim 10 and transferring the herbicide tolerant trait to the progeny of the said transgenic parent plant involving known plant breeding techniques.

28. A method of producing a DNA molecule encoding a protein from a plant having APRT activity comprising

(a) establishing a cDNA library from a suitable eukaryotic source;

(b) identifying cDNA clones encoding a APRT enzyme based on their ability to supply

APRT enzymatic activity to a mutant host organism deficient in this activity.

29. A method of producing a DNA molecule encoding a protein from a eukaryote having APRT activity comprising

(a) establishing a genomic or a cDNA library from a suitable eukaryotic source;

(b) probing the said library with a probe molecule according to claim 24.

30. Use of a DNA molecule according to claim 5 to confer tolerance to a herbicide in amounts which inhibit naturally occurring protox activity from a parent plant to its progeny comprising first stably transforming the parent plant with a DNA molecule according to any one of claims 3to 4 by stably incorporating the said DNA molecule into the plant genome of the said parent plant and second transferring the herbicide tolerant trait to the progeny of the said transgenic parent plant involving known plant breeding techniques.

31. An assay system for screening chemicals for herbicidal activity using APRT.

32. A method for assaying a chemical for the ability to inhibit the activity of a APRT enzyme from a plant comprising

(a) combining said APRT enzyme and phosphoribosyl pyrophosphate (PRPP), ATP and other enzymes of the His biosynthetic enzymes under conditions in which said APRT enzyme is capable of catalyzing the conversion of said phosphoribosyl pyrophosphate (PRPP) to 5-amino-1 -ribosyl-4-imidazole carboxamide (AICAR)

(b) combining said chemical, said APRT enzyme, said phosphoribosyl pyrophosphate (PRPP), ATP and said other enzymes of the His biosynthetic enzymes in a second reaction mixture under the same conditions as in said first reaction mixture;

(c) comparing the absorbance of said first and said second reaction mixture at about 550nm, wherein said chemical is capable of inhibiting the activity of said APRT enzyme if the absorbance of said second reaction mixture is significantly less than the absorbance of said first reaction mixture.

33. A method for assaying a chemical for the ability to inhibit the activity of a APRT enzyme from a plant comprising (a) combining said APRT enzyme and phosphoribosyl pyrophosphate (PRPP) and ATP under conditions in which said APRT enzyme is capable of catalyzing the conversion of said phosphoribosyl pyrophosphate (PRPP) to PRATP,

(b) combining said chemical, said APRT enzyme, said phosphoribosyl pyrophosphate (PRPP) and ATP in a second reaction mixture under the same conditions as in said first reaction mixture;

(c) comparing the absorbance of said first and said second reaction mixture at about 290 nm, wherein said chemical is capable of inhibiting the activity of said APRT enzyme if the absorbance of said second reaction mixture is significantly less than the absorbance of said first reaction mixture.

34. A bag of seeds comprising seed of a transgenic plant comprising a DNA encoding a protein having a plant ATP-phosphoribosyl transferase activity together with lable instructions.

35. A bag of seeds comprising seed of a transgenic plant comprising a DNA encoding a DNA molecule encoding a modified ATP-phosphoribosyl transferase comprising a plant ATP-phosphoribosyl transferase having at least one amino acid modification, wherein said modified plant ATP-phosphoribosyl transferase is tolerant to a herbicide in amounts which inhibit said plant ATP-phosphoribosyl transferase together with lable instructions.