CA2217367A1 - Transgenic plants expressing acetyl coa carboxylase gene - Google Patents

Abstract

The present invention provides the complete cDNA sequence of maize acetyl CoA
carboxylase and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA
carboxylase gene in plant cells. The method of imparting herbicide tolerance to a plant includes the steps of introducing an expression cassette encoding a plant acetyl CoA carboxylase or an antisense DNA sequence complementary to the sequence for a plant acetyl CoA carboxylase gene operably linked to a promoter functional in plant cells, into the cells of a plant tissue and expressing the plant acetyl CoA carboxylase gene in an amount effective to render the acetyl CoA carboxylase and/or plant cell tolerant to the herbicides. The method of altering the oil content in a plant includes the steps of introducing an expression cassette into plant cells and expressing the acetyl CoA carboxylase gene in an amount effective to alter the oil content of the cells. The expression cassette can also be introduced into other host cells to increase yield of a plant acetyl CoA carboxylase so that crystallized enzyme can be used to screen and identify other herbicides that bind to and inhibit the enzyme.

Description

W O96/31609 . W PCTrUS96104625 TRANSGENIC PLANTS EXPRESSING
ACETYL CoA CARBOXYLASE GENE
B~ckground of the Invention Acetyl CoA carboxylase (ACCase) is an enzyme involved in many important metabolic pathways in plant, animal and bacterial cells. The enzyme is especially important in fatty acid synthesis in plants and is sensitive to inhibition by some types of herbicides. Structurally, ACCases are biotinylated and are quite large enzymes consi~tin~ of one or more subunits. For example, most ACCases of ~nimslls, higher plants, and yeast are dimers of 420 to 700 kD
native MW and contain subunits of 200 to 280 kD. Diatom and algal ACCases are 700 to 740 kD tetramers of 160 to 180 kD subunits. Bacterial ACCase l 5 consists of three dissociable proteins; biotin carboxylase (5 l kD), biotin carboxyl carrier protein (22.5 kD), and biotin transcarboxylase ( l 30 kD).
Acetyl CoA Carboxylase (ACCase) catalyzes the forrnation of malonyl-CoA from acetyl-CoA and bicarbonate in animal, plant, and bacterial cells. Malonyl-CoA is an essential substrate for (i) de novo fatty acid (FA) synthesis, (ii) fatty acid elongation, (iii) synthesis of secondary metabolites such as flavonoids and anthocyanins, and (iv) malonylation of some amino acids and secondary metabolites. Synthesis of malonyl-CoA is the first committed step of flavonoid and fatty acid synthesis and current e- idence suggests that ACCase catalyzes the rate-limiting step of fatty acid synthesis. Formation of malonyl-CoA by ACCase occurs via two partial reactions and requires a biotin prosthetic group:
(i)E-biotin + ATP + HCO3--> E-biotin-CO, + ADP + Pi (ii)E-biotin-CO7 + Acetyl-CoA --> E-biotin + malonyl-CoA
(NET)Acetyl-CoA + ATP + HCO~ --> malonyl-CoA + ADP + Pi ", 30 In E. coli, these reactions are catalyzed by three distinct components, biotin carboxylase, biotin transcarboxylase~ and biotin carboxyl carrier protein. whichcan be separated and yet retain partial activity. Plant and animal ACCases contain all three activities on a single polypeptide.

W O96131609 . PCTrUS96/04625 In plants, most ACCase activit~r is located in plastids of green and non-green plant tissues including leaves and oil seeds. Leaf ACCase activity is prirnarily located in mesophyll cells, but lesser arnounts have been found in C-4 bundle sheath cells and in epidelmal cells. The subcellular location of ACCase 5 activity in epidermal cells is unknown, but since synthesis of very long-chainfatty acids (VLCFA) for formation of waxes, cutin, and suberin occurs on the endoplasrnic reticulum (ER), malonyl-CoA might also be derived from a cytosolic ACCase. In contrast, rat ACCase is primarily cytosolic or associated with the outer mitochondrial membrane.
De novo fatty acid synthesis in chloroplasts involves successive

2-carbon additions to acetate, using malonate as the 2-C donor. All intermediates are attached to acyl carrier protein (ACP). Synthesis in plastids resembles that in E. coli in that the fatty acid synthesis complex can be dissociated into separate enzymes: ~-ketoacyl-ACP synthase (KAS), ~-ketoacyl-ACP reductase, ~-hydroxyl-ACP dehydratase, and enoyl-ACP reductase, acetyl-CoA:ACP transacylase, and malonyl-CoA:ACP transacylase. A highly active KASIII iso~;yme catalyzes the condensation of acetyl-CoA and malonyl-ACP. Successive additions of malonyl-CoA to acvl-ACPs catalyzed by KAS I form Cl 6 acyl-ACP, some of which is converted to C 18 acyl-ACP by KAS II and then to C18:1-ACP. Fatty acid metabolism thell diverges; de-esterification allows movement to the cytoplasm (eukaryotic path) where fatty acids may be further unsaturated and/or elongated by additions of malonyl-CoA
in the ER. Alternatively, fatty acids are linked to glycerol-3-phosphate (prokaryotic path), further unsaturated, and used for synthesis of chloroplast lipids. A portion of cytoplasmic lipids returns to the chloroplast. The relativecontributions of these two paths are species-specific but appear to be relatively flexible in mutants blocked in either path. In oil-storing organs such as cotyledons and monocot embryos, the triacylglycerides are stored in cytoplasmic oil bodies surrounded bv a single unit membrane.
Conden~tion of malonyl-CoA with phenylpropionyl-CoAs or acetyl-CoA leads to svnthesis of flavonoids, anthocyanins, or to polyacetates.

W O 96/31609 . PCTrUS96104625 Con~l~n.~tion is increased by light, elicitors, or pathogens and may be the rate-limiting step in synthesis of some phytoalexins. In addition to the secondary metabolites derived by de novo synthesi~, malonyl conjugates of flavonoid glycosides, formed by malonyl-CoA:flavonoid glycoside malollyll~ sferase~
5 D-arnino acids and 1-arnino-carboxyl-cycloplol)~le (ethylene precursor) are found in plants. Malonylated compounds accllmlll~te in vacuoles, probably after synthesis in the cytoplasm.
An important property of ACCase is the central role it plays in fatty acid synthesis and accurnulation in plants and seeds. Available evidence 10 supports the idea that ACCase activity is the rate-limiting step for de novo fatty acid synthesis in plants. High rates of ACCase activity in vitro parallel or slightly precede high rates of lipid deposition or [l4C]acetate incorporation into lipids in developing leaves and oil seeds. Significant changes in plant ACCase activity occur during chloroplast development and increase in ACCase activity 15 correlates with lipid deposition in developing oil seeds. Turnham et al., Biochem. J.. 212:223 (1883); and Beittenmiller et al., Plant Physiol.. 100:923 (1992).
Among other properties, ACCase in most monocots is also inhibited bv several herbicides. ['4C]acetate incorporation into maize lipids is20 strongly inhibited by fluazifop and sethoxydim due to inhibition of plastid ACCase. In barley, however, fluazifop had little effect on ['~C]acetate incorporation into very long-chain fatty acids. Since synthesis of very long-chain fatty acids occurs in the cytosol on the ER. and de novo fatty acid synthesis occurs in the plastids, cytosolic malonyl-CoA might be supplied by a 25 herbicide insensitive ACCase isozyme.
There are three general mech~ni~m~ by which plants may be resistant to. or tolerant of, herbicides. These mechzlnicm~ include insensitivity at the site of action of the herbicide (usually an enzyme). rapid metabolism ,, (conjugation or degradation) of the herbicide, or poor uptake and translocation of 30 the herbicide. Altering the herbicide site of action from a sensitive to an insensitive form is the preferred method of conferring tolerance on a sensitive W O96~1609 . PCTrUS96/04625 plant species. This is because tolerance of this nature is likely to be a dominant trait encoded by a single gene, and is likely to encompass whole families of compounds that share a single site of action, not just individual chemicals.
Therefore, detailed information concerning the biochemical site and mechanism S of herbicide action is of great importance and can be applied in two ways. First, the information can be used to develop cell selection strategies for the efficient identification and isolation of a~ o~l;ate herbicide-tolerant variants. Second, it can be used to characterize the variant cell lines and regenerated plants that result from the selections.
Tissue culture methods have been used to select for resistance (or tolerance) using a variety of herbicides and plant species (see review by Meredith and Carlson, 1982, in Herbicide Resistance in Plants. eds. Lebaron and Gressel, pp. 275-291, John Wiley and Sons, NY). For example, P. C. Anderson et al., in U.S. Patent No. 4,761,373, disclose the use of tissue culture methods to produce maize plants resistant to herbicidal imidazolidones and sulfonamides.
The re~i~t~nce is due to the presence of altered acetohydroxy acid synthase which is resistant to deactivation by tllese herbicides.
Certain 1,3-cyclohexanediones exhibit general and selective herbicidal activity against plants. One such cyclohexanedione is sethoxydim {2-[1 -(ethoxyimino)-butyl]-5-[2-(ethylthio)propyl]-3 -hydroxy-2-cyclohexen- 1 -one}. Sethoxydim is commercially available from BASF (Parsippany, New Jersey) under the designation POASTTM.
Other herbicidal cyclohexanediones include clethodim, (E,E)-(~)-2-[1-[[(3-chloro-2-propenyl)oxy] imino]propyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one; available as SELECTTM from Chevron Chemical (Valent) (Fresno, California); cloproxydim, (E,E)-2-[1-[[(3-chloro-2-propenyl)oxy]imino] butyl]-5-[2-(ethylthio)propyl]-3 -hydroxy-2-cyclohexen- 1 -one: available as SELECTONETM from Chevron Chemical (Valent) (Fresno, California); and tralkoxydim, 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-mesitylcyclohex-2-enone, available as GRASPTM from Dow Chemical USA
(Midland~ Michigan).

W O~6~1609 . PCTrUS~
.

For purposes of reference in the present specification, the ~, herbicides described in the two prece-ling paragraphs and other structurally related herbicidal compounds, are collectively referred to as the cyclohexanedione family of herbicides.
Certain aryloxyphenoxypropanoic acids exhibit general and selective herbicidal activity against plants. In these compounds, the aryloxy group may be phenoxy, pyridinyloxy or quinoxalinyl. One such herbicidal aryloxyphenoxypropanoic acid is haloxyfop, ~2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]- propanoic acid}, which is available as VERDICTTM from Dow Chemical USA (Midland, Michigan). Another is diclofop, {(+)-2-[4-(2,4-dichlorophenoxy)-phenoxy]propanoic acid}. available as HOELONTM from Hoechst-Roussel Agri-Vet Company (Somerville, New Jersey).
Other members of this family of herbicides include fenoxyaprop, (~)-2-[4-r(6-chloro-2-benzoxazolyl)oxy] phenoxy]propanoic acid; available as WHIPTM from Hoechst-Roussel Agri-Vet Company (Somerville, New Jersey);
fluazifop, (~t)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid; available as FUSILADETM from ICI Americas (Wilmington, Delaware);
fluazifop-P, (R)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid; available as FUSILADE 2000TM from ICI Americas (Wilmington~
Delaware); and quizalofop, (1~)-2-[4[(6-chloro-2-quinoxalinyl)-oxy]phenoxy]propanoic acid; available as ASSURETM from E. I. DuPont de Nemours (Wilmington, Delaware).
For purposes of reference in the present specification, the herbicides referred to in the two prece~ling paragraphs and other structurally related herbicidal compounds, are collectively referTed to as herbicidal aryloxyphenoxypropanoic acids.
Thus, there is a need for methods to develop plants that are resistant or tolerant to herbicides. There is also a nee-d to increase the oil and/or fatty acid content of the plants and seeds, as well as for methods to increase their resistance to herbicides. Moreover, there is a need to identify and clone genes W O96131609 . - PCT/U~G,'~S~25 important in conferring herbicide tolerance and in increasing the oil content of plants. ~, Summa~v of the Invention The present invention provides an isolated and purified DNA
5 molecule compri~ing a DNA segment encoding a maize acetyl CoA carboxylase gene and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in the plant cells. The DNA molecule encoding a plant acetyl CoA
carboxylase can encode an unaltered plant acetyl CoA carboxylase or an altered 10 plant acetyl CoA carboxylase substantially tolerant to inhibition by cyclohexanedione or aryloxyphenoxypropanoic acid herbicides as well as encoding an antisense DNA sequence that is substantially complementary to a plant acetyl CoA carboxylase gene or to a portion thereof. A DNA molecule of the invention can also further comprise an amino terminal plant chloroplast 15 transit peptide sequence operably linked to the maize acetyl CoA carboxylase gene.
The method of imparting cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant includes the steps of introducing a chimeric DNA molecule comprising a gene coding for a plant 20 acetyl CoA carboxylase or an altered or a functional mutant thereof operably linked to a promoter functional in a plant cell into cells of a susceptible plant, and regenerating the transformed plant cells to provide a differentiated plant.
The promoter can be an inducible or tissue specific promoter or provide for overexpression of at least about a 2-fold amount of a native plant acetyl CoA
25 carboxylase. The functional linkage of a promoter to the chimeric DNA
molecule results in an expression cassette. Expression of the chimeric DNA
molecule is in an amount effective to render the acetyl CoA carboxylase and/or the plant tissue substantially tolerant to the herbicides relative to the native acetyl CoA carboxylase present in said plant. Herbicide tolerance can be achieved in 30 the plants by at least two methods, including increasing the level of gene expression of a native or unaltered acetyl CoA carboxylase. or by introducing an W O96/31609 . PCTrUS9610462S

altered gene coding for an acetyl CoA carboxylase that is less sensitive to herbicide inhibition. The level of gene exl~les~ion can be increased by either combining a plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression, such as a 35S cauliflower mosaic virus S promoter (CaMV), or by introducing multiple copies of the gene into the cell so that the multiple copies of the gene are integrated into the genome of transformed plant cells. The preferred plant cells into which to introduce the expression c~se~te of the invention, to achieve herbicide tolerance, are monocotplant cells. Once transformed cells exhibiting herbicide tolerance are obtained,10 transgenic plants and seeds can then be regenerated th~l~r~olll, and evaluated for stability of the inheritance of the herbicide tolerance trait.
The invention also provides a method for altering, preferably raising, the oil content in a plant. The method includes the steps of introducing a chimeric DNA molecule comprising a gene coding for a plant acetyl CoA
15 carboxylase or an altered or a functional mutant thereof operably linked to apromoter functional in a plant cell into the cells of plant tissue and expressing the gene in an amount effective to alter the oil content of the plant cell. An alteration in oil content can include a change in total oil content over that normally present in that type of plant cell or a change in the type of oil present in 20 the cell. An alteration in oil content in the plant cell. according to tlle method of the invention. can be achieved by at least two methods including:
( 1 ) an increase or decrease in expression of an altered plant acetyl CoA carboxylase gene; or (2) by introducing an altered or functional mutant plant acetyl CoA carboxylase gene.
The level of gene expression of an unaltered plant acetyl CoA carboxylase gene can be increased by either combining an unaltered plant acetyl CoA carboxylase with a promoter that provides for a high level of gene expression, or by introducing multiple copies of an expression cassette into cells so that multiple 30 copies of the gene are integrated into the genome. When an altered or a functional mutant plant acetyl CoA carboxylase gene codes for an-enzyme that W O96~1609 . PCTrUS96/04625 exhibits an increase in specific activity, it can lead to an increase in total oil content of the plant cell. When an altered or a functional mutant acetyl CoA
carboxylase gene codes for an en_yme having a decrease in specific activity, it may lead to a decrease in the total oil content of the plant cell. Preferably, the S expression c~ette is introduced into dicot plants such as soybeans, canola, and surlflower. In an especially ~lc;rell~d version, transformed cells exhibiting about a 1.2- to S-fold increase in total oil content and/or expression or specific activity of acetyl CoA carboxylase are selected for and used to generate transgenic plants and seeds exhibiting a substantial increase in oil content. A substantial increase 10 in oil content depends on the oil content normally present in the plant or seecl and can range from about a 1.2 to a 20-fold increase.
The invention also provides for a method of producing plant acetyl CoA carboxylase in a host cell. The method includes the steps of introducing an expression c~ette comprising a chimeric gene encoding a plant 15 acetyl CoA carboxylase or an altered or a functional plant acetyl CoA
carboxyiase operabiy iiniced io a promoter inio a host cell and explessing the gene in an amount sufficient to permit cryst~lli7~tion of the plant acetyl CoA
carboxylase. An expression c~sette can include a promoter that is functional in either a eukaryotic or a prokaryotic cell. Preferably, the expression cassette is 20 introduced into a prokaryotic cell, such as E. coli. that is routinely used for production of recombinantly produced proteins. Recombinantly produced and crystallized plant acetyl CoA carboxylase can then be used to identify other herbicides and that bind to and inhibit acetyl CoA carboxylase in plants. In addition. the availability of large amounts of purified enzyme can permit the 25 screening of the efficacy of such herbicides in terms of their ability to bind to, or otherwise inhibit. the activity of the enzyme.
The present invention also provides an isolated and purified DNA
molecule of at least seven nucleotide bases which hybridizes under high stringency conditions to a DNA molecule comprising a DNA segment encoding 30 a plant acetyl CoA carboxylase and provides a hybridization probe comprising an isolated and purified DNA molecule of at least seven nucleotide bases, which W O96/31609 . PCTrUS96/04625 is detectably labeled or which binds to a detectable label, which DNA molecule hybridizes under high stringency conditions to the non-coding strand of a DNA
molecule comprising a DNA segment encoding a plant acetyl CoA carboxylase.
High stringency conditions are defined as: hybridization at 65 ~C for at least 16 hours in 5xSSC, lx Denhardt's solution, 50 mM Tris-HCl, pH 8, 0.2% SDS, 10 mM EDTA, 0.1 mg/ml salmon sperm DNA, followed by washing twice for 5 min~tes in 2xSSC, 0.5% SDS at 25~C, once for 10 minutes in 0.2xSSC, 0.1%
SDS at 25~C and twice for 30 minutes in 0.2xSSC, 0.1% SDS at 65~C.
The present invention also provides a method of introducing an exogenous plant acetyl CoA carboxylase gene into a host cell comprising transfolming host cells in vitro with an expression cassette comprising a chimeric DNA molecule encoding a plant acetyl CoA carboxylase gene operably linked to a promoter functional in the host cell, exr~ncling the transformed host cells in vitro, and identifying a transformed host cell which expresses the chimeric DNA molecule.
The term "consists essentially of" as used with respect to the present DNA molecules is defined to mean that a major portion of the nucleotide sequence encodes an ACCase. and that nucleotide sequences are not present which encode proteins other than ACCase or functional equivalents thereof.
l~rief Description of the Figures Figure 1 is a schematic depiction of the fatty acid biosynthesis pathway in plants.
Figure 2 is a graph depicting the effect of sethoxydim on the growth of mutant maize callus.
Figure 3 is a graph depicting the shoot length growth of maize see~llin~ seven days after treatment with sethoxydim.
Figure 4 is a graph depicting the shoot length growth of maize seedlings fourteen days after treatment with sethoxydim.
Figure 5: Total soluble and biotinylated polypeptides in ACCase purification fractions from ~ee~llin~ leaves of maize inbred A619. Proteins wereseparated by SDS-PAGE in 7.5% gels and then silver-stained (Panel A). An W O 96~1609 . PCTrUS96/04625 identical gel was Western-blotted and a longitudinal section of each lane was probed with avidin (Panel B). Lanes were 1: crude (10 llg); 2: (NH4)2SO4 (10 ~Lg); 3: S-300 (5 ~lg); 4: Blue Sepharose (2 ,ug); 5: Mono-Q ACCase II
(5 ,ug); and 6: Mono-Q ACCase I (5 llg). Diagonal lines between lanes indicate position of molecular weight markers shown on the left.
Figure 6: Immunoprecipitation of ACCase activity from B73 leaf, embryo, endosperm, and BMS suspension cultured cells. Equal activities (0.58 nmol min~') were incubated with 16 ~LL serum (immune plus preimmune), immune complexes were precipitated with Protein A-agarose, and ACCase activity rçm~ining in the resulting supernatant fraction was measured relative to the preimmune serum control.
Figure 7: Herbicide inhibition of acetyl-(AcCoA) or propionyl-CoA (Prop-CoA)-dependent Hl4CO3-incorporation into acid-stable product by ACCase I and II Mono-Q fractions. Activities in the presence of haloxyfop (1 ,~LM) are expressed relative to the minus herbicide control.
Figure 8: Comparison of the peptide sequence of maize cDNA
clones #15-14 and #18-5 with chicken ACCase. The approximate locations of the biotin carboxylase, biotin binding site, and biotin transcarboxylase functional domains are indicated for the chicken sequence. Tlle percentages of amino acid identity are indicated by cross-hatched boxes for the maize coding sequence.
Regions of genomic DNA Type I and Type II clone sequel1ces that ali,~n with cDNA #18-5 are indicated by solid heavy lines. The appro~imate locations of subclone #28 and #16 from genomic Type 1 and subclone #34 from genomic Type II clones are indicated.
Figure 9: Northern blot of total RNA from leaf. imm:~tllre embryo and endosperm tissue (16 days after pollination), and Black Mexican Sweet corn (BMS) cells. Lanes contain 10 ~Lg total RNA and were probed with the 2 kb EcoRI fragment of lambda clone # 15- 14.
Figure 10: DNA sequence (SEQ ID NO. 1) of a 2 kb EcoRI
fragment of lambda clone #15-14 including a portion of a maize ACCase gene located at bases 2883 to 83 from the 3' stop codon.

W O ~6/31609 . PCTrUS96/04625 Figure 11: Graph of ACCase activity during seed development in -,~ two high oil soybean cell lines and one low oil soybean cell line.
Figure 12: Cloning strategy to obtain the complete coding - sequence of the maize ACCase gene.
Figure 13: DNA sequence (SEQ ID NO:5) of a 7470 base pair cDNA of a maize ACCase gene. (Genbank Accession No. U19183).
Figure 14: Predicted amino acid sequence of the complete ACCase gene of maize (SEQ ID NO:6).
Figure 15: Restriction map of ACCase genomic clones.
Figure 16: Partial nucleotide sequence of a Type A~ ACCase genomic clone (SEQ ID NO:12).
Figure 17: Partial nucleotide sequence of clone 5A, a Type A
ACCase genomic clone (SEQ ID NO:13).
Figure 18: Partial nucleotide sequence of six Type A~ ACCase genomic clones (A-F) (SEQ ID NOs 14, 15, 16. 17, 18, and 19).
Figure 19: Partial nucleotide sequence ofthree Type B ACCase clones (SEQ ID NOs 20, 21 and 22).
Figure 20: SDS/PAGE analysis of chloroplast importation of 35S-labeled ACCase polypeptides. (A) Pea and maize chloroplasts incubated with 35S-labeled ACCase polypeptides for 30 minutes. (B) A time course analysis of the importation of 35S-labeled ACCase polypeptides into maize chloroplasts.
Detailed Descril~tion o~ the Invention The present invention provides a DNA molecule encoding a plant acetyl CoA carboxylase gene and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in the plant cells. In plants. acetyl CoA
carboxylase plays a central role in regulating fatty acid synthesis and in the sensitivity of monocots to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides.
In accord with the present invention, a plant acetyl CoA
carboxylase gene is identified, isolated and combined with a promoter functional W O96131609 PCTrUS96/04625 12 in a plant cell to provide a recombinant ~x~lession cassette. A plant acetyl CoAcarboxylase gene can be introduced and expressed in a plant cell. Depending on the type of plant cell, the level of gene t:x~ ;ssion, and the activity of the enzyme encoded by the gene, introduction of a plant acetyl CoA carboxylase gene into 5 the plant cell can confer herbicide tolerance and/or alteration of the oil of the plant cell.
In monocots, an exogenously introduced plant acetyl CoA
carboxylase gene can be expressed at a level effective to render the cells of the plant tissue s1-bst~nti~11y tolerant to cyclohexanedione or lO aryloxyphenoxypropanoic acid herbicide levels which normally inhibit a nativeor endogenous acetyl CoA carboxylase. A native acetyl CoA carboxylase ii an enzyme that is normally encoded and expressed in the plant cell prior to transformation. An exogenously introduced plant acetyl CoA carboxylase gene is a gene which has been isolated and amplified from either the same or different l 5 type of cell. Exogenous introduction and expression of a plant acetyl CoA
carboxylase gene in both monocots and dicots can result in alteration of the oilcontent and quality of plant tissue and seeds. Exogenous introduction and expression in a host cell, such as a bacteria, can provide for sufficient amounts of plant acetyl CoA carboxylase to allow for crystallization and isolation of the 20 enzyme. Cryst~11i7~ plant acetyl CoA carboxylase is useful to identify other herbicides that bind to and can inhibit plant acetyl CoA carboxylases. The enzyme could also be used to screen potential herbicidal compounds for efficacy.A. Formation of an Expression Cassette An expression c~ssette of the invention can comprise a chimeric 25 DNA molecule encoding a plant acetyl CoA carboxylase gene or an altered or functional mutant thereof operably linked to a promoter functional in a plant cell.
~he gene can code for a plant acetyl CoA carboxylase that is substantially tolerant to herbicides, preferably cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides. An expression r~sett~ of the 30 invention can also include an antisense DNA sequence that is substantially W O~6/31609 . ' PCTnUS96/04625 complementary to an acetyl CoA carboxylase gene or a portion thereof operably linked to a promoter functional in a plant cell.
1. Isolation and Identif cation of a Gene Collin~ for a Plant Acetyl CoA Carboxylase A gene encoding a plant acetyl CoA carboxylase can be identified and isolated by standard methods, as described by Sambrook et al., Guide to Molecular Clonin~: A Laboratory Manual. Cold Spring Harbor, NY (1989). The gene can be obtained either from monocot or dicot plant cells. When the gene encoding a plant acetyl CoA carboxylase is obtained from a dicot plant, the 10 enzyme encoded by the gene exhibits tolerance to cyclohexanedione or aryloxyphenoxypropanoic acid herbicides. The gene can also be obtained from herbicide-tolerant maize cell lines, prepared as described in U.S. Patent No.
5, 1 62,602.
A gene encoding a plant acetyl CoA carboxylase can be identified 15 by screening of a DNA or cDNA library generated from plant cells. Screening for DNA frz~gment.c that encode all or a portion of the gene encoding a plant acetyl CoA carboxylase can be accomplished by complementation of an auxotrophic mutant of acetyl CoA carboxylase in E. coli (fabE) (Bachman, Microbiolo~ical Reviews~ 47:180 (1983)) or yeast (accl) (Michionada, Eur. J.
20 Biochem.~ 79 (1980)) or by screening of plaques for binding to antibodies that specifically recognize a plant acetyl CoA carboxylase. DNA fragments that can restore ACCase activity in E. coli or yeast and/or plaques carrying DNA
fragments that are immunoreactive with antibodies to a plant ACCase can be subcloned into a vector and sequenced and/or used as probes to identify other 25 cDNA or genomic sequences encoding all or a portion of a plant acetyl CoA
carboxylase gene.
Specific examples of cDNA sequences encoding a portion of a plant acetyl CoA carboxylase gene include DNA fragments that include a DNA
sequence that substantially corresponds to the coding sequence for the 30 transcarboxylase active site of a plant acetyl CoA carboxylase, DNA fragmentsthat include a DNA sequence that substantially corresponds to a coding sequence for the biotin binding site of a plant acetyl CoA carboxylase, a DNA fragment W 096/31609 . PCT/U~G/O~

encoding the 5' transcriptional start sequence of a plant acetyl CoA carboxylasegene, and a DNA fragment encoding the 3' transcriptional stop sequence for the acetyl CoA carboxylase gene. Substantially corresponding DNA sequences share about 90% to about 100% DNA sequence homology. Especially preferred cDNA probes can be obtained from lambda clone #18-5 which include DNA
sequences corresponding to the transcarboxylase active site domain and the biotin binding site domain. Lambda clone #18-5 includes EcoRI subclones of

3.9 kb, 1.2 kb, or 0.23 kb. Lambda subclone #18-SI is an 3.9 kb EcoRI subclone.
The lambda subclone #1 8-5I has been deposited with the American Type Culture Collection, Rockville, MD, and given Accession No. 69236.
In a preferred version, a plant acetyl CoA carboxylase gene is identified and isolated from an herbicide tolerant maize cell line prepared as described in Example II. A cDNA library can be prepared by oligo dT priming.
Plaques contz~ining DNA fr~gment~ can be screened with antibodies specific for maize acetyl CoA carboxylase. DNA fragments encoding a portion of an acetyl CoA carboxylase gene can be subcloned and sequenced and used as probes to identify a genomic acetyl CoA carboxylase gene. DNA frzlgmçnt~ encoding a portion of a maize acetyl CoA carboxylase can be verified by deterrnining sequence homology with other known acetyl CoA carboxylases, such as chicken or yeast acetyl CoA carboxylase, or by hybridization to acetvl CoA carboxylase specific messenger RNA. Once DNA fragments encoding portions of the 5', middle and 3' ends as well as the transcarboxylase active site or biotin bindingsite of a plant acetyl CoA carboxylase are obtained~ they can be used to identify and clone a complete genomic copy of a maize acetyl CoA carboxylase gene.
To isolate a complete copy of a maize acetyl CoA carboxylase gene, a maize genomic library can then be probed with cDNA probes prepared as described above. Portions of the genomic copy or copies of a plant acetyl CoA
carboxylase gene can be sequenced and the 5' end of the gene are identified by standard methods including either DNA sequence homology to other acetyl CoA
carboxylase genes or by RNAase protection analysis, as described by Sambrook et al., Molecular Clonin~: A Laboratorv Manual. Cold Spring Harbor Press, Cold W O ~6~1609 . PC'~rUS96/04625 Spring Harbor, New York (1989). Once portions of the 5' end of the gene are identified, complete copies of a plant acetyl CoA carboxylase gene can be obtained by standard methods, including by cloning or by polymerase chain reaction (PCR) synthesis using oligonucleotide primers complementary to the 5 DNA sequence at the 5' end of the gene. The presence of an isolated full-length copy of a plant acetyl CoA carboxylase gene can be verified by hybridization, partial sequence analysis, or by expression of a plant acetyl CoA carboxylase enzyme. The maize acetyl CoA carboxylase gene cloned and expressed from a maize herbicide tolerant cell line can be assessed for tolerance to l 0 cyclohexanedione or aryloxyphenoxypropanoic acid herbicides by standard methods, as described in Example I.
An expression cassette of the invention can also contain an antisense DNA sequence. A antisense DNA sequence is a sequence that is substantially complement~ry to all or a portion of a coding sequence of a plant 15 acetyl CoA carboxylase gene. A subst~n~ y complementary sequence has about 90% to about 100% DNA sequence homology with that of the coding sequence of all or a portion of a plant acetyl CoA carboxylase. The antisense DNA sequence when expressed can act to inhibit the synthesis and expression of a native plant acetyl CoA carboxylase. Antisense sequences are preferably about 20 200 to 1000 nucleotides long in order to provide sufficient inhibition of synthesis and/or expression of a native acetyl CoA carboxylase. The inhibition of acetyl CoA carboxylase synthesis and gene expression by antisense DNA sequences can be confirmed in a transformed plant cell bv standard methods for measuring the presence and/or activity of the enzyrne such as described in Examples I and 25 V.
An expression cassette of the invention can also include a functional mutant of a plant acetyl CoA carboxvlase gene. Mutants of a plant acetyl CoA carboxylase gene are substantially homologous to a plant acetyl CoA
carboxylase gene and are functional if the acetyl CoA carboxylase expressed 30 retains significant enzyme activity. A mutant substantially homologous to a plant acetyl CoA carboxylase can share about 90% to 99.99% DNA sequence CA 022l7367 l997-lO-03 W O96131609 . PCTrUS~

with that gene. For example, a mutant acetyl CoA carboxylase gene can code for a herbicide tolerant acetyl CoA carboxylase, or for an acetyl CoA carboxylase with altered substrate specificity so that the total amount of oil content in the plants or seeds is increased, or for an enzyme with an altered substrate specificity 5 so that synthesis of secondary metabolites such as flavonoids or anthocyanins is decreased. A pl~f~llc;d mutant is a gene coding for an acetyl CoA carboxylase that is subs~nti~lly tolerant to cyclohexanedione or aryloxyphenoxypropanoic acid herbicide Altered or functional ml-t~nt~ of a gene coding for a plant acetyl 10 CoA carboxylase can be obtained by several methods. The alteration or mutation of the ACCase gene can be accomplished by a variety of means including, but not limited to, the following methods.
1. spontaneous variation and direct mutant selection in cultures, 2. direct or indirect mutagenesis procedures on tissue culture of all cell types, seeds or plants; and 3. mutation of the cloned acetyl CoA carboxylase gene by methods such as site specific mutagenesis (Sambrook et al. cited supra), transposon mediated mutagenesis (Berg et al., Biotechnology.
1:417 (1983)) and deletion mutagenesis (Mitra et al., Molec. Gen.
Genetic.. 215:294 (1989)).
Mutants can be identified by a change in a functional activity of the enzyme encoded by the gene or by detecting a change in the DNA sequence using restriction enzyme mapping or partial sequence analysis.
In a pler~ d version, a functional mutant gene encoding for a plant acetyl CoA carboxylase tolerant to cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides is isolated from a maize herbicide tolerant cell line. The maize herbicide tolerant cell line was obtained as described in U.S. Patent No. 5,162,602, issued November 10, 1992, the disclosure of which is incorporated in Examples I-III. Briefly, partially 30 differentiated cell cultures are grown and subcultured with continuous exposures to low herbicide levels. Herbicide concentrations are then gradually increased W O~6/31609 . PCTrUS96/04625 over several subculture intervals. Maize cells or tissues growing in the presence of normally toxic herbicide levels are repeatedly subcultured in the presence ofthe herbicide and characterized. Stability of the herbicide tolerance trait of the cultured cells may be evaluated by growing the selected cell lines in the absence of herbicides for various periods of time and then analyzing growth after exposing the tissue to herbicide.
Maize cell lines which are tolerant by virtue of having an altered acetyl CoA carboxylase enzyme can be selected by identifying cell lines having enzyme activity in the presence of normally toxic levels of sethoxydim or 10 haloxyfop. The tolerant maize cells can be further evaluated for whether acetyl CoA carboxylase is altered to a less sensitive form or increased in its level of expresslon.
Maize cell lines with a acetyl CoA carboxylase less sensitive to herbicide inhibition can be used to isolate a functional mutant gene of a plant 15 acetyl CoA carboxylase. A DNA library from a maize cell line tolerant to herbicides can be generated and DNA fragments encoding all or a portion of an acetyl CoA carboxylase gene can be identified by hybridization to a cDNA probe encoding a portion of the maize ACCase gene. A complete copy of the altered gene can be obtained either by cloning and ligation or by PCR synthesis using 20 appropriate primers. The isolation of the altered gene coding for acetyl CoA
carboxylase can be confirmed in transformed plant cells by determining ~ hether the acetyl CoA carboxylase being expressed retains enzyme activity when exposed to normally toxic levels of herbicides.
2. Promoters Once a plant acetyl CoA carboxylase gene or functional mutant thereof or an ~nticellce DNA sequence is obtained and amplified, it is combined with a promoter functional in a plant cell to form an expression cassette.
Most genes have regions of DNA sequence that are known as promoters and which regulate gene expression. Promoter regions are typically 30 found in the fl7lnkinE DNA sequence upstream from the coding sequence in bothprocaryotic and eukaryotic cells. A promoter sequence provides for regulation W O96/31609 . PCTrUS96104625 of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous genes, that is a gene different from the native or homologous gene. Promoter sequences are also known to be strong or weak or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for turning on and off of gene 10 expression in response to an exogenously added agent or to an environmental or developmental stimulus. Promoters can also provide for tissue specific or developmental regulation. An isolated promoter sequence that is a strong promoter for heterologous genes is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of15 transformed cells and provides for a high level of gene expression when desired.
The promoter in an ~x~lt~ion cassette of the invention can provide for overexpression of acetyl CoA of a plant acetyl CoA carboxylase gene or functional mutant thereof. Overexpression of the gene is that amount of gene expression that results in an increase in tolerance of the plant cells to an 20 herbicide or that results in an increase in the total oil contel1t of the cells.
Overexpression of an acetyl CoA carboxylase gene is preferably about a 2- to 20-fold increase in expression of an acetyl CoA carboxylase over the expression level of the native acetyl CoA carboxylase. The promoter can also be inducible so that gene expression can be turned on or off by an exogenously added agent.
25 For example~ a bacterial promoter such as the Pt~C promoter can be inc~uced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transforrned bacterial cells. It may also be preferable to combine the gene with a promoter that provides tissue specific expression or developmentally regulated gene expression in plants.
Specific promoters functional in plant cells include the 35S
cauliflower mosaic virus promoter, nopaline synthase (NOS) promoter and the W O96/31609 . PCTrUS96104625 like. Currently, a ~.erel,~d promoter for expression in monocots is the 35S
. cauliflower mosaic virus promoter.
An acetyl CoA carboxylase gene can be combined with the promoter by standard methods as described in Sambrook cited supra. Briefly, a S plasmid conf~ining a promoter such as the 35S cauliflower mosaic virus promoter can be constructed as described in Jefferson, Plant Molecular Biology Reporter~ 5,387 (1987) or obtained from Clontech Lab in Palo Alto, CA (e.g.
pBI121 or pBI221). Typically these plasmids are constructed to provide for multiple cloning sites having specificity for different restriction enzymes 10 do~,vnstream from the promoter. A gene for plant acetyl CoA carboxylase can be subcloned downstream from the promoter using restriction enzymes to ensure that the gene is inserted in proper orientation with respect to the promoter so that the gene can be expressed. In a ~l~felled version, a maize acetyl CoA
carboxylase is operably linked to a 35 S CaMV promoter in a plasmid such as 15 pBI 12 l or pBI22 1. Once a plant acetyl CoA carboxylase gene is operably linked to a promoter and the plasmid, the expression cassette so formed can be subcloned into other plasmids or vectors.
3. Optional Sequences in the Expression Cassette The expression cassette can also optionally contain other DNA
20 sequences. The expression cassette can further be comprised of a chloroplast transit peptide sequence operably linked between a promoter and a plant acetyl CoA carboxylase gene. If the expression cassette is to be introduced into a plant cell, the expression cassette can also contain plant transcriptional terminationand polyadenylation signals and translational signals linked to the 3' terminus of 25 a plant acetyl CoA carboxylase gene. The expression cassette can also optionally be further comprised of a plasmid.
Because one site of action for biosynthetic pathways involving plant acetyl CoA carboxylase is the chloroplast, an expression cassette of the invention can be combined with an exogenous DNA sequence coding for a 30 chloroplast transit peptide, if necessary. An exogenous chloroplast transit peptide is one which is not encoded within the plant acetyl CoA carboxylase W O96~1609 . PCTrUS96/04625 gene. A chloroplast transit peptide is typically 40 to 70 amino acids in length and functions post-translationally to direct the protein to the chloroplast. Thetransit peptide is cleaved either during or just after import into the chloroplast to yield the mature protein. The complete copy of a gene encoding a plant acetyl CoA carboxylase may contain a chloroplast transit peptide sequence. In that case, it may not be necessary to combine an exogenously obtained chloroplast transit peptide sequence into the ~ ion cassette.
Exogenous chloroplast transit peptide encoding sequences can be obtained from a variety of plant nuclear genes, so long as the products of the genes are expressed as preproteins comprising an amino tçrrnin~l transit pep~ideand transported into chloroplast. Examples of plant gene products known to include such transit peptide sequences are the small subunit of ribulose biphosphate carboxylase, ferredoxin, chlorophyll a/b binding protein, chloroplast ribosomal proteins encoded by nuclear genes, certain heatshock proteins, arnino l S acid biosynthetic enzymes such as acetohydroxy acid synthase, 3-enolpyruvylphospho~hikim~te synthase, dihydrodipicolinate synthase, and the like. ~ltern:~tively, the DNA fragment coding for the transit peptide may be chemically synthesized either wholly or in part from the known sequences of transit peptides such as those listed above.
Regardless of the source of the DNA fra,~~ment coding for the transit peptide, it should include a translation initiation codon and an amino acid sequence that is recognized by and will function properly in chloroplasts of thehost plant. Attention should also be given to the amino acid sequence at the junction between the transit peptide and the plant acetyl CoA carboxylase enzyme where it is cleaved to yield the mature enzyme. Certain conserved amino acid sequences have been identified and may serve as a guideline. Precise fusion of the transit peptide coding sequence with the acetyl CoA carboxylase coding sequence may require manipulation of one or both DNA sequences to introduce, for example, a convenient restriction site. This may be accomplished ~0 by methods including site directed mutagenesis, insertion of chemically synthe~i7~d oligonucleotide linkers and the like.

W O96t31609 . PCTIU~C~01C25 21 Once obtained, the chloroplast transit peptide sequence can be a~ opliately linked to the promoter and a plant acetyl CoA carboxylase gene in an ~x~ s~ion c~eeett~ using standard methods. Briefly, a plasmid cont~ininp a ~ promoter functional in plant cells and having multiple cloning sites downstream 5 can be constructed as described in Jefferson cited supra. The chloroplast transit peptide sequence can be inserted downstream from the promoter using restriction enzymes. A plant acetyl CoA carboxylase gene can then be inserted immediately downstream from and in frame with the 3' terminus of the chloroplast transit peptide sequence so that the chloroplast transit peptide is 10 linked to the amino terminus of the plant acetyl CoA carboxylase. Once formed, the expression cassette can be subcloned into other plasmids or vectors.
When the expression cassette is to be introduced into a plant cell, the expression czleeette can also optionally include 3' nontr:~n.el~ted plant regulatory DNA sequences. The 3' nontr:~n.el~t~-l regulatory DNA sequence 15 preferably includes from about 300 to 1,000 nucleotide base pairs and contains plant transcriptional and translational termination sequence. Specific examples of 3' nontranslated regulatory DNA sequences functional in plant cells include about 500 base pairs of the 3' fl~nkin~ DNA sequence of the pea ribulose biphosphate carboxylase small subunit E9 gene, the 3' fl~nking DNA sequence 20 of the octopine synthase gene, and the 3' flanking DNA sequence of tlle nopaline synthase gene. These 3' nontranslated re~ulatory sequences can be obtained as described in An, Methods in Enzymolo~y. 153:292 (1987) or are already present in plasmids available from commercial sources such as Clontech, Palo Alto, CA.
The 3' nontr~n.el~tt--l regulatory sequences can be operably linked to the 3' 25 terminus of a plant acetyl CoA carboxylase gene by standard methods.
An expression cassette of the invention can also be further comprised of a plasmid. Plasmid vectors included additional DNA sequences that provide for easy selection, arnplification and transformation of the expression cassette in procaryotic and eukaryotic cells. The additional DNA
30 sequences include origins of replication to provide for autonomous replication of the vector, selectable marker genes, preferably encoding antibiotic resistance, W O 96/31609 . PCTrUS96104625 unique multiple cloning sites providing for multiple sites to insert DNA
sequences or genes encoded in the expression cassette, and sequences that enhance transformation of prokaryotic and eukaryotic cells. The ~ ed vectors of the invention are plasmid vectors. The especially ~l~f~lled vector is5 the pBI121 or pBI221 vector formed as described by Jefferson cited supra.
Another vector that is useful for expression in both plant and procaryotic cells is the binary Ti vector PGA582. This binary Ti vector has beenpreviously characterized by An, cited supra., and is available from Dr. An. Thisbinary Ti vector can be replicated in procaryotic bacteria such as E. coli and 10 Agrobacterium. The Agrobacterium plasmid vectors can be used to transfer the expression cassette to plant cells. The binary Ti vectors preferably include thenopaline T DNA right and left borders to provide for efficient plant cell transformation, a selectable marker gene, unique multiple cloning sites in the Tborder regions, the colEl replication of origin and a wide host range replicon.
15 The binary Ti vectors carrying an expression cassette of the invention can beused to transform both prokaryotic and eukaryotic cells~ but is preferably used to transform plant cells.
B. Method for Screenin~ for Expression and/or O~ erexpression of a Plant Aceh~l CoA Carboxylase Gene A method for screening for expression or overexpression of a plant acetyl CoA carboxylase gene is also provided b tlle invention. Once formed, an expression cassette comprising an acetyl CoA carboxylase gene can be subcloned into a known expression vector. The screening method in the 25 invention includes the steps of introducing an expression ~rector into a host cell and detecting and/or qll~"~ g expression of a plant acetyl CoA carboxylase gene. This method of screening is useful to identify expression cassettes providing for an overexpression of a plant acetyl CoA carboxylase gene, antisense molecules that effectively inhibit acetyl CoA carboxylase synthesis, 30 and expression of an acetyl CoA carboxylase in the chloroplast of a transformed plant cell.

W O ~6/31609 . PCTrUS~6/0 Suitable kno~,vn expression vectors include plasmids that autonomously replicate in prokaryotic and eukaryotic cells. Specific examples include plasmids such as the pBI121 or pBI221 plasmid constructed as described by Jefferson cited supra, a binary Ti vector such as PG582 as described by An 5 cited supra, PUCl 19, or PBR322. The ~l~fc~ d expression system is a pBI121 orpBI221 plasmid.
An expression cassette of the invention can be subcloned into an expression vector by standard methods. The expression vector can then be introduced into prokaryotic or eukaryotic cells by standard methods including 10 protoplast transformation, Agrobacterium mç~ t~-l transformation, electroporation, microprojectiles and liposomes. The expression vector can be introduced into plant cells such as tobacco, Brassica, Black Mexican sweet corn,and Arabidopsis cells. The vector can also be introduced into procaryotic cells such as E. coli or Agrobacterium. Transformed cells can be selected typically 15 using a selection marker encoded on the expression vector.
Transient ~x~.cs~ion of a plant acetyl CoA carboxylase gene can be detected and quantitated in the transformed cells. Gene expression can be quantitated by a quantitative Western blot using antibodies specific for the cloned acetyl CoA carboxylase or by detecting an increase in specific activity of 20 the enzyme. The tissue and subcellular location of the cloned acetyl CoA
carboxylase can be determined by immunochemical staining methods using antibodies specific for the cloned acetyl CoA carboxylase. Sensitivity of the cloned acetyl CoA carboxylase to herbicides can also be assessed. Expression cassettes providing for OVC~ ssion of a plant acetyl CoA carboxylase or 25 acetyl CoA carboxylase tolerant to herbicides can then be used to transform monocot and/or dicot plant tissue cells and to regenerate transformed plants andseeds.
C. Method of Impartin~ Cvclohexanedione or Arvloxyphenoxypropanoic Acid Herbicide Tolerance to a Plant The invention provides a method of conferring cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant. The method W O96/31609 . PCTrUS9''0 includes the steps of introducing an ~ ;ssion c~sse~te compr~ing a gene coding for a plant acetyl CoA carboxylase or a functional mutant thereof operably linked to a promoter into the cells of plant tissue and ~ cs~illg the gene in an amount effective to render the cells of the plant tissue substantially 5 tolerant to herbicides. An effective amount of gene ~x~les~ion to render the cells of the plant tissue substantially tolerant to the herbicide depends on whether the gene codes for an unaltered acetyl CoA carboxylase gene or a mutant or altered form of the gene that is less sensitive to the herbicides. Expression of an unaltered plant acetyl CoA carboxylase gene in an effective amount is that 10 amount that provides for a 2- to 50-fold increase in herbicide tolerance and preferably increases the amount of acetyl CoA carboxylase from at least about 2-to 20-fold over that amount of the native enzyme. An altered form of the enzyme can be expressed at levels comparable to that of the native enzyme or less if the altered form of the enzyme has higher specific activity. Acetyl CoA
15 carboxylase substantially tolerant to herbicides is an enzyme that is tolerant of levels of herbicide which normally inhibit a native acetyl CoA carboxylase and preferably can function in concentrations of herbicide of about 2- to 20-fold greater than are toxic to the native enzyme.
Herbicide tolerance can be achieved by at least two methods 20 including: 1 ) by increasing the level of gene expression of a native or unaltered acetyl CoA carboxylase gene; or 2) by introducing an altered gene coding for an acetyl CoA carboxylase that is less sensitive to herbicide inhibition. The level of gene expression can be increased by either combining a plant acetyl CoA
carboxylase gene with a promoter that provides for a high level of gene 2~ expression such as the 35S CaMV promoter or by introducing the gene into the cells so that multiple copies of the gene are integrated into the genome of the transformed plant cell. Formation of an expression cassette comprised of a plantacetyl CoA carboxylase gene operably linked to a promoter that can be expressed in an effective amount to confer herbicide tolerance has been 30 described previously.

W O96/31609 . PCTrUS96/04625 Most monocots, but not dicots, are sensitive to cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides. The ~le~ll.,d plant cells for introducing an expression cassette of the invention to achieve herbicide tolerance - for the plant cells then are monocot plants. Monocot plants include corn, wheat, S barley, sorghum, rice, and others. An expression cassette of the invention can be introduced by methods of transformation, especially effective for monocots including biolistic transformation of Type II embryogenic suspension cells as described by W.J. Gordon-Kamm et al., Plant Cell. 2, 603-618 (1990), M.E.
Fromm et al., Bio/Technolo~y. 8, 833-839 (1990) and D.A. Walters et al., Plant 10 Molecular Biolo~y. 18, 189-200 (1992) or by electroporation of type 1 embryogenic calluses described by D'Hafluin et al., The Plant Cell. 4, 1495 (1992). Transformed cells can be selected for the presence of a selectable marker gene. Transient expression of a plant acetyl CoA carboxylase gene can be detected in the transgenic embryogenic calli using antibodies specific for the 15 cloned plant acetyl CoA carboxylase. Transformed embryogenic calli can be used to generate transgenic plants that exhibit stable inheritance of either thealtered acetyl CoA carboxylase gene or ov~ plession of the acetyl CoA
carboxylase gene. Maize cell lines exhibiting satisfactory levels of tolerance to herbicide are put through a plant regeneration protocol to obtain mature maize 20 plants and seeds expressing the tolerance traits such as described in D'Hafluin~
cited supra., or An, cited supra. The plant regeneration protocol allows the development of somatic embryos and the subsequent growth of roots and shoots.
To determine that the herbicide-tolerance trait is expressed in differenti~ted organs of the plant, and not solely in undifferentiated cell culture, regenerated 25 plants are exposed to herbicide levels which will normally inhibit shoot and root formation and growth.
Mature maize plants are then obtained from maize cell lines that are known to express the trait. If possible. the regenerated plants are self-pollin~ted Otherwise, pollen obtained from the regenerated plants is crossed to 30 seed-grown plants of agronomically important inbred lines. Conversely, pollenfrom plants of these inbred lines is used to pollinate regenerated plants. The W O 96~1609 . PCTrUS96/04625 genetics of the trait are then characterized by evaluating the segregation of the trait in the first and later generation progeny. Stable inheritance of ov~ ssion of a plant acetyl CoA carboxylase or a functional mutant of a plant acetyl CoA carboxylase conferring herbicide tolerance to the plant is achieved if the plants mzlints~in herbicide tolerance for at least about three to six generations.
Seed from transformed monocot plants regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants. Progenies from these plants become true breeding lines 10 which are evaluated for herbicide tolerance in the field under a range of environmental conditions. Herbicide tolerance must be sufficient to protect the monocot plants at the maximum labeled delivery rate under field conditions which cause herbicides to be most active. Appropriate herbicide concentrations and methods of application are those which are known and have been developed 15 for the cyclohexanedione and/or aryloxyphenoxypropanoic acid herbicides in question.
In a plerell.,d version, an expression cassette comprised of a maize acetyl CoA carboxylase gene isolated from a maize cell line tolerant to sethoxydim and haloxyfop and linked to the 35S CaMV promoter is introduced 20 into an herbicide sensitive monocot tissue using biolistic transformation.
Transformed calli are selected and used to generate trans~enic plants.
Transformed calli and transgenic plants can be evaluated for tolerance to sethoxydim and haloxyfop and for stable inheritance of the tolerance trait.
D. Method for Altering the Oil Content in a Plant The invention also provides a method of altering the oil content in a plant. The method include the steps of introducing an expression cassette comprising a gene coding for plant acetyl CoA carboxylase or functional mutant thereof operablv Iinked to a promoter functional in a plant cell into the cells of plant tissue and expressing the gene in an amount effective to alter the oil 30 content of the plant cell. An alteration in the oil content of a plant cell can include a change in the total oil content over that normally present in that type of W O 96~1609 . PCTrUS96/04625 plant cell, or a change in the type of oil from that normally present in the plant cell. Expression of the gene in an amount effective to alter the oil content of the gene depends on whether the gene codes for an unaltered ~acetyl CoA
carboxylase or a mutant or altered form of the gene. Expression of an unaltered 5 plant acetyl CoA carboxylase gene in an effective amount is that amount that may provide a change in the oil content of the cell from about 1.2- to 20-fold over that normally present in that plant cell, and preferably increases the amount of acetyl CoA carboxylase about 2- to 20-fold over that amount of the enzyme normally present in that plant cell. An altered form of the enzyme can be 10 expressed at levels comparable to that of the native enzyme or less if the altered form of the enzyme has higher specific activity.
An alteration in oil content of the plant cells according to the method of the invention can be achieved in at least two ways including:
(1 ) an increase or decrease in expression of an unaltered plant acetyl CoA carboxylase gene, or (2) by introducing an altered or functional mutant plant acetyl CoA carboxylase gene coding for an enzyme that exhibits a change in specific activity.
The level of gene expression of an unaltered plant acetyl CoA carboxylase gene 20 can be increased by either combining an unaltered plant acetyl CoA carboxylase gene with a promoter that provides for a high level of gene expression, such as the 35S cauliflower mosaic virus or by introducing the expression cassette and/or selecting for plant cells having multiple copies of a plant acetyl CoA carboxylase gene integrated into the genome. A decrease in expression of an unaltered acetylCoA carboxylase can be achieved by transformation with an ACCase antisense gene containing an expression c~ette. When an altered or functional mutant plant acetyl CoA carboxylase gene codes for an enzyme that has an increase in specific activity, it may lead to an increase in total oil content of a plant cell even if the level of gene expression is comparable to that of the native enzyme. When30 an altered or functional mutant acetyl CoA carboxylase gene codes for an W O96~1609 . PCTrUS96/04625 enzyme having a deerease in specifie activity, it may lead to a deerease in the total oil content of the plant cell compared to that normally present.
An expression eassette as deseribed above ean be introdueed into either monocots or dicots. Preferably, the ~x~l~;s~,ion cassette is introduced into 5 dicot plants such as soybean, canola, and sunflower. An expression cassette can be introduced by standard methods including protoplast transformation, Agrobacterium-mediated transforrnation, microprojeetiles, electroporation, and the like. Transformed cells or tissues ean be seleeted for the presence of a selectable marker gene.
Transient expression of a plant acetyl CoA carboxylase genc ean be detected in transformed eells or tissues by immunoreactivity with antibodies specifie for the eloned acetyl CoA earboxylase. O~ x~lession of a plant acetyl CoA earboxylase ean be deteeted by quantitative Western blots. A ehange in speeifie activity of the enzyme ean be detected by measuring enzyme activity in 15 the transformed eells. A change in total oil eontent can also be examined by standard methods, as deseribed in Clark & Snyder, JACS, 66:1316 (1989).
Transgenie plants and seeds can be generated from transformed cells and tissues showing a change in oil content or in the amount or specific activity of a plant acetyl CoA carboxylase using standard methods. It is 20 especially preferred that the oil content of the lea~es~ seeds~ or fruits is increased.
In a preferred version, a maize acetyl CoA carbo~ylase gene is combined with a 35S cauliflower mosaic virus promoter in a vector such as pBI121 or pBI221 and introduced into soybean cells using the microprojectile method. Transformed soybean cells showing an increase in expression of acetyl 25 CoA carboxylase of at least about 2-fold or at least a 1 .2-fold increase in oil content are seleeted. Transformed soybean eells exhibiting overexpression of acetyl CoA carboxylase or showing an increase in total oil content are used to generate transgenic plants and seeds.

W Og6/31609 . PCTrUS96/04625 E. Method of Producin~ Plant Acetyl CoA Carboxylase The invention also provides a method of producing plant acetyl CoA carboxylase in a host cell. The method includes the steps of introducing an expression c~sett~ comprised of a gene encoding a plant acetyl CoA
S carboxylase or functional mutant thereof into a host cell and expressing the gene in an amount sufficient to allow for cryst:~lli7~tion of the plant acetyl CoA
carboxylase. An amount sufficient to allow for cry~t~ tion of a plant acetyl CoA carboxylase is about 20- to 1 00-fold increase over the amount of plant acetyl CoA carboxylase that can normally be purified from plant cells, preferably about 2 to 10 mg protein. Cryst~11i7~1 plant acetyl CoA carboxylase can be used to identify other herbicides that can bind to and inhibit acetyl CoA carboxylasefunction. In addition, the availability of large amounts of purified enzyme provides for screening of the efficacy of such herbicides.
An expression cassette can include a promoter that is functional in either a eukaryotic or prokaryotic cell. The expression cassette can be introduced into a prokaryotic cell such as E. coli, or a eukaryotic cell such as a plant or yeast. The ~ rt~ d cell is a prokaryotic cell used routinely in producing recombinant proteins such as E. coli. The expression cassette can be introduced and transformed cells selected by standard methods.
The plant acetyl CoA carboxylase L~ene can be expressed in an prokaryotic cell until sufficient amount of the enzyme is produced so that it can be crystallized. Plant acetyl CoA carboxylase can be isolated from bacterial cells using standard methods~ including those described in Example V. The purified acetyl CoA carboxylase can then be crystallized and characterized by standard methods.
EXAMPLE I
Identification of Herbicide Mech~lni~m and Site of Action The objective of this Example was to identify the mechanism whereby sethoxydim and/or haloxyfop inhibit fatty acid synthesis in maize. The results, reported in J. D. Burton et al., Biochem. Biophys. Res. Comm.~ 148, W O96/31609 . PCTrUS96/04625 1039 (November 13, 1987), show that both sethoxydim and haloxyfop inhibit acetyl-coenzyme A carboxylase (ACCase) ~EC 6.4.1.2) in maize chloroplasts.
A. Chemicals Buffers and cofactors were purchased from Sigma Chemical Company (St. Louis, Missouri), [2-'4C]acetate was purchased from Research Products Tntern~tional; [2-'4C]pyruvate and ['4C]NaHCO3 were purchased from New England Nuclear, and [2-'4C]malonyl coenzyme A was purchased from Amersham. Sethoxydim was a gift from BASF (Parsippany, New Jersey), and haloxyfop was provided by Dow Chemical USA (Midland, Michigan).
10 B. Plant Growth Conditions Corn (Z. mays L., 'B37 x Oh43') seeds were germin~ted in darkness for 96 hours in vermiculite in an incubation chamber m~int~ined at 30~C, 80% RH. Seedlings were then transferred to a growth chamber with a 16 hour light (25~C) and an 8 hour dark (20~C~ cycle, 90% relative humidity (RH).
15 After greening 48 hours, seedlings were returned to the dark incubation chamber for 12 hours to deplete chloroplast starch reserves. Seedlings were harvested 6 days afterplanting. Pea ~P. sativum L., 'Pl 9901-C') seedlings were grown in vermiculite in a growth chamber with a 16 hour ligl1t (21 ~C) and 8 hour dark (16~C) cycle, 80% RH. Peas were harvested 10 to 13 da~s after planting. Black 20 Mexican Sweet (BMS) corn suspension cultures ucre maintained in a supplemented Murashige-Skoog (MS) medium (C. E. Green. Hort. Sci., 1~ 7-10 (1977)), and subcultured weekly by 20-fold dilution ofthe suspension culture into fresh medium.
C. Chloroplast Isolation Chloroplasts from corn and pea seedlings were isolated at 4~C (K.
Cline et al., J. Biol. Chem., 260, 3691-3696 ( l 985)). Seedlin~gs (50 g of shoots) were homogenized in 200 ml buffer A (50 mM HEPES-NaOH pH 7.5, 330 mM
sorbitol, 0.1% w/v BSA, 1 mM MgCl~, 1 mM MnCl~, 2 mM EDTA, 5 mM
isoascorbate, 1.3 mM glutathione) in an omnimixer (five~ 3-second bursts at full30 speed). The homogenate was filtered through six layers of cheesecloth and twolayers of miracloth. and then centrifuged at 3000 g for 3 minutes with hand-W O 96/31609 . PCTrUS96/04625 braking. The pellet was gently resuspended in buffer A and layered onto a pl~rulllled linear Percoll gradient (50 mM HEPES-NaOH pH 7.5, 330 mM
sorbitol, 1.9 mM isoascorbate, 1.08 mM glutathione, 0.1% w/v BSA, 50%
Percoll) which was centrifuged at 3000 g for 20 minutes in a Sorvall HB-4 rotor.5 The lower band in the gradient, cont~ining intact chloroplasts, was washed twice by gently resuspending it in 20 ml of buffer B (50 mM HEPES-NaOH, pH 7.5, and 330 mM sorbitol) followed by repelleting (3000 g, 5 minutes). The final pellet, con.~i~tin~; of intact chloroplasts, was resuspended in 2 to 3 ml of buffer B
and stored on ice in the dark until use.
10 D. Fatt~ Acid Synthesis ['4C]acetate and ['4C]pyruvate were used as precursors to measure fatty acid biosynthesis in isolated chloroplasts (B. Liedvogel et al., Planta, 169, 481-489 (1986)). t'4C]acetate incorporation was assayed in a 0.5 ml-volume cont~inin,~J: 50 mM HEPES-NaOH (pH 7.5), 330 mM sorbitol, 5 mM KH7PO4, 10 mM NaHCO3, 1 mM MgCl7, I mM ATP, 0.1 mM CoA, 0.15 mM
['4C]acetate (3.33 mCi/mmol), and chloroplasts (20 to 50 ~g chlorophyll).
['4C]pyruvate incorporation into fatty acids was assayed in the same medium except that it included 2 mM TPP, 1 mM NAD~, 0.15 mM ['4C]-pyruvate (1.33 mCi/mmol). but no acetate. Assay suspensions were ill~lmin~te-l ~ ith 1400 20 ,uE/m' second PAR at 25~C. Assays were initiated by the addition of the labeled substrate and stopped by the addition of 0.5 ml of 40% KOH. To determine the incorporation of radiolabel into a non-polar (fatty acid) fraction, each treatment was saponified at 90~C for 30 minutes in capped vials (P. B. Hoj et al., Carlsberg Res. Commun., 47, 119-141 (1982)). The vials were acidified with 0.5 ml 40%
25 H7S04. and carrier fatty acids (20 ,ug each of C 14:0, C 16:0, and C 18:0) were added. The assay mixture was extracted twice with 4 ml hexane. The extracts were combined. dried under N., and redissolved in 0.3 ml hexane. Aliquots (50 ~-1) were counted for radioactivity by liquid scintillation spectrometry.
Incorporation of ['4C]malonyl-Coenzyme A into fatty acids (P. B.
~0 Hoj et al., supra; and J. B. Ohlrogge et al., Proc. Natl. Acad. Sci. USA. 76, 1194-1198 (1979)) was assayed using cell-free preparations from BMS tissue culture.

W O96/31609 . PCTrUS96/04625 Cells harvested during logarithrnic growth phase were frozen in liquid nitrogen,ground with a mortar and pestle, and thawed in a medium co~ inp: 0.1 M
HEPES-KOH, pH 7.5, 0.3 M glycerol, and S mM DTT (burr~ issue, 2:1, v/w).
The homogenate was centrifuged at 12,000 g for 20 minlltçs The supernatant S was filtered through miracloth and centrifuged (125,000 g) for 60 minlltes andthen filtered through miracloth and assayed. Assays were conducted at 25~C in a 0.4 ml volume cont~ining: 1.0 mM ATP, 0.32 rnM NADPH, 0.38 mM NADH, 25 ,uM CoA, 10 ~LM acetyl-CoA, 25 ,ug acyl-carrier protein, and 12 !lM malonyl-CoA (11.54 IlCi/llmol). Reactions were initiated by addition of [l4C]malonyl 10 CoA and stopped by addition of 0.4 ml 40% KOH. Label incorporation into fatty acids was determined as above. Chlorophyll (D. I. Arnon, Plant Physicl., 24, 1-15 (1949)) and protein (P. K. Smith et al., Anal. Biochem., 150, 76-85 (1985)) were deterrnined as described therein.
E. Acetvl-Coenzyme A Carboxylase (ACCase) Activity Maize chloroplasts, isolated as described above, were suspended in buffer C (0.1 M Tricine-KOH, pH 8.0; 0.3 M glycerol, and 1 mM DTT) and homogenized in a glass tissue homogenizer. The disrupted chloroplast fraction was centrifuged at 16,000 g for 15 minutes. The supernatant was desalted on a Sephadex G-25 column (1.5 x 5 cm equilibrated with 0.1 M Tricine-KOH~ pH
8.0: and 0.3 M glycerol) and assayed directly. ACCase activity (B. J. Nikolau etal., Arch. Biochem. Biophys., 211, 605-61' (1981)) was assayed at 30~C in a 0.2 ml volume which contained 1 mM ATP, 3 mM acetyl coenzyme A, 2.5 mM
MgC12, 50 mM KCl, 0.5 mM DTT, and 15 mM ['4C]NaHCO3 (0.17 mCi/mmol).
Reactions were initiated by addition of acetyl coenzyme A and stopped by addition of 25 ~11 of 12 N HC1. Product formation was determined by the radioactivity found in an acid stable fraction by liquid scintillation spectrometry.
Enzyme activity was linear for 15 minutes.
F. Results To probe for the site of herbicidal activity of sethoxydim and haloxyfop, labeled acetate, pyruvate, and malonyl-CoA were used individually as precursors for fatty acid synthesis. Isolated chloroplasts from corn seedlings W O96/31609 . PCTrUS96/04625 incorporated ['4C]acetate and [i4C]pyruvate into a non-polar fraction (fatty acids). Acetate incorporation was linear for 30 min after a 5 min lag period, and dependent upon the addition of free acetyl coenzyme A. Addition of either 10 ,uM sethoxydim or 1 ~M haloxyfop inhibited [l4C]acetate incorporation into fatty5 acids by 90% and 89%, respectively, as shown in Table I, below. Sethoxydim (10 ~) and haloxyfop (1 ~M) also iilhibited the incorporati;~P. of [l4C]pyrùvateinto fatty acids by 98% and 99%, respectively.

TABLE I

Inhibition of [l~C]acetate and [l~C]pyruvate Incorporation into Fatty ~cids in Corn Seedling Chloroplasts by Sethoxydim (lO ~M) and Haloxyfop (l ~M), 15 lO minute asRay time Acetate Pyruvate ---Activity (nmol/mg chl-min)---20 Control4.4 + 0.41 lO.8 + 2.3 ----------% Inhibition----------- -Sethoxydim 90 + 2.5 98 + l.l Haloxy~op 89 + 3.l 99 + 0.3 1Results are expressed as mean of two experiments + standard error.

The effect of 10 ~M sethoxydim and 1 IlM haloxyfop on ['4C]malonyl-CoA incorporation into fatty acids was determined using cell-free 30 extracts from corn suspension cultures. Neither sethoxydim (10 ,uM) nor haloxyfop (1 ,uM) inhibited fatty acid synthetase activity. Thus. both herbicides inhibited fatty acid synthesis in intact chloroplasts from corn seedlings with either acetate or pyruvate as a precursor, but did not inhibit incorporation of malonyl-CoA into fatty acids. This suggests that ACCase which catalyzes the 35 formation of malonyl-CoA is the site of action of these herbicides.

W O96/31609 . PCTrUS96/04625 EXAMPLE II
Selection and Characterization of Herbicide-tolerant Cell Lines A selection protocol to identify and isolate herbicide-tolerant 5 maize cells was developed to minimi7~ the adverse effects of high herbicide concentrations on somatic embryo development and plant regeneration capacity.
The procedure involved exposing tissue to gradually increasing concentrations ofherbicide beginnin, with a sethoxydim concentration representing 1/20th of lethal dose and doubling the herbicide concentration at approximately two-week 10 intervals until the lethal dose (10 !lM sethoxydim) was reached. In this way, the herbicide was allowed to take effect slowly witl1 continuous selection pressure,thus permitting herbicide-tolerant cells to accumulate o-~er time while not affecting the potential for plant regeneration.
A. Selection of a Sethoxvdim-Tolerant Cell Line Many selections were carried out utilizing the selection protocol described in the preceding paragraph. The selection of one such sethoxydim-tolerant cell line that was identified and characterized is described below in detail.
Approximately 100 grams of ~igorously ~rowing. regenerable, 20 friable, embryogenic maize callus tissue established from an F, immature embryo resulting from the cross A188 x B73 were transferred to agar-solidified m~int~?n~nce medium (Armstrong and Green, Planta. 164, 207 (1985)) in petri plates cont~ining 0.5 ,uM sethoxydim (BASF) (Parsippany, New Jersey). This callus line was de~ign~t~cl 2167-9/2160-154. Forty plates were prepared and five25 clumps of callus tissue weighing about 0.5 grams each were placed on each plate. The 0.5 ~LM sethoxydim concentration was cllosen from growth inhibition studies to provide less than 10-20% growth inhibition during the first two weeksof herbicide exposure. After 14 days, 0.25-0.5 gram pieces of tissue showing vigorous growth rate and retention of embryogenic morphology (i.e., presence of 30 somatic embryos) were subcultured on fresh medium cont~ining 1.0 ,~ M
sethoxydim. Eighty plates contz~ining five pieces of tissue per plate were prepared. For each subsequent transfer? all callus tissue showing growth and W O96/31609 . PCTrUS96/04625 somatic embryo forming ability was placed on fresh media contzlinin~ a two-fold increased sethoxydim concentration. Therefore, callus was transferred at t~,vo-week intervals to petri plates cont~ining 0.5, 1.0, 2.0, 5.0 and 10.0 ~lM
sethoxydim. During the course of the selection process, the total number of lines decreased as the herbicide-mediated growth inhibition became more intense.
Cell lines exhibiting growth on 10 ,uM sethoxydim were designated as herbicide-tolerant and given an identification number. Two sethoxydim-tolerant lines were recovered that exhibited uninhibited growth at 10 ~lM sethoxydim. These lines were design~tecl 2167-9/2160-154 S-l and 2167-9/2160-154 S-2.
10 B. Characterization of Herbicide-Tolerant Maize Cell Line Tolerant cell line 2167-9/2160-154 S-2 ("S-2") was characterized to evaluate: (1) the magnitude of sethoxydim tolerance; (2) cross-tolerance of haloxyfop; and (3) the biochemical basis for the tolerance. Callus tissue from S-15 2 that had been m~int~ined on 10 IlM sethoxydim was transferred to media containing up to 100 ~M sethoxydim. One-half gram of S-2 tissue was plated on a 7 cm filter paper as a lawn overlaying 50 ml agar-solidified culture medium containing 0. 0.5, 1.0, 2.0, 5.0, 10.0, 50.0 and 100 IlM sethoxydim. and cultured fortwo weeks. Control cell line 2167-9/2160-15~ was plated similarly on 20 medium cont:~ining the same levels of sethoxydim. The results of this study are summarized in Figure 2. The control cell line gro~th after two weeks was inhibited 50% at 1 ,uM sethoxydim. Growth of S-2 was not inhibited at 100 ~LM
sethoxydim. indicating that S-2 was at least 100-fold more tolerant than the control callus line.
Growth of S-2 was inhibited witll 0.65 ,uM haloxyfop, whereas the control cell line was inhibited 50% with 0.02 ~lM. indicating approximately a 30-fold increase in tolerance.
C. Acetvl-Coenzyme A Carboxylase (ACCase) Activity of Maize Cell Line S-2 Assays were conducted to determine if ACCase extracted from cell line S-2 was altered with respect to herbicide activity. ACCase activity ofcontrol tissue was 50% inhibited either by 1.5 ~lM sethoxydim~ or by 0.25 ~LM

W O96~1609 . PCTrUS96/04625 haloxyfop. ACCase activity of S-2 tissue was inhibited 50% either by 70 IlM
sethoxydim, or by 1.8 ,uM haloxyfop, indicating at least 40-fold and 7-fold decreases in herbicide sensitivity on concentration basis, respectively.
EXAMPLE III
Plant Re~eneration and Production of Herbicide-Tolerant Seed A. Plant Re~eneration Protocol Sixteen ca. 150 mg clumps of S-2 callus were transferred per 25 x 100 mm petri plate cont~ining agar-solidified N6 basal salts and 6% sucrose and incubated 7-14 days in low light (20 ~E m~2 s-l). Several plates cont~ining callus on plant regeneration medium were prepared. Callus was transferred to agar-solidified Murashige-Skoog (MS) medium without hormones and incubated in high intensity light (200 ,~LE m-2 s~') for shoot elongation. Developing plants (1-3 cm long) were isolated from the callus surface and transferred to magenta boxes containing agar-solidified MS salts, 2% sucrose with no hormones for two weeks of further growth. When plants reached the 2-3 leaf stage, they were transplanted to peat pots contS~inin~ potting soil, and were incubated in the growth room until growing stably. Surviving plants were transferred to soil in 4"
diameter plastic pots and grown in the greenhouse.
B. Expression of Herbicide Tolerance in Plants Re~enerated from S-2 Callus Tissue Groups of eight control (2167-9/2160- 154 unselected) and eight S-2 plants were sprayed with either 0.0, 0 01, 0.05, 0.11, 0.22 or 0.44 kg/ha sethoxydim to determine whole plarlt sethoxydim-tolerance of greenhouse-grown plants. Control plants were killed by 0.05 kg/ha or more sethoxydim.
Plants regenerated from the S-2 cell line survived the 0.44 kg/ha sethoxydim treatment, indicating that S-2 plants exhibit at least 20-fold more tolerance ofsethoxydim than control. Figure 3 shows the growth response of the regenerated plants seven days after treatment with 0.44 kg/ha sethoxydim. As shown in Figure 4, shoot height of regenerated S-2 plants was only slightly reduced 14 days after treatment with 0.44 kg/ha sethoxydim.

W O 96/31609 . PCTrUS~6/0 C. Seed Production from S-2 Plants Plants surviving sethoxydim treRtment~ of up to 0.44 kg/ha were transplanted to the genetics plot on the University of Minnesota campus, St.
- Paul, Minnesota. Additional S-2 plants were transplanted to the field that had 5 not been sprayed. Sixty-five 2167-9/2160-154 control plants and ninety-five S-2 plants were grown to maturity in the field. Plants were either self-pollinRter1 or cross-pollinated to inbred maize lines A188, A619, A641, A661, A665, B~7, B73, R806, and W153R. Control seed were produced by selfing 2167-9/2160-154 regenerated plants, or by crossing them with the inbreds listed above.
10 D. Expression of Herbicide Tolerance in Pro~eny of Regenerated Plants Seeds obtained by the crossing procedure described above were viable and germinRted normally. Seeds from thirty S-2 selfed plants and fifteen 2167-9/2160-154 control plants were planted in 25 x 50 cm trays of soil (28 seeds from each plant in one tray) and grown in the greenhouse. Seedlings at the15 3-4 leaf stage were treated with 0.1, 0.44, and 1.1 kg/ha sethoxydim and evaluated for visual herbicide damage and shoot height. Based on visual rating of herbicide damage two weeks after treatment, selfed progeny of S-2 plants segregated approximately 1 :2: 1 for healthy, uninjured plants: to plants showin~
partial injury: to dead plants, respectively~ at 0.44 and 1.1 kgil1a sethoxydim 20 treatments. All control progeny of 2167-9/2160-154 control plal1ts were killed by 0.1 kg/ha and greater levels of sethoxydim. These results demonstrate dominant expression of sethoxydim tolerance indicating that sethoxydim tolerance in S-2 plants is a heritable trait. Similar tests were conducted on progeny of S-2 plants crossed to the other inbreds. In all cases. these test cross 25 progeny treated with 0.44 kg/ha sethoxydim segregated 1: 1 for growing shootsversus dead shoots whether S-2 plants were used as male or female parents.
These results confirm that sethoxydim tolerance is controlled by a single dominant nuclear gene. In all cases, control plants crossed to the other inbredswere killed and therefore sethoxydim-sensitive.

W O96/31609 . PCTrUS96/04625 E. I~Iethod for Obt~inin~ Uniform Herbicide-Tolerant Seed Progeny of S-2 plants surviving sethoxydim tre~trnent~ of 0.44 and 1.1 kg/ha and showing no herbicide injury were transferred to the greenhouse and grown to maturity. These plants may be selfed and their progeny evaluated for sethoxydim and haloxyfop tolerance to identify pure breeding herbicide-tolerant maize lines.
Progeny of S-2 plants crossed to inbred lines and exhibiting sethoxydim tolerance may be recurrently backcrossed to the same inbreds.
Progeny of each cross may be screened for sethoxydim-tolerance, and tolerant plants grown to maturity and again crossed to the recurrent parent. After six orseven cycles of backcrossing, sethoxydim-tolerant plants may be selfed and progeny screened for tolerance to produce homozygous sethoxydim tolerant maize inbreds.
EXAMPLE IV
Selection of Additional Herbicide-Tolerant Maize Cell Lines One primarily sethoxydim-tolerant maize cell line. 2167-9/2160-154 S-l, and two haloxyfop-tolerant maize cell lines. 2167-9/2160- 154 H- 1 and 2167-9/2160-154 H-2~ were selected and characterized as follows:
A. Selection of Maize Cell Line 2167-9/2160-1~
Maize cell line 2167-9/2160- 154 S- I ~ as selected from maize cell culture using the protocol described in detail abo~e for the selection of Line 2167-9/2160-154 S-2. Approximately 70 plants were regenerated from Line 2167-9/2160- 154 S- 1, and either self-pollinated or cross-pollinated to the inbred maize lines A188~ A619, A641, A661, A665, B37, B73. R806. and W153R.
B. Selection of Maize Cell Line 2167-9/2160-1~4 H-l Line 2167-9/2160-154 H-l was selected from maize cell culture using a similar protocol described in detail above except maize callus tissue was selected using the herbicide haloxyfop. Maize callus tissue was initially platedon 0.0l ,uM haloxyfop. At two-week intervals, surviving tissue was subcultured onto 0.05, 0.10 and 0.20 ~LM haloxyfop. Approximately 50 plants were regenerated from Line 2167-9/2160-154 H-l, and were self-pollin~te~l W O~6/31609 . PCTrUS96/0 C. Selection of Maize Celi Line 2167-9/2160-154 H-2 Line 2167-9/2160-154 H-2 was selected from maize cell culture using a similar protocol described in detail for line 2167-9/2160- 154 H- 1. No - plants have been successfully regenerated from this line.
D. Characterization of Lines 2167-9/2160-154 S-l. H-l and H-2 The tolerant callus cultures were characterized to determine the magnitude of sethoxydim and haloxyfop tolerance. Callus tissue from these lines was evaluated in experiments as described above in the characterization ofline 2167-9/2160- 154 S-2. Table II summarizes the results of these studies.
Line 2167-9/2160-154 S-l and Line 2167-9/2160-154 H-2 showed a four-fold increase in haloxyfop tolerance, while Line 2167-9/2160-154 H-l exhibited approximately a 60-fold increase in haloxyfop tolerance. Neither haloxyfop selected line showed a significant degree of sethoxydim tolerance, while the sethoxydim selected line S-l exhibited approximately a 100-fold increase in sethoxydim tolerance.
TABLE II
Herbicide Tolerance of Cell Lines S-1, H-l and H-2 Herbicide Cell Line Haloxyfop Sethoxydim 2167-9/2160-154 S-1 ~l 100 2167-9/2160-154 H-1 61 o lThe numbers represent the ~old increase in herbicide concentration that results in a 50~ reduction in growth of the selected cell lines compared to the unselected control cell line 2167-9/2160-154.

CA 022l7367 l997-lO-03 W O96/31609 . PCTrUS96/0 E. Herbicide Inhibition of Acetyl (Coenzyme A Carboxylase of Maize Cell Lines S-1~ H-l and H-2 Acetyl Coenzyme A Carboxylase (ACCase) was extracted from cell lines S-1, H-l and H-2 and assayed as described in detail for maize cell line 5 S-2, above. Table III below sllmm~ri7~c the results of these studies. The ACCase from line S-l was more tolerant of both sethoxydim and haloxyfop, while the ACCase from line H-1 was more tolerant of haloxyfop, but not of sethoxydim. The ACCase from line H-2 showed no difference from the unselected parent line 2167-9/2160-154 in sensitivity to either herbicide.
However, cell line H-2 exhibited approximately a five-fold h gher level of ACCase activity as compared to the unselected parent line 2167-9/2160-154. Thus, selection for sethoxydim or haloxyfop tolerance resulted in a less sensitive ACCase in cell line S-l and H-l, as well as a higher level of ACCase activity in cell line H-2.
TABLE III
Herbicide Inhibition of ACCase of Maize Cell Lines S-l, H-l and H-2 Herbicide Cell Line Haloxvfop Sethoxvdim lThe numbers represent the fold increase in herbicide concentration that inhibits ACCase activity of the selected cell lines by 50~ compared to the unselected parent cell line 2167-9/2160-154.

Deposit of Seeds Seeds from representative S-2 plants (Ex. III (B)) and H-1 plants (Ex. IV(B)) have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA on October 25,1988 and assigned accession numbers ATCC 40507~ and ATCC 40508. respectively.

CA 022l7367 l997-lO-03 W O96/31609 . PCTrUS96/04625 EXAMPLE V
Formation of cDNA Clones Encodin~ ACCase A.ACCase Purification 5The acetyl CoA carboxylase enzyme was isolated and purified from plant tissues and characterized. The purified enzyme was used to generate antibody reagents useful in identifying cDNA clones encoding the gene or portions of the gene for ACCase.
ACCase was extracted from frozen shoots of 7-d-old maize (Zea 10 Mavs L. inbred A619 or B73) see-llin~s grown in a growth chamber (24~C, 90%
RH, 16-h daylength at 210 ,uE m~~ s~'). The outermost leafand blade were removed and the rem~inder of the shoot was frozen in liquid N7. Embryos and endosperm tissue from developing kernels were harvested from field-grown ears at 36 to 40 days after pollination (DAP). Black Mexican Sweet corn (BMS) 15 maize suspension cells were obtained from cultures as previously described (W.B. Parker et al., Plant Physiol.. 92, 1220-1225 (1990)). Tissues were stored in liquid N~ until used.
Extraction and purification steps were performed at 0 to 4~C.
Crude extracts of leaf, bundle sheath strands, embryo, endosperm. and BMS
20 cells were prepared from frozen tissue as described by W.B.Parker et al., Proc.
Nat'l Acad. Sci. USA. 87, 7175-7179 (1990), except that extraction buffer contained 0.1 M Tricine-KOH, pH 8.3, 0.3 M glycerol, 5 mM DTT, 2mM
Na~EDTA, and 0.5 mM phenyl methonyl sulfonyl fluoride (PMSF). Triton X-100 (0.01% v/v) was added to bundle sheath strand extracts and to some whole 25 leaf extracts. For some experiments, additional protease inhibitors (leupeptin, 2 ,ug mL, pepstatin A, 100 ~Lg mL~'; benzamidine. 1 mM; ~-amino-n-caproic acid, 5 mM; and soybean trypsin inhibitor~ 10 ~g mL-I) were included. Filtered homogenates were centrifuged 20 minutes at 30,000g. A portion ofthe crude supern~t~nl fraction was immediately boiled 5 minutes in 1 volume of SDS
30 sample buffer (W.B. Parker et al., Plant Physiol.. 92, 1220-1225 (1990)) for SDS-PAGE analysis; the rem~incler was desalted on a 10-mL Sephadex G-25 column into extraction buffer minus PMSF.

W O 96/31609 . PCT~US96/04625 ACCase was purified from the crude extract supem~t~nt in four steps. This fraction was brought to 30% saturation with solid (NH4)7SO4, stirred15 mimltes, and centrifuged 20 minute at 20,000g. The sUpern~t~nt was then brought to 40% saturation with (NH4)2SO4 solution, stirred 30 minutes, and centrifuged. The pellet was dissolved in 5 mL extraction buffer, microfuged S minutcs, and the resulting sUpern~t~nt was applied to a Sephacryl S-300 gel filtration column (Ph~rrn~rizl 2.5 x 46 cm) equilibrated with S-300 buffer (0.1 M
Tricine-KOH, pH 8.3, 0.5 M glycerol, 0.5 mM DTT, 2 mM Na50 mM KCI). In later exp~"" ~çnts a Sephacryl S-400 column was used. Fractions (2.5 mL) were eluted at 0.75 mL min~l . ACCase activity eluted shortly after the void A780 peak (V0 = 75 mL). Active fractions were pooled, brought to 4.25 mM MgCl, (from a 0.5 M solution), and applied at 0.2 mL min-l to a Blue Sepharose CL-6B
(Ph~rrn~ 1.5 x 15 cm) equilibrated with Blue sepharose buffer (S-300 buffer cont~ining 4.25 mM MgCl7 and 10 mM NaHCO3). The column was washed overnight with 150 mL buffer (0.45 mL min~'). ACCase activity was then eluted with 50 mL buffer plus 10 mM ATP (0.45 mL min-'). Active fractions were pooled and applied to an FPLC Mono-Q HR 5/5 anion-exchange column (Phàrmacia) equilibrated with S-300 buffer minus KCl. The column was washed with 30 mL S-300 buffer minus KCl and then with a 48-mL. 0 to 500 mM KCl gradient in S-300~buffer (0.25 mL min~'). Fractions ( l n L) from the two peaks of ACCase acti~ ity were pooled separately All purification fractions were desalted into S-300 buffer and assayed for ACCase activity and protein.
ACCase was also analyzed from mesophyll chloroplasts and bundle sheath strands. Mesophyll chloroplasts from homogenates of 7- to 8-day-old see-llings that were kept in the darl~ 24 hours prior to harvesting were isolated on a linear Percoll gradient according to J.D. Burton et al., PesticideBiochemistrv and Physiology. 34, 76-85 (1989), except that buffers contained 0.6 M sorbitol and centrifugation g-forces were reduced by 25%. Intact chloroplasts were taken from the discrete lower green band present after Percollgradient centrifu~ation (G. Morioux et al., Plant Phvsiol.. 67, 470-473 (1981)).
Pelleted chloroplasts were lysed by resuspending them in ACCase extraction W O 96/31609 . PCTAUS~"0 buffer plus PMSF and 0.01 % (v~v) Triton X- 100. Bundle sheath strands were obtained from the original leaf homogenate material retained on a 70-,um filter after re-homogenizing the lc~r~ five times in a total of 2 L buffer. Triton - X-treated, desalted leaf, mesophyll chloroplast, and bundle sheath strand extracts 5 were assayed for activities of Rubisco (G. Zhu et al., Plant Physiol.. 97, 1348-1353 (1991)), NADP-dependent malate dehydrogenase (M.D. Hatch et al., Biochem. Biophys. Res. Commun..34, 589-593 (1969)), phosphoenolpyruvate carboxylase (R.C. Leegood et al., "Isolation of Membranes and Organelles from Plant Cells," Academic Press, New York, 185-210( 1983)), ç~tz~ e (Worthington Biochemicals, 1972), and fumarase (R.L. Hill et al., Methods Enzymol.~ 13, 91-99 (1969)), and for total chlorophyll (D.E. Arnon, Plant Physiol., 24, 1-5 (1949)). Mesophyll chloroplast pl~aldLions were judged to be relatively free of cont~minzltion by bundle sheath chloroplasts because they contained 3-fold greater NADP-dependent malate dehydrogenase and one-tenth as much Rubisco activity (mg~l chlorophyll) than bundle sheath strand extracts.
Mesophyll chloroplast ~,ep~dlions also contained ~2.6% as much c~t~ e, fumarase, and phosphoenolpyruvate carboxylase activities (mg-' chlorophyll) as did whole-leaf extracts, indicating they were relatively free of peroxisomal, mitochondrial, or cytoplasmic components.
ACCase activity as measured by acetyl-CoA-dependent H'4CO3-(ICN. 2.07 GBq mmol~') incorporation into acid-stable product previously shown to be malonyl-CoA (J.D. Burton et al., Pesticide Biochemistry and Physiology.
34, 76-85 (1989)). Assays of desalted purification fractions or crude, desalted tissue extracts contained up to 50 and 250io (v/v) enzyme, respectively. In someexperiments methylcrotonyl-CoA or propionyl-CoA were substituted for acetyl-CoA (E.S. Wurtele et al., Archives of Biochemistrv and Biophysics. 278, 179-186 (1990)). Avidin (10 U mL~I) was included in some assays. Herbicide inhibition assays contained 1 % (v/v) ethanol plus or minus 1 ,uM haloxyfop (2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy] propanoic acid, Dow Chemical Co. analytical grade racemic mixture) or 10 !lM sethoxydim (2-[1 [(ethoxylmino)butyl]-5-[2-(ethylthio)-propyl]-3-hydroxy-2-cyclohexene - 1 -W O96/31609 PCTrUS96/04625 one, Li salt, BASF Corp. technical grade). Data are means plus standard error ofthree assays.
Protein concentrations were determined in duplicate with the Bio-Rad Coomassie blue dye-binding assay as described by the m~nllfz~ctllrer, using 5 BSA as the standard.
Centrifuged crude extracts and proteins in purification fractions and immunoprecipitation supern~tzlnt~ were separated by SDS-PAGE in 6 or 7.5% gels as previously described (W.B. Parker et al., Plant Physiol.. 92, 1220-1225 (1990)). Purification fractions were precipitated in 10% (v/v) TCA, washed with 80% (v/v) acetone, and air-dried 10 minlltes prior to eleckophoresis. Proteins in gels were stained with silver (J. Heukeshoven et al., Electrophoresis. 6, 103-112 (1985)). High molecular weight protein standards for SDS-PAGE (Pharmacia) were used to estimate polypeptide masses.
The four-step purification procedure shown in Table IV typically yielded 30 to 190 ,ug of highly purified ACCase from 50 grams (fresh weight) of maize inbred A619 or B73 see~linp; leaves. ACCase activity in the crude supern~t~nt fraction precipitated between 30 and 40% saturation with (NH~)7SO4, which appeared to increase total ACCase activity approximately 38%. Crude extract components might have depressed the reaction rate shown in Table IV
because the assay mixture contained 50% enzyme (v/v). In tests of fractions from another purification, enzyme velocity was proportional to enzyme concentration in assay mixtures cont~ining up to 25% (v/v) crude extract. but 50% (v/v) mixtures were not tested. ACCase activity eluted from the Sephacryl S-300 gel filtration column slightly after the green void peak. Approximately 56% of the S-300 fraction ACCase activity was recovered from the Blue Sepharose column, primarily in the initial ATP-cont~ining fractions (12.5 mL).
Both 10 mM NaHCO3 and 4.25 mM MgCl7 (1- and 0.85-fold standard assay concentrations, respectively) were included in the Blue Sepharose buffer becausethey improved the total and specific ACCase activity rem~inin~ after batch absorption to Blue Sepharose beads, elution with ATP, and desalting into extraction buffer minus PMSF. Neither NaHCO3 nor MgCl7 improved enzyme W O96/31609 PCTrUS96/0 stability of crude extracts. Mono-Q anion-exchange chromatography resulted in separation of two ACCase activity peaks which eluted at approximately 210 mM
(~lçsj~n~t~l ACCase II) and 250 mM KCI (~lesign~te~l ACCase I), as previously observed for a hybrid maize variety (J.L. Howard et al., FEBS Lett.. 261, 5 261-264 (1990)). ACCase I comprised about 85% ofthe total activity recovered from the column (29% of the original crude extract activity) and had high specific activity (Table IV). The specific activity of ACCase II was less than 30% that of ACCase I. Both activities were inhibited >90% by avidin, as previously reported (J.L. Howard et al., FEBS Lett.~ 261, 261-264 (1990)). The 10 mass of native ACCase I was çstim~tecl to be approximately 490 kD by gel filtration on Superose 6.

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W O 96/31609 PCTrUS96/04625 B. Formation and Specificity of Antibodie~ to AC~ase Antibodies are sensitive reagents that allow for the identification of gene products from cDNA and other cloned genes. Antibodies to purified ACCase were prepared and used to screen for cDNA clones encoding all or a 5 portion of a gene for ACCase.
Antiserum to maize ACCase was obtained by immunizing a female New 7e~1~n~1 White rabbit (Egli et al., Plant Physiol.. 101, 499 (1993)).An intramuscular injection of 100 ,ug of Mono-Q-purified, SDS-denatured ACCase I in Freund's complete adjuvant was followed by subcutaneous 10 injections of 20 to 100 ,ug of gel-purified ACCase I polypeptide in acrylamide plus incomplete adjuvant every 4 to 6 weeks, for a total of six injections. Serum was stored at -20~C in 0.02% (w/v) NaN3.
For Western blots, proteins in SDS gels were electrophoretically transferred to Immobilon (W.B. Parker et al., Plant Phvsiol.~ 92, 1220-1225 15 (1990)) for 1 hour at 20 V in a Bio-Rad Transphor semi-dry blotter and then stained with Ponceau S (E. Harlow et al., "Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1988)).
Destained blots were blocked with Tris-buffered saline plus 0.5% (v/v) Tween-20 (Bio-Rad). and 10% (w/v) bovine serum (for antiserum blots only). ACCase 20 and biotinylated proteins were detected with immune serum (1/10.000) plus goat anti-rabbit I~G-alkaline phosphatase conjugate or with avidin-~lk:~line phosphatase (W.B. Parker et al., Plant Physiol.. 92, 1220-1225 (1990)). Blots were repeated at least three times.
For immunoprecipitations, equal ACCase activities (0.58 nmol 25 min~1) in crude extracts were desalted into S-300 buffer cont~inin?~ 0.1 M KCl and incubated 1 hour at 25~C with 16 ~LL buffer or with 16 ~lL serum con~i~ting of 0 to 100% ACCase antiserum in ~ hlllllune serum. Immune complexes were incubated 1 hour at 25 ~C with a 2-fold (IgG binding) excess of Protein A-a,~arose and then microfuged S min~ltes to obtain immunoprecipitation 30 supernatant fractions. ACCase activity of supernatants was expressed as a W 096/31609 . PCT~US9~'0162 percent of the 100% plei., -,--llne serum control. Data are means plus SE of three replicate assays for each of two sets of extracts.
Western blots and silver-stained gels of purification fractions separated by 7.5% SDS-PAGE showed that neither ACCase I nor ACCase II
S Mono-Q fractions contained biotinylated polypeptides smaller than 212 kD. A
polypeptide > 212 kD was the primary protein component of the ACCase I
Mono-Q fragment (Fig. 5). The ACCase II fraction contained a biotinylated polypeptide > 212 kD and a large amount of a 55 kD non-biotinylated polypeptide. Fractions from earlier purification steps contained additional 10 biotinylated proteins of apprc-~imz~1ely 74, 75, and 125 kD (Fig. 5).
To better compare the biotinylated polypeptides > 212 kD in ACCase fractions I and II, we used 6% SDS-PAGE, which showed that the mass of ACCase II was approximately 8 kD less than that of ACCase I. Molecular masses were estim~te-l to be 219 kD (ACCase II) and 227 kD (ACCase I). based 15 on comparisons with polypeptide standards and the observation (N.R. Palosaari, Plant Physiol.. 99(S), 359 (1992)) that, on Phastgels (Ph~rm~ ), ACCase I
polypeptide was slightly smaller than dodecameric horse spleen ferritin (238 kD;M. Heu~,L~ ule et al., FEBS Lett.. 129, 322-327 (1981)). All purification fractions through the Blue Sepharose step contained both ACCase I and II
20 polypeptides. Rapid extraction of leaves in buffer cont~ining five additionalprotease inhibitors, or a 4 hour incubation of extracts at 25 ~C, had little or no effect on the relative amounts of the two polypeptides, suggesting that ACCase II is not a breakdown product of ACCase I.
Antiserum to ACCase I strongly recognized the ACCase I
25 polypeptide in crude extracts and showed little or no recognition of ACCase II
polypeptides. No bands were recognized by preimmune serurn. ~ ming that avidin binds similarly to ACCase I and II polypeptides, it appears that the amount of ACCase II on the Western blot was slightly less than the amount of ACCase I. However, the relative staining with antibody compared to avidin 30 indicated that the antibody had significantly less affinity for ACCase II than ACCase I.

W O 96/31609 . PCTrUS~G~
49 . .
To determine whether the same ACCase polypeptides were expressed in diL~ GllL maize cell types, proteins in mesophyll chloroplasts and crude extracts of leaves, endosperm tissue, embryos, and BMS cells were separated by SDS-PAGE. All ~l~al~lions cont~ined a predominant biotinylated polypeptide of approximately 227 kD (ACCase I) that was strongly recognized by ACCase arltiserum or avidin. Similar 227 kD band densities were observed when gel lanes were probed with either avidin or ACCase antiserum. The 219 kD ACCase II polypeptide was readily detected in leaves only by avidin binding, but was in low ablln-l~nce or not detected in extracts from other tissues.
Only the 227 kD ACCase I polypeptide was detected in purified mesophyll chloroplasts, however, suggesting that the 219 kD ACCase II polypeptide i:, localized elsewhere in mesophyll cells or in other cell types of young leaves.
ACCase activity and a > 212 kD biotinylated polypeptide(s) were also found in bundle sheath strand extracts, but low yields prevented us from detP~nining the type of ACCase present. Two other major biotinylated polypeptides of 75 and 74 kD were found in all tissues. Other non-biotinylated proteins of 66 kD (faint) and 55 kD were also recognized by ACCase antiserum. The 55 kD polypeptide was only found in leaves; it was also present in both ACCase I and II Mono Q
fractions (Fig. 5) and was identified as the Rubisco large subunit based on its comigration with protein immunoprecipitated by spinach Rubisco antiserum.
ACCase antiserum immunoprecipitated at least 75% of ACCase activity from crude, desalted extracts of leaves, endosperm tissue~ embryos, andBMS cells (Fig. 6), indicating that most of the ACCase activity in these tissues is immunologically related to the ACCase I polypeptide of leaves. Less activity was precipitated from leaves (75%) than from other tissues, particularly embryos(98%). Compared to immunoprecipitation, inhibition of ACCase activity by antiserum in solution was less than 20% as effective in reducing ACCase activity.
7 The substrate specificity of ACCase from different purification 30 fractions was examined to compare ['4C]HCO3- incorporation in the presence ofdifferent acyl-CoA substrates. Both ACCase I and II utilized propionyl-Co-A 40 W O96/31609 . PCT~US96/04625 to 50% as rapidly as acetyl-CoA at 50 to 500 ,uM substrate even though they contained no biotinylated polypeptides (~igure 5) the size of known propionyl CoA carboxylases (70 to 75 kD, see E.S. Wurtele et al., Archives of ~iochemistry and Biophysics. 278, 179-186 (1990)). Activities in the presence 5 of both acetyl-CoA and propionyl-CoA (250 or 500 ~LM each) were approximately 90 (ACCase I) to 130% (ACCase II) that of 500 ,uM acetyl-CoA
alone. Crude leaf extracts utilized propionyl-CoA and methylcrotonyl-CoA 60%
as efficiently as acetyl-CoA. Methylcrotonyl CoA carboxylase activity was reduced 85% by gel filtration and was completely removed by Blue Sepharose 10 affinity chromatography.
ACCase I and II differed significantly in their inhibition by either haloxyfop or sethoxydim (Fig. 7). Acetyl-CoA or propionyl-CoA-dependent Hl4C03- incorporation by ACCase I was strongly inhibited (65 to 80%) by 1 ,uM
haloxyfop or 10 ~LM sethoxydim, while ACCase II activity was inhibited less 15 than 50% for all herbicide/substrate combinations examined.
C. Cloning and Identification of Maize cDNA Clones Encoding ACCase Maize cDNA clones encoding a portion of the ACCase game were identified by screening a DNA library generated from maize. The cDNA
clones were used to identify the sequence of the ACCase gene and to identify the20 genomic DNA fragments encoding the gene or genes for ACCase.
A ~ gtl 1 cDNA library from maize inbred A188 see-llings was prepared by standard method for oligo-dT priming, as described for pea cDNA.
(Gantt and Key, Eur. J. Biochem.. 166:119-125 (1987). Plaque lifts ofthe maize cDNA library were screened with maize ACCase antiserum (Egli et al., Plant 25 Phvsiol.. 101, 499 (1993)) to identify plaques expressing ACCase-like proteins.
as described by Sambrook et al., cited supra. (1989). The initial screen of 800,000 plaques yielded 120 positives. Rescreening and plaque purification reduced the number of positives to 14. All 14 clones bound ACCase antibodies that, when eluted from plaque lifts (J. Hammarback et al., J. Biol. Chem..
30 265:12763 (1990)), recognized a 227-kD biotinylated polypeptide on SDS-PAGE western blots of embryo and leaf crude extracts. The strongest western W O~6/31609 . - PCT/Ub~C~'~1625 blot reaction was obtained with cDNA clone # 15- 14. The six best clones were digested with EcoRI to excise maize cDNA inserts. Total insert sizes ranged from 1.2 to 5.1 kb indicating the clones most likely did not contain the full coding sequences for the mature 219-kD and 227-kD ACCase polypeptides (mi-,i-,--l--l ç~tim~tes of 6.1 and 6.3 kb, respectively).
(~lQE~tl 5-14 ~r.tain~d three EcoRI i~l~mer;t~ of 2.0, 1.~ ~ld 0.23 kb shown in Figure 8. Southern blots showed that the 1.2 and 2.0-kb fr~ment~ of clone #15- 14 each hybridized to ~irre~ fragments in the other five clones, with the exception of clone #4-4 which only contained a 1.2-kb fragment. The six maize cDNA clones contained EcoRI fragments that hybridized to a large transcript (ca. 7.8 kb) on Northern blots of total RNA from maize leaves, embryos and endosperm (Fig.9). BMS cell culture RNA also contained a 7.8 kb transcript, but the hybridization signal is not evident on this exposure (Fig. 9). The relative abundance of the 7.8-kb transcript in embryos was higher than the other sources which is consistent with their ACCase activity.
The three EcoRI fragments were subcloned from cDNA clone #15-14 into BlueScript vector and sequenced by the dideoxy chain termination method (Sequenase 2.0 USB) initially using T3 and T7 primers and then oligonucleotide primers based on insert sequence. A clone #16-6 was also sequenced in a similar manner. Clone #16-6 included three EcoRI fragments of 3.1 kb, 1.2 kb, and 0.23 kb and had additional sequence located upstream from that of clone # 15- 14. After comparing the sequence and determining that the sequence was the same, the additional 1.2 kb sequence at the 5' end was sequenced.
Clone #18-5 was sequenced in a similar manner. Clone #18-5 included 3.9 kb, 1.2 kb, and 0.23 kb EcoRI fragments and contains an additional 1.9 kb 5' sequence ~L.ealll from clone #15-14. Subclone #18-5I (3.9 kb EcoRI
fragment) has been deposited with the American Type Culture Collection and given Accession No. 69236.
GenBank, PIR-29, and Swiss-Prot 19 data banks have been searched for amino acid homology with the corresponding amino acid sequences CA 022l7367 l997-lO-03 W O96~1609 . PCTrUS~6/Ol'~

of the three subclones of clone #18-5. Peptide sequences corresponding to the maize cDNA subclones had higher similarity to chicken, rat, and yeast ACCases than to any other peptide sequence in the data banks. Figure 8 illu~ ~es the relative org~ni7~tion of the 3.9, 1.2 and 0.23-kb EcoRI fragments of clone #18-55 and their co-linearity and extent of amino acid identity with chicken ACCase cDNA sequence. This comparison shows that the maize clone #18-5 has a large region near the 3' end with high amino acid identity (40 to 61%) to chicken ACCase, a longer region with 23% identity in the middle of the 3.9-kb sequence, and a short region with 52% identify near the 5' of the 3.9 kb sequence.
Portions ofthe sequence ofthe #18-SI subclone have been identified as encoding domains of the ACCase enzyme of functional significance. Those functional regions include a fragment that spans the presumed transcarboxylase active site in the enzyme having the following presumed sequence SEQ ID NO: 2:

GTT CCT GCA AAC ATT GGT GGA CCT CTT CCT ATT ACC A~A CCT CTG GAC
CCT CCA GAC AGA CCT GTT GCT TAC ATC CCT GAG AAC ACA TGC GAT CCA
CGT GCA GCT ATC TGT GGT GTA GAT GAC AGC CAA GGG AAA TGG TTG GGT
20 GGT ATG TTT GAC A~A GAC AGC TTT GTG GAG ACA TTT GAA GGA TGG GCA
A~A ACA GTG GTT ACT GGC AGA GCA AAG CTT GGA GGA ATT CCT GTG GGC
GTC ATA GCT GTG GAG ACA

This functional domain is contained in the sequence 1112 to 856 base pair from 25 the 3' stop codon or carboxy terminus region of the ACCase coding sequence ofmaize. This transcarboxylase active sequence is also present in clone #15-14.
Another functional region that has been identified spans the 12 base pair sequence encoding the biotin binding site having the following peptide sequence SEQ ID NO: 3:

5' GTT ATG AAG ATG 3' Val Met Lys Met W O~6/31609 . PCTrUS96/04625 The biotin binding site is encoded approximately 30% in from the 5' (N-terminus) end of rat, chicken and yeast ACCase genes. These functional domains are useful in mapping and further identifying other cDNA and/or genomic frzlgm~nt~ encoding ACCase genes.
The cDNA clones encoding portions of the acetyl CoA
carboxylase genes are useful to identify the sequence of the gene or genes and are useful as probes to locate the genomic copies of the gene or genes. Because the ACCase antibodies used to screen the ~ gtl l library recognize both the 219 and 227 kD ACCase polypeptides, it has not been determined which polypeptide 10 is encoded by these less than full length clones. It is likely that the majorit~ of the clones encode the 227 kD polypeptide since that polypeptide is more abundant in the leaf tissue source of the DNA library and the antibodies have a higher affinity for the 227 kD ACCase polypeptide.
EXAMPLE VI
Isola~ion and Sequencin~ of Genomic Encoded ACCase Genes and a Coml)lete cDN~ Seq-len~e of a M2ize ACCase Gene The maize genome has been analyzed to identify copy number and location of the genomic copies of ACCase gene or genes. Four distinct types of maize ACCase genomic clones have been identified, termed A1, A2, B 1 and B2 (see below).
To obtain genomic copies of ACCase genes, a maize B73 genomic library (Clontech, Palo Alto, CA) was screened with the 2 kb subclone from #15-14 and several clones of about 15 kb were identified as having homology to the ACCase cDNA. Restriction mapping and partial sequence analysis revealed two types of genomic clones (Type A and Type B) that differed in restriction sites and in their position relative to the ACCase partial cDNA
sequence as shown in Figure 8.
The 2.~ kb EcoRI-SaII fragment (#16) from the Type A genomic clone and the 3.0 kb EcoRI-EcoRI fragment (#34) from the Type B genomic clone were shown to hybridize to the 3.9 kb probe from #18-5 and were subcloned into the Bluescript vector and sequenced. Approximately 1.5 kb of DNA sequence from the genomic Type A 2.5 kb fragment were 100% identical W O96/31609 . PCT/U~,G~OIC25 to coding sequence from the 3.9 kb cDNA subclone #1 8-5I described in Example V, the rem~ining sequence exhibited no identity with the cDNA clone and presumably represents a noncoding intron sequence. A 350 nucleotide sequence derived from the genomic Type B 3.0 kb fragment was about 95% identical to 5 the cDNA clone inf1ic~ting that its coding sequence differs from that of genomic Type A. These results also indicate that the maize genome encodes at least two different genes encoding a polypeptide having acetyl CoA carboxylase activity.
To identify and clone the rem~in~ler of the gene representing the amino-terminll~ of maize ACCase, additional regions from the Type A genomic 10 clone have been subcloned and partly sequenced. The 3.5 kb HindIII-HindIII
fragment (#28) has been sequenced for about 400 nucleotides from each end.
The 3' end of #28 shows significant homology to the amino acid sequence from the chicken sequence located about 0.5 kb from the start of the chicken gene.
The complete sequence for fragment #28 can be obtained and 15 analyzed to determine whether it contains the 5' end of the ACCase coding region. The start of the transcribed region, and thus the likely start of the coding region for ACCase, can be identified by using the genomic clones in RNAse protection analysis (J. Sambrook et al., "Molecular Cloning - A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)).
20 Based on sequence data from the genomic clone~ alignment, as shown in Figure 8, with sequences of other ACCases and identification of potential open reading frames, oligonucleotide primers can be constructed to synthesize cDNA
molecules representing the amino t~rminll~ of the ACCase gene. These molecules can be hybridized to genomic Type A DNA fragments such as #28 25 and the nonhybridizing portions digested with S 1 nuclease. The end of the protected fragment are ~let~rmined by analysis on a DNA sequencing gel.
To synthesize the remslining coding region between the end of the cDNA clone #18-5 and the start of transcription, two oligonucleotide primers were synthesized. Primer 1 is complementary to the DNA sequence: (SEQ ID
30 NO:4) 5' GCCAGATTCC ACCAAAGCAT ATATCC 3' W O96/31609 . PCTrUS_~'01'~

near the 5' end of cDNA subclone #18-5I and can be used as a primer for synthesis of cDNA molecules from maize see~11ing, leaf or embryo RNA.
A primer corresponding to a DNA sequence near the transcription - start site can be used in combination with primer 1 for the amplification of DNA
5 by the polymerase chain reaction (PCR). Several independent clones are then sequenced and their sequences compared to the known sequence of the Type A
genomic clone to determine the exact coding sequence corresponding to that maize gene for ACCase. A similar strategy can be used to obtain the complete coding sequence for genomic Type B ACCase.
The rem~ining cDNA sequence was obtained by three successive rounds of RT-PCR using oligonucleotide primers based on genomic a~pa~ exon (5') and known cDNA (3') sequences. The primers used to amplify nucleotides 1 -240 of the cDNA were 28sst-a5+ (SEQ ID NO. 7) and 28sst-6at3+ (SEQ ID NO:8), nucleotides 217-610 ofthe cDNA were 28sst-5+
15 (SEQ ID NO:9) and 28-2t3+ (SEQ ID NO: 10), and nucleotides 537-2094 of the cDNA were ACCPCR5' (SEQ ID NO:l l) and I55- (SEQ ID NO:4) (Table V).
PCRproducts corresponding to nucleotides 1-240, 217-610? and 537-2094 ofthe final sequence were cloned into PCR-script (Stratagene).

W O 96~1609 . PCTrUS96/04625 _ 56 ~

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The original 5.4-kb cDNA clone # 18-5 and PCR products from at least three individual PCR per oligonucleotide pair were sequenced in both directions by the dideoxy chain-t~?rmin~tion method, using either Sequenase II
(U.S. Biochemicals) or ABI 373 (Applied Biosystems, Inc.) protocols. No 5 sequence differences were found in regions of clone overlaps. The complete e~quer.~{~ft.~ c~NA of ~naize ACCase (nucl~otides 1-7~70 SEQ ID NO:5~and its corresponding amino acid sequence (amino acids 1-2325 SEQ ID NO:6) are shown in Figures 13 and 14. The 7470 bp cDNA includes a 459 nucleotide 3' untr~n.cl~te-l region and 36 nucleotides of 5' untranslated sequences.
The first Met codon in the cDNA (nucleotides 37-39) was identified as the start codon based on its similarity to consensus initiation sequences (Kozak, J. Cell. Biol.. 108, 229 (1989), Lutcke et al., Embo. J.~ 6, 43 (1987)). An in-frame stop was found in the genomic sequence 6 nucleotides upstream of the sequenced cDNA, and RT-PCR analysis of this region suggested 15 that the in frame stop codon was also present in the cDNA. The 3' end of the coding sequence was defined by two stop codons present in the large open reading frame after nucleotide 7011. The translated coding sequence predicted a polypeptide of 2325 amino acids (257 kD; SEQ ID NO:6) which was 79 to 81 %
identical to the multifunctional (MF) ACCases from alfalfa (Shorrosh et al., 20 Proc. Nat'l. Acad. Sci., 91, 4323 (1994)) and wheat (Gornicki et al., Proc. Nat'l Acad. Sci.. 91, 6860 (1994)), and to a 118-amino acid predicted polypeptide of arice e.Ypressed sequence tag (Genbank accession # D39099, T. Sasaki), but only 53 to 55% identical to ACCase from other eukaryotes.
In a pileup alignment of plant ACCases (Genetics Computer 25 Group. Madison, Wisconsin), Met 1 of both maize and Brassica napus ACCases was located about 130 amino acids upstream of the conserved sequence VDEFCKALGG, compared to only 25 amino acids upstream for other plant ACCases. The predicted 2325 amino acids of maize ACCase contains a biotinylation site at position 806, within the conserved MKM motif (Ton et al., 30 Eur. J. Biochem.~ 215, 687 (1993)). The arrangement and amino acid sequence of binding sites (Shorrosh et al., Proc. Nat'l. Acad. Sci.. 91, 4323 (1994)) for CA 022l7367 l997-lO-03 W O96/31609 . PCTIU~,-'01C25 58 ATP (amino acids 318-333), biotin (amino acids 799-811, biotin at 806), acetyl-CoA (amino acids 1952-1961), and carboxybiotin (amino acids 1662-1711) were highly conserved among all MF ACCases.
EXAMPLE VII
S Chara~le. ~alion of other Genomic Clones The initial restriction fragment length polymorphism (RFLP) analysis of EcoRI-digested total DNA from three maize inbred lines showed one band when probed with the 2 kb subclone from #15-14 (internal to gene) and two bands when probed with the 1.2 kb subclone (near the 3' end of the gene).
Fragments homologous to the 2 kb probe were monomorphic and the more intense of the two bands hybridizing with the 1.2 kb probe was dimorphic. As discussed in Example V, these results support the view that maize contains at least two distinguishable ACCase genes and that they may be quite similar for much of the coding region. Additional genomic Southern blots of a set of recombinant inbred lines were used to map polymorphisms for the ACCase probes to maize chromosomes. One polymorphism was mapped to the short arm of chromosome 2; other polymorphisms were not evident in these initial tests to identify a chromosomal location for other maize ACCase genes.
The isolation and restriction mapping of additional genomic clones from a B73 genomic library (Clontech) resulted in the identification of four different types of clones termed Al. A2, B I and B2 (Figures 16- 19) which had 96% nucleotide sequence identity. Types A and B correspond to previously published pA3 and pA4 cDNAs (Ashton et al., Plant Mol. Biol.~ 24, 35 (1994)) and differ from pA3 and pA4 by ~ 4% in their coding sequences.
Type A and B genomic clones have linear sequence homology except for an insertion in an intron of the Type B genes about 1400 bp 3 ' of the Al(SEQ ID NO:5) translation start site. Analysis ofthe insert boundaries revealed a 3-bp target site duplication and a 6-bp direct repeat, and further sequence analysis showed the presence of two new and unique LINE elements (Long Interspersed Nuclear Elements) in Bl and B2. ~mm~ n LINE
elements are highly abundant (10~ to 105 copies), 6 to 7 kb long~ and have W O 96/31609 . PCTrUS~-'01'~

frequent 5'-end deletions and an A-rich 3' terrninll~ They are fl~nkerl by shortdirect repeats, and contain two ORFs, one encoding a reverse transcriptase.
Three LINE elements (Cin4, 50-100 copies in maize; del2, 250,000 copies in - lily; BNRl, 2-5% of genome in sugarbeet) have been described in plants (Leeton 5 et al., Mol. Gen. Geneti~ Z, 97 (1993), Schmidt et al., Chromo. Res.~ 3, 335 (1995); Schwarz-Sommer et al., EMBO J.~ 6, 3873 (1987)). Maize ACCase B1 has one unique LINE element and B2 has two. The two B2 LINE elements were characterized by differences in their reverse transcriptase sequence. The B
genomic clone inserts have characteristic LINE features including cysteine motifs and a possible polyA tail, and high abundance. The LINE insert also has been found in an intron of the maize Shrunken-2 gene (Hannah et al., Plant Phvsiol.~ 98, 1~14 (1992)).
The partial nucleotide sequence (3489 nucleotide) of a Type Al ACCase genomic clone is shown in Figure 16 (SEQ ID NO:12). The clone is a HindIII fragment which includes nucleotides 1-931 of the cDNA in Figure 13 (SEQ ID NO:5), and the first four introns within the coding region, at positions240 (460 nucleotides), 296 (480 nucleotides), and 872 (76 nucleotides) of SEQ
ID NO:5. The clone also has 1395 nucleotides 5' to the cDNA of SEQ ID NO:5 (i.e., 1431 nucleotides 5' ofthe translational start at nucleotide position 1432).
The partial nucleotide sequence ( 13 '8 nucleotide) of another Type A clone is shown in Figure 17 (SEQ ID NO: 13) The partial sequence is all 5' untr~n~ .od sequence and contains a 7 base insert between nucleotides 279-290, but is otherwise identical to SEQ ID NO: 12.
The partial nucleotide sequence of six Type A2 clones is shown in Figure 18 (1565, 1168, 638, 558, 976 and 852 nucleotides, SEQ ID NOs 14, 15, 16, 17, 18 and 19, respectively).

W O 96/31609 . PCTÇUS96/04625 Within the A1-A2 clone pair, identified differences are in introns and 5 ' UTR sequences. The A2 genomic clone is weakly amplified with Type A1 PCR primers specific for the 5' UTR if the 3 ' primer employed is for a conserved amino acid sequence found in all ACCases (e.g., 28sst-l lOF, ACTGTGCGTTTGAGAAGGTC, SEQ ID NO: 23, and 28sst-2T3+, CCTCTACGTAATTGGTCAGC, SEQ ID NO:24). The A2 amplified product is the same size as that from the Type Al genomic clone, and restriction analysis indicated a difference in sequence from Al. Sequence differences in the 5' region should provide a means to distinguish between expression of A1 and A2 10 ACCase genes and to determine whether A2 also encodes a CTP.
The partial nucleotide sequence (231, 207 and 180 nucleotides) of three Type B clones is shown in Figure 19 (SEQ ID NOs 20, 21 and 22, respectively).
The cDNAs corresponding to genomic clones A2, B 1 and B2 are 15 cloned and sequenced in a manner similar to that described above. The derivedamino acid sequences are aligned with known ACCase sequences. If putative CTP sequences are identified, functionality is tested as described below. Also if the tissue specificity and developmental timing of expression differ for different ACCase genes. the sequences of the promoter regions of the corresponding 20 genomic clones are compared. Gene-specific probes for specific ACCase genes can provide more information on their roles in lipid synthesis (plastid and cytoplasmic isoforms), secondary metabolism (cytoplasmic isoforms), and herbicide resistance (likely plastid isoforms).
A 3' Type Al ACCase cDNA probe mapped to chromosome 2S
25 (Egli et al., Maize Genetics Newsletter~ 68, 92 (1994)) and IO lOL (Caffrey et al., Maize Gen Coop.~ ~, 3 (1995)). Two 5' Type Al cDNA probes which span the transit peptide mapped to chromosome 2S in the same location as the 3 ' probe (see maize genetic map, 1996 version, Maize Genomic Database). PCR primers 28sst-97F (C~''l-l''l"l-l'ATGGCACTGTGCG, SEQ ID NO:25) and 28sst-6t3+;
30 (CATCGTAGCCTATATGAGGACG, SEQ ID NO:26) located in non-coding regions of A 1 that span the chloroplast transit peptide were used to amplify a W O96/31609 . PCTrUS96/04625 B73 chromosome-specific product which segregated with the resict~nce trait. A
nearby 5' primer (28sst-aS+, SEQ ID NO:7) amplified all genotypes and functioned as a positive conkol. Herbicide resistance due to the Accl-S3 mutation segregates (29/29 individuals to date) with production of a Type A 5 ' 5 end-specific PCR product derived from the mutant parent while herbicide sensitive plants lack the transit sequence (15/17 progeny). Two individual plants which contained B73-specific DNA at this location died of unknown causes while grown in the presence of herbicide.
Mutations in maize that confer recict~nce to cyclohexanedione 10 and aryloxyphenoxypropionate herbicides by means of an altered ACCase target are found at two non-allelic loci, Accl and Acc2. Al and A2 appear to encode plastidic ACCases and correspond to the Accl and Acc2 herbicide resistance loci. Acc2 has been mapped to 10 L (VanDee, M.S. Thesis, University of Minnesota (1994)). Accl is the site of five allelic mutations including Accl-S2 15 and -S3 (Marshall et al., Theor. Appl. Genet.. 83,435 (1992)), and has been mapped to chromosome 2.
Only one plastidic ACCase polypeptide was identified by SDS-PAGE of maize leaf extracts, although 2-D gel analyses might provide evidence for a second. highly similar isoform. Of the two ACCase isoforms, only 20 ACCase I shows altered herbicide inhibition in Acc1-.~2 mutants~ and most of the ACCase activity in leaves and developing embryos is herbicide-resistant and thus attributed to the Accl-S2 gene product.
Although a 3 ' ACCase probe has been mapped both to 1 OL near Acc2-S5 and to 2S, the conserved sequence of ACCase genes and lack of 25 polymorphism in multiple bands complicates identification of genes encoded atthese loci. The Type Al ACCase gene is probably located on chromosome 2, since (i) 5' untr~n~l~ted and chloroplast transit peptide probes from Type Al hybridize to two bands (dark and light) in maize inbreds, and (ii) analysis of maize-oat addition lines carrying maize chromosomes 2 through 9 indicates the 30 dark band is on chromosome 2 and the light band is on chromosome 1 or 10.

W O96/31609 . PCTrUS96/04625 Type B ACCase genes are likely to encode cytosolic isoforms.
Given that cytosolic malonyl-CoA is a precursor in the synthesis of many secondary metabolites including flavonoids (e.g. maysin, a corn silk component associated with corn ear~,vorm resistance), these cytosolic ACCases can have agronomic utility.
Northern blot analysis of total maize RNA with an ACCase probe (nucleotides 3400-5932) showed a single 8.3 kilobase band. To determine whether the ~ es~,ion of ACCase RNAs was developmentally regulated, blots of total RNA from 16 to 42 DAP (days after pollination) embryos were probed with an ACCase cDNA fragment. Transcript abundance peaked about 23 DAP
and the steady state pattern was similar to in vitro ACCase enzyme activities and protein measured from developing embryos. Type A- and B- specific 3'P-CTP-labeled antisense transcripts were 780 nt long (662 nt of ACCase sequence ~ 118 nt of vector/~lollloter sequence) and were identical except for 15 base mi~m~tches scattered along their length. Each antisense transcript was hybridized to total RNA from embryos at 16, 20, 23, and 42 DAP and digested with RNAse A/TI mixture to yield a 662-base fragment specific to the probe used. The results showed that the Type A transcript was more abundant than Type B at all tested stages, and that only Type A remained hi h in older embryos. Types A and B had similar expression patterns and peaked around 20-23 DAP. The ratio of Type A:B mRNA in leaves was about 2: 1, similar to its relative abundance in cDNA expression libraries.
EXAMPLE VIII
Expression of the Maize ACCase Chloroplast Transit Peptide The N-terminus of the predicted maize ACCase polypeptide is longer than that of predicted cytosolic ACCase isoforms and has several characteristics typical of chloroplast transit peptides within the first approximately 73 amino acids of the predicted N-t~rmin~l sequence. The CTP
cleavage site motif is not found in the putative maize ACCase CTP, although only about 30% of known CTPs contain this consensus sequence (Gavel and von Hejne, FEBS Lett.. 261, 455 (1990)). However, the maize ACCase N-tellllhlus W O 96/31609 . PCT~US96/04625 appears to have several other pLO~)~l lies typical of known CTPs: (1 ) a lack ofacidic residues in amino acids 1-49, (2) high Ser + Thr content (69% within amino acid residues 23-35),(3) an R-rich region between S- and D-rich regions - in amino acid residues 36-49, and (4) a predicted turn -,~ sheet within amino 5 acid residues 58-73 (von Hejne and Nishikawa, FEBS Lett.. 278,1(1991)).
The ability of the amino acid sequence contained within the N-terminal 100 amino acids of the tr~n.~ tecl maize acetyl-CoA carboxylase (ACCase) cDNA to direct the N-terrninzll portion of the maize ACCase biotin carboxylase domain into chloroplasts was tested in vitro by methods used 10 extensively in the liL~ldlul~ (see Cline et al., J. Biol. Chem.. 260,3691(1985);
Lubben and Keegstra, Proc. Nat'l. Acad. Sci.. 83,5502(1986)). The criteria for import was that (1) in vitro-synthesized, 35S-labeled protein was imported into chloroplasts, and (2) the transported protein was smaller than the original kanslation product, by an amount which corresponds to the removal of the 15 expected CTP. Import studies utilized either maize or pea chloroplasts. Pea chloroplasts have been reported to correctly import proteins from many differentspecies, including maize (Nieto-Sotelo et al.. Plant Physiol.. 93.1321(1990)).
Alternatively, the function of the putative maize ACCase CTP is tested by inserting the first 258 coding nucleotides of maize ACCase in frame with and 5' of a GUS reporter gene in pBI221(Clontech). This construct and the pBAR
plasmid are used to co-transform maize "Black Mexican Sweet" suspension cells by particle bombardment. Basta-resistant transformants are selected. and GUS
activity and/or protein is assayed in surviving cultures or in plasmids isolatedfrom kansformants.
A partial ACCase construct consisting of nucleotides 1-833 of SEQ ID NO: 5 including the putative CTP (nucleotides 37 to 256) and the first domain within the biotin carboxylase region (identified by amino acid sequence comparison with E. coli biotin carboxylase, see Waldrop et al.. Biochem.. 33, 6249(1994)) was amplified by PCR and cloned into the EcoRV site of PCR-script (Stratagene) to create the plasmid pBCN1. A corresponding plasmid W O96/31609 . PCT~US96/04625 64 lacking CTP sequences (nucleotides 278-833) was also made (-pBCNl). The protein encoded by -pBCNl begins at amino acid residue 83 (Val - Met). ,.
Constructs were transformed into E. coli SURE cells (Stratagene).
Restriction analysis of pBCN 1 with BamH 1 and HindIII indicated that the 5' end5 of the ACCase was located ~ cçnt to the T7 RNA polymerase binding site in PCR-Script. A partial sequence of pBCN1 obtained by using the T7 seqllencing primer and the ABI373 automated sequencing protocol confirmed this orientation and showed that the pBCNl insert sequence was identical to maize ACCase cDNA for at least the first 300 nucleotides and that it included the 10 maize ACCase Met 1 ATG. An acyl carrier protein clone conl~inin~ a CTP
(spinach ACPII, a gift of Dr. John Ohlrogge, Michigan State University) can be used as a positive control. These constructs are used for Zn vitro transcription, translation, chloroplast import, and SDS-PAGE analysis of products in the same manner as pBCNl.
Purified pBCNl was digested with EcoRl to linearize the plasmid at the 3' end ofthe BCNl insert, electrophoresed in 1.5% agarose, and the plasmid band at approximately 3.8 kb was excised and Gene-Cleaned (BioLab 101). The purified band was digested with 20 ,ug proteinase K to remove any residual RNAse, extracted with phenol and then chloroforrn under RNAse-free 20 conditions. DNA content was estimated by ethidium bromide fluorescence in droplets, relative to ~ DNA standards (Sambrook et al., Molecular Clonin~: A
Laboratory Manual. 2nd ed. (1989). One ,ug of pBCNl DNA was transcribed into capped RNA with the T7/mMessage mMachine kit (Ambion). Uncapped transcripts (Sp6 RNA polymerase, Promega) of pea RUBISCO small subunit 25 (SSU, Anderson et al., Biochem. J.. 240, 709 (1986)) were also transcribed.
RNA yield was estimated by determining the % incorporation of a 3'P-ATP
(Amersham) into a prec;pitable product, according to the Ambion kit instructions. Electrophoresis and autoradiography of 3~P-labeled product showed that it contained a single RNA band of approximately 895 nucleotides, as 30 expected.

W O96/31609 . - PCTnUS96/04625 The RNA trans==cripts were tr~n~l~tecl into 35S-labeled polypeptides 7 with Ambion's wheat germ IVT kit and approximately 45 ,uCi 35S-methionine (Amersham; 37 TBq/mmol) in a 50-,ul reaction. Labeled proteins were held on ice 6 hours prior to their use in chloroplast import experiments.
Pea (cv. "Little Marvel") and maize (inbred A188) plants were grown in a growth chamber at 25 ~C, 16 hour day length. Chloroplasts were isolated from pea and maize leaves 7 days after planting, respectively, as previously described (Burton et al., Pestic Biochem. and Physiol.. 34, 76 (1989);
Egli et al., Plant Physiol.. 101, 499 (1993)). Intact mesophyll chloroplasts were 10 washed in resuspension buffer [50 mM HEPES-KOH, pH 7.8 plus 0.33 M (pea) or 0.66M (maize) sorbitol] in preparation for import assays. Suspensions were diluted to obtain 75 ,ug chlorophyll/0.3 ml (Arnon, Plant Physiol., 24, 1 (1949)).
Import experiments were carried out essentially as described by Cline et al.(J. Biol. Chem.. 260, 3691 (1985)). Import reactions cont~ining 0.3 15 ml pea or maize chloroplast suspension, 40 ~11 35S-translation mixture, 3 mM
Mg-ATP and 10 mM Met were incub~tecl under light for 1-30 minutes at 25~C.
Unimported proteins were digested for 30 minutes with 40 ,ug of thermolysin, and proteolysis was stopped with 10 mM EDTA.
Chloroplasts were re-isolated by centrifuging them through l-ml 20 40% v/v Percoll gradients in the presence of resuspension buffer plus 3 mM Mg-ATP, 10 mM Met, and 20 mM EDTA, washed twice in the same buffer, and resuspended in 65 !11 of 1 mM MgCl,/10 mM Tris buffer, pH 8Ø Chloroplasts were Iysed by three cycles of freeze-thawing in liquid N" microfuged, and aliquots of the supern~tz~nt.~ and of the original in vitro-trzln~l~tP~l proteins were 25 analyzed by SDS-PAGE in 8-25% gradient Phast gels (Pharmacia), followed by direct detection of radiolabeled proteins in the wet gels (AMBIS) (Figure 20).
As estimated by SDS-PAGE, a 30 minute import converted the original 32-kD BCNl polypeptide to a -doublet of 27.2 and 27.5 kD in maize and produced an additional 30-kD band in pea (Figure 20A). Neither maize or pea 30 chloroplasts imported -pBCNl-derived polypeptides. Formation ofthe 27.2-kD
polypeptide likely resulted from cleavage after amino acid #47-49, a likely W O96/31609 . PcT/ub9~ c25 cleavage site because it lies between S- and D-rich regions, and R residues are located at -2, -7, and -8 (S. Gavel and G. Von Heijne, FEBS Lett.. 261, 455 (1 990)).
Time-dependence of BCN l import was further ex~min~
5 30 minlltes) (Figure 20B) to dett-rrnine if any imported polypeptides were a result of incomplete processing or proteolysis. Import was maximal after 15 min~1tec, but import time had no effect on the relative amounts of different-sized import products. Higher amounts of ATP (5 mM) stimulated import relative to lower amounts of ATP (~ 0.2 mM). The data suggest that, in maize, efficient 10 cleavage of BCN1 occurs at two closely adjacent sites and that partially processed products are also formed during BCNl import by pea chloroplasts.
Therefore, nucleotides 1-833 of the maize ACCase gene encode a CTP.
EXAMPLE IX
Expression of a cDNA Clone or Genomic Clones Encoding the ACCase 1 5 Gene The cDNA and genomic clones encoding all or a portion of the ACCase gene can be subcloned into a known expression system and the gene products reactive with the antibodies specific for maize ACCase can be identified using a Western blot. For example, the ACCase cDNA clones are 20 inserted into two transformation plasmids: (i) Glblexp which contains the embryo-specific maize Globulinl (Glbl ) promoter and 3 ' regions (Belanger et al., Genetics~ 129, 863 (1991)), and (ii) pAHCI 7 which contains the maize ubiquitin (Ubi-1) constitutive promoter and first exon and intron, and the NOS 3 ' terminator (Christensen et al., Plant Mol. Biol.. 18, 675 (1992), Toki et al., Plant 2~ Physioh 100, 1503 (1992)). The 3' end ofthe A, cDNA has a unique Sall site just 3 ' of the stop codon which is used to ligate into a SalI site in both plasmids ahead of the construct terminator. Other cloning sites will be added as needed to the plasmids or cDNA to complete the ligation of the S ' end. The gene products can also be further characterized structurally and/or enzymatically. This will 30 ensure that the genomic and cDNA clones that encode acetyl CoA carboxylase can be screened for promoters that provide for overproduction of the native or herbicide tolerant ACCase enzyme in plants.

W O96/31609 . PCTrUS96/04625 For example, the 2 kb EcoRI fragment from clone #15-14 can be subcloned into a plant transformation plasmid pBI 121 or pBI221 downstream from the 35S CaMV promoter and upstream from the nopaline 3' polyadenylation signal sequence, as described in Jefferson, Plant Molec. Biol.
5 Reptr., 5, 387-405 (1987). This plasmid can then be used to transform plant cells such as tobacco, Brassica and Arabidopsis cells using protoplast or biolistic transformation, as described by W.J. Gordon-Kamrn et al., Plant Cell. 2, 603-618(1990); M.E. Fromm et al., Bio/Technolo~y. 8, 833-839 (1990); An, Methods in Enz,vmolo~y. 153, 292 (1987); and D'Hafluin, The Plant Cell~ ~, 1495 (1992).
10 An increase in transient expression can be detected using quantitative Western blotting with antibodies specific for the ACCase enzymes. Polyclonal antibodies to maize ACCase most likely do not substantially crossreact with ACCase from dicots like tobacco or Arabidopsis.
Alternatively, the ACCase gene can be subcloned along with the 15 35S CaMV promoter into a binary Ti vector pGA482, as described in An, cited supra., which is a binary Ti vector system and can be used to transform plant cells by Agrobacterium-mediated transformation. Stably transformed plants can be generated by standard methods as described in Example III~ and levels of expression of ACCase genes can be determined by quantitati~ e Western blots. as 20 described in Harlow and Lane, Antibodies~ Cold Spring Harbor Laboratories (1988). The ability to monitor expression of cloned ACCase genes will permit the identification of promoters that provide for enhanced expression of the ACCase gene. The expression system can be used to screen for those promoters that enhance gene expression of the ACCase gene at least about 5 to 10-fold over25 the endogenous levels of ACCase produced normally in the plant cells. Becausethe 35S CaMV promoter is known as a strong promoter, it is likely this promoter will provide for at least a 5-fold increase in the expression of ACCase over that normally produced in the plant cell.
In addition. this ~u~ ion system can be used to screen antisense 30 DNA sequences. For exarnple, an antisense sequence can be obtained that is complementary to an about 0.5 kb region of the maize ACCase cDNA that has -W 096/31609 . PCTrUS96tO4625 high homology with a portion of the chicken ACCase gene and contains the sequence for the presumed transcarboxylase active site domain, as shown in FIG.
8. The ~nti~çn~e sequence could be subcloned into a pBI121 or pBI221 expression under the control of an inducible plant promoter, such as nitrite re~ r.t~e promoter (Back et al., Plant Molec. Biol.. 17:9-18 (1991)). The ability of the antisense sequence to inhibit expression of the native ACCase gene can be evaluated in transformed cells, for example as described in Hamilton et al., Nature, 346:284-287 (1990).
EXAMPLE X
Identification and Clonin~ of the Gene From Herbicide Resistant Maize Cell Lines Herbicide resistant maize cell lines were generated as described in Examples I, II, and IV. These herbicide resistant cell lines have been shown to produce an ACCase enzyme that is less sensitive to inhibition by sethoxydim or haloxyfop. The genes encoding the herbicide resistant forms of the ACCase will be identified and cloned using standard methods as described in Sambrook et al.,~uide to Molecular Cloning: A Laboratorv Manual (1989). The genes encoding the herbicide resistant forms of ACCase can then be introduced into herbicide sensitive plant species by standard methods to confer herbicide resistance . Forexample, the ACCase enzyme in the maize cell line 2167-9/2160-154 S-l is at least 100-fold less sensitive to sethoxydim than the wild-type.
DNA from the cell line or plants will be obtained and digested with EcoRI and/or other ~ l;ate restriction enzymes. according to standard methods. The restriction enzyme digest will be separated out by agarose gel electrophoresis and probed with either the 2 kb or the 3.9 kb cDNA ACCase probe described in Example V. Fragments hybridizing to the 2 kb or 3.9 kb probe will be subcloned into a Bluescript vector and portions of the gene will be sequenced. as described in Example V, to verify that the entire ACCase gene has been isolated.
To confirm that the clone encodes the ACCase gene, it will be subcloned into the pBI121 or pBI221 expression vector~ as described in Example VIII. The ACCase gene product expressed by the clone in either Black Mexican W O96/31609 . PCT/U~51~625 sweet corn cells or tobacco cells will be evaluated for reactivity with ACCase specific antibodies, by enzyme activity, and/or r~Si~t~nre of the enzyme activity to irlhibition with sethoxydim and/or haloxyfop. It is likely that the cloned gene will encode an ACCase which is resistant to inhibition by sethoxydim and 5 haloxyfop. This gene can then be introduced into an herbicide-sensitive embryogenic plant cell or an embryo, including maize cells or immZlh1re embryos. to confer herbicide resistance to that plant species upon regeneration.The complete coding sequence encoding the herbicide resistant form of the ACCase enzyme will be cloned into a plant transformation vector 10 such as pBI121 or pBI221 as described in Jefferson, Plant Molec. Biol. Rep:~rter~
5:387-405(1987). This vector contains the 35S CaMV constitutive promoter, the ,B-glucuronidase structural gene, and the nopaline synthase 3' polyadenylation signals. The ~-glucuronidase gene is replaced with a cloned ACCase gene.
Optionally, the cloned ACCase gene can be combined with natural or 15 synthetically produced chloroplast transit peptide sequence from pea, as described in Keegstra & Olsen, Ann. Rev. Plant. Physiol./Mol. Biol..40:471-501 (1989) and/or unique restriction sites introduced so the cloned gene can be distinguished from the endogenous maize ACCase gene. Standard methods of subcloning will be utilized as described in Sambrook et al.. cited supra.
For transformation of maize cells, type 11 calli can be transformed using biolistic transformation, as described by W.J. Gordon-Kamm et al., Plant Cell, 2, 603-618(1980); M.E. Fromm et al., Bio/Technologv. 8,833-839(1990);
and D.A. Walters et al., Plant Molecular Biologv. 18,189-200(1992).
Alternatively, type I embryogenic calli can be transformed using electroporation25 after mechanically or enzymatically wounding calli, as described by D'Hafluin et al., The Plant Cell. 4:1495(1992). Once the cloned gene is introduced into thesecells and transformants are selected, typically by antibiotic resistance, fertile transgenic maize plants can be regenerated, as described by D'Hafluin et al. cited supra. Fertile transgenic plants can be evaluated for herbicide tolerance, as 30 described in Example III. It is likely that the fertile transgenic plants having and expressing a cloned ACCase gene as an ACCase resistant to sethoxydim and/or W O96~1609 . . PCTrUS~G

haloxyfop will exhibit herbicide tolerance as compared to the corresponding untransformed plant.
EXAMPLE XI
Generation of Tr~n~S~enic Plants Hav;n~ an Increase in Oil Content S Once identified and cloned, the gene or genes from maize acetyl CoA carboxylase can be introduced into monocot or dicot plant species, including maize, under the control of a promoter that provides for overexpression of the ACCase enzyme. The overexpression of the ACCase enzyme is likely to lead to an increase in the oil content of the plants and seeds.
Naturally occurring soybeans that have a high oil content and soybeans that have a low oil content have been identified. The acetyl CoA
carboxylase from both types of soybeans was isolated, as described in Example V. The activity of the enzyme was measured as a function of the time of seed development and the results are shown in Figure 11.
The results in the Figure 1 1 indicate that higher oil content soybean is associated with a 2-fold increase in the ACCase activity during earlyto mid stages of development when compared with a low oil content soybean.
Thus, increased expression of the ACCase gene correlates with an increase in theoil content of the seed. Total oil content of the seed was also measured at maturity (60 days). The high oil producing cell lines, Anoka and PI28C. 134, have a total oil content of 21.8% and 19.9%, respectively. In contrast. the low oil soybean line of M76-395, has an oil content of 13.6% oil. Thus, the increaseof ACCase expression early in seed development correlates with a higher total oil content in the seed at maturity.
A gene encoding a genomic maize acetyl CoA carboxylase can be isolated, as described in Example V, and used to transform plant species by protoplast or biolistic transformation. If the gene is combined with a strong promoter, such as the 35S cauliflower mosaic virus promoter, overexpression of the ACCase gene is likely. Alternatively, selecting transformed cells with multiple copies of the gene can also result in transformed cells o~ielc~x~ ssingthe ACCase gene. The gene can be cloned into a vector such as pBI121 or pBI221, as described by Jefferson, cited supra. This vector contains the 35S

-CA 022l7367 l997-lO-03 W O 96~1609 . PCTrUS9~'01f~

cauliflower mosaic virus promoter, the ~-glucuronidase structural gene, and the nopaline synthase 3' polyadenylation si~n~l.C The cloned ACCase gene can replace the ~-glucuronidase gene and then be used to transform plant cells, including maize, as described in Example VIII.
S Transformed cells can be screened for overproduction of ACCase.
The presence of the cloned gene can be verified by identifying the unique restriction enzyme sites incorporated into the cloned gene. ACCase levels can be assessed by standard enzyme assay methods and quantitative Western blots using antibodies specific for maize ACCase. Fatty acid and lipid content in cells 10 lines overproducing ACCase are likely to be elevated and can be assessed using standard methodologies, as described in Clark & Snyder, JACS, 66:1316 (1989).
Transformed cell lines overproducing ACCase and having increased total oil content will be used to regenerate fertile transgenic plants and seeds, as described in D'Hafluin, cited supra.
EXAMPLE XII
Generation of Tr~n.~enic Plants Havin~ an Increase in Plastidic ACCase Activity Maize embryos are transformed with sense and antisense cDNA
20 constructs encoding the plastidic Al ACCase. Selected transgenic cultures andregenerated transgenic plants and progeny are subjccted to detailed analyses of:ACCase transcript levels: activity of ACCase I (plastidic) and ACCase II
(presumably cytosolic) in various plant tissues; fatty acid synthesis; lipid accumulation in kernels (primarily embryos); and other plant traits. The 25 clllmin~tion of these tests indicates whether plastidic ACCase activity can be modified via transformation and whether fatty acid synthesis is affected.
1. Transformation of maize embryos and plant re~eneration Tmm~tnre maize embryos of the Hi-II genotype are transformed by particle bombardment according to slight modifications from previously 30 described procedures (Fromm et al., Biotech.. 8, 833 (1990); Koziel et al., Biotech., 11, 194 (1993)). This procedure has been employed in transformation studies for bombardment of approximately 15,000 embryos which were then W O 96/31609 . PCTrUS96/04625 selected for Basta-resistant callus (bar selectable marker gene ~ es~ion), and regener~ted into plants. Transformed (Basta-resistant) plants are obtained from 1-2% of the initial embryos and, when s~ plasmids are used for co-transformation, the nonselected transgene is recovered in about 50% of the 5 Basta-resistant plants.
Basta-reci~t~nt, hemizygous transformed (To = Fl) plants will be tested by PCR or Southern blots for the presence of A1 ACCase sequences unique to the transformation vector, grown to maturity in growth chambers, greenhouse or field, and self-pollinated when possible, or backcrossed to a 10 nontransformed parent. F2 or backcross progeny are grown in the greenhouse and field and tested for Basta resistance and presence of the A1 ACCase transgene to identify homozygous transgenic plants. Homozygous inbred transgenic lines are then developed.
2. Analvsis of ACCase A 1 transformants Plants recovered from at least 100 independent transformation events (i.e., from dirrelelll bombarded embryos) for both the UBI 1 and GLB 1 vectors are recovered. Regenerated plants are tested for the presence of the intact ACCase Al transgene and its cosegregation with the Basta-re~i~t~nce marker. Homozygous and heterozygous transgenic lines are assayed for total 20 ACCase activitv in leaves for UBI 1 transformants and in developing; embryos (22-26 DAP) for both UBI 1 and GLB 1 transformants. Sethoxydim and haloxyfop inhibition are used to quickly dete~nine the levels of herbicide-sensitive ACCase I (plastidic) and the herbicide-insensitive ACCase II activity in both leaves and embryos. Increased expression of the ACCase A 1 transgene 25 contributes to plastidic ACCase activity and not to ACCase II activity. Kernel fatty acid and oil content are analyzed at maturity and their relationship with ACCase I determined by methods well known to the art.
Transformants that differ in kernel ACCase activity and/or fatty acid and oil content are then selected for more detailed analysis of embryos 30 throughout development (4-day intervals from 16 DAP to maturity). These analyses include RNAse protection assays to determine total Al + A2 transcript W O 96/31609 . PCT/U~ 25 levels using a non-specific probe and to ~lett?rrnine relative levels of endogenous A1 versus transgene A 1 transcripts by use of antisense riboprobes spanning the 5' UTR region of the A1 transgene constructs. Western blots of total proteins ~ separated by SDS-PAGE gels are probed with the ACCase I-specific antibody described hereinabove or with avidin and analyzed by densitometry to distinguish changes in the 227-kD ACCase I and 21 9-kD ACCase II isoforrns.
ACCase I activity, fatty acid and lipid content are determined in embryos at each stage of development. These analyses clete~nine whether expression of an additional gene(s) for plastidic ACCase increases ACCase activity and 1 Q consequently fatty acid and oil content in maize tissues, especially in embryos.
3. Transformation with maize ACCase Al ~ntis.?n~e transformation veetors Antisense transformation vectors were constructed by blunt-end ligation of nucleotides 1-833 of SEQ ID NO:5 in reverse orientation into multicloning sites of both the GLB 1 and UBI 1 plasmids. A sense construct with the same 833-bp cDNA sequence also was made with the GLB 1 plasmid to serve as a transformation control. Insert orientations were verified by restriction mapping. UBI1 ~nti~nse, GLB1 antisense, and GLBl sense constructs were introduced into > 2100, > 2900 and > 2000 embryos. respectively, and Basta-resistant callus were selected.
If antisense expression results in significant reduction in ACCase activity, it may not be possible to obtain viable callus or plants from the constitutive UBI 1 antisense transformants. Similarly, plants transformed with the embryo-specific GLB1 ~ntic~nce construct may exhibit deleterious effects on embryo development. Thus, failure to obtain transgenic progeny cont~ining the antisense ACCase gene from these transformations may indicate that ACCase activity cannot be downregulated without loss of viability.

4. Analysis of ACCase Al antisense transformants All Basta-resistant cultures will be regenerated. The presence of the UBI 1 and GLB 1 antisense constructs will be determined by PCR analysis for uniquetransgenesequencessuchasthe Ubi-l intron/ACCaseA1 junctionor Glbl 5' UTR/ACCase A1 junction, or by Southern blotting to detect unique W O96~1609 . PCTrUS9''0 fr~gment~ Plants and lines homozygous or heterozygous for the ~nti~enee transgene are analyzed for steady state level of the ACCase A1 ~nti~n~e transcripts in applo~,iate tissues/organs (such as leaves, tassels, ears, embryos and endosperm for UBIl; leaves and embryos for GLB1) by using ACCase A1

5 sense riboprobes for hybri~li7~tion on RNA blots or for RNAse protection assays.
Total ACCase activity (both ACCase I and ACCase II isoforms) and fatty acid and lipid content are determined for the ~nti~çn~e transgenic lines and for corresponding tissues from nontransformed control plants. These analyses show whether ACCase Al antisense transgenes are expressed in plants and, if so, 10 whether expression is associated with reduced ACCase activity and altered fatty acid and lipid content in maize.
EXAMPLE XIII
Expression of Plastidic and Cvtosolic ACCases during Plant Development Intact embryos are isolated from developing kernels of field-grown inbred B73 at 2 to 4 day intervals between 16-42 DAP and frozen immediately in liquid nitrogen. Samples also are saved for fresh and dry weight det~rmin~tions. Subsamples from each stage are analyzed for total lipid and 20 fatty acid content Seedling leaves are sampled along the leaf blade ranging from the etiolated, meristematic basal region to the fully expanded. green tip.
Leaves and other tissues (e.g., epidermis) of maize genotypes that accumulate anthocyanin pigments are also analyzed to assess whether a specific ACCase (such as a cytosolic ACCase) is more highly expressed in tissues in which 25 malonyl-CoA also is required as a substrate for chalcone synthase in the flavonoid pathway leading to anthocyanin synthesis.
Gene-specific antisense riboprobes in RNAse protection assays are employed to determine Al, A2, Bl and B2 transcript levels. The corresponding sense transcripts are produced in vitro and used as standards to 30 verify specificity and quantitate the sample transcript levels. Quantitation is done on an AMBIS radioanalytic image system. Herbicide inhibition of total ACCase activity provides an assessment of levels of herbicide-sensitive ACCase W O ~6~1609 . PCTnUS96104625 I (plastidic) and the herbicide-insensitive ACCase II activity in these tissues.ACCase I and II isoforms are separated by ion-exchange chromatography. Total plotehls are separated by SDS-PAGE and Western blots probed with avidin to detect the biotinylated 227-kD ACCase I and 21 9-kD ACCase II isoforms or 5 probed with ACCase I-specific antibodies.
While the present invention has been described in connection with the pl~rc~ d embodiment thereof, it will be understood many modifications will be readily apparent to those skilled in the art, and this application is int~nde~l to cover any adaptations or variations thereof. It is manifestly intPnc1~-1 this10 invention be limited only by the claims and equivalents thereof.

W O96/31609 . 76 PCTrUS96/04625 ~Uu~N~ LISTING
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(ii) TITLE OF THE lNV~YllVN: TRANSGENIC PL~NTS EXPRESSING ACETYL
COA ~R~YT.~qR G
(iii) NUMBER OF ~Uu~N~s: 26 (iv) ~u~K~uN~N~ Ann~R.q.q (A) AnD~Rq~qRR S~-~y~ , Tlln~h~g, h'~-gRn~ & Kluth, P. A.
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(A) LENGTH: 2001 base pairs (B) TYPE: nucleic acid (C) STR~NIJ~IIN~ .q single (D) TOPOLOGY: linear (ii) M~T~R~lT~R TYPE: cDNA
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RECTIFIED SHEET (RULE 91~
ISA/EP

W O 96/31609 ~ 77 PC~rrUS95/0 (Xi) ~YU~N~ DESCRIPTION: SEQ ID NO:1 AGAGATGAAG CTCGCATGCC AATGCGCCAC ACA'~'L'~ ~lL'W ATGA CAAGAGTTGT 60 TATGAAGAAG AGCAGATTCT CCGGCATGTG GAG~.~L-L- T ~ CTArArT TCTTGAATTG 120 - GATAAGTTGA AGGTGAPAGG AT~r~ATGAA ATGAAGTATA ~ ,~ TGACCGCCAA 180 TGGCATATCT Ar~r~rT~r. APATACTGAA APrCCrAAAA ~1aL~ATAG W ~ L-' 240 CGAACTATTG TCAGGCAACC CAATGCAGGC AACAAGTTTA GA.~ W ~CA GATCAGCGAC 300 GCNAAGGTAG GA1~CL~A AGAATCTCTT TCATTTACAT r~AATAr-CAT CTTAAGATCA 360 TTGATGACTG CTATTGAAGA ATTAGAGCTT CATGCAATTA Gr~rArGTCA TTCTCACATG 420 TATTTGTGCA TACTGA~AGA GCAAAAGCTT CTTGACCTCA TTCCATTTTC AGGGAGTACA 480 A11~1~ATG ~ C~AAGA TGAAGCTACC GL1~ ~AC TTTTAAAATC AA~LL ~ ~a 540 AAGATACATG AG~'1''1'~G TGrA~rr.~TG CATCATCTGT CTGTATGCCA GTGGGAGGTG 600 A~ACTCAAGT T WACTGTGA 1~CCL~aLA AGTGGTACCT W AGAGTTGT AArTArAAAT 660 AAGTTAGTGT ACCATTCAGC CAL~--~-~A GCTGr~rrAT TGCATGGTGT TGCACTGAAT 780 AATCCATATC AACCTTTGAG TGTGATTGAT CTAAAGCGCT G~LUAG rA~r~ArAGA 840 ACAACATATT GCTATGATTT ~CCG~.aGCC TTTGAAACTG CACTGCAGAA GTCATGGCAG 900 TCCAATGGCT CTA~L~111-~ TGAAGGCAAT GAPAATAGTA AATCCTACGT GAAGGCAACT 960 GAGCTAGTGT 11~1~A,AAA ACA1~W LCL ~GGGLACTC rTAT~TTCC GATGGAACGC 1020 CCTGCTGGGC TCAACGACAT TGGTATGGTC GL~1~ATCA TGGAGATGTC AAr~rCTGAA 1080 111CCLAATG GCAGGCAGAT TA~11~1-~-A GCAAATGATA TCACTTTCAG AGCTGGATCA 1140 TTTGGCCCAA GGGAAGATGC A~ ~AA ACTGTCACTA ACLLW ~1~ CGAAAGGAAA 1200 ~CL~11A TATACTTGGC AGCAAACTCT G~1~1AGGA TTGGCATAGC TGATGAAGTA 1260 APATCTTGCT l'~l~l'L~ A1~1~1~AC GAAGGCAGTC CTGAACGA W GTTTCAGTAC 1320 GAGCTAGATA GTGGTGAAAT TAGGT W ATT ATTGACTCTG 11~L~GGCAA Gr~A~r~TGGG 1440 ~11~1~1CG AGAACATACA TGGAAGTGCT GCTATTGCCA GTGCTTATTC TAGGGCATAT 1500 GAGGAGACAT TTACACTTAC A1~1~ACT ~&GC W ACTG TAGGAATAGG AGCTTATCTT 1560 TCTGCCCTGA ACAAGCTCCT 1~G~L~;~AA GTGTACAGCT CCrArATGCA G~1 ~L~'L 1680 CCTAAGATCA TGGCGACCAA ~1~11~1C CACCTCACTG TTCCAGATGT CCTTGAAGGT 1740 ~111C~AATA TATTGA W TG GCTCAGCTAT ~1-C~CAA ACA.1~1~ A~1~CL'1' 1800 ATTACCAAAC CTCTGGACCC TCCAGACAGA ~'1'~-G~- ACA'1CC~1~A r~A~r~rATGC 1860 GATCCACGTG CAGCTATCTG 1~AGAT GACAGCCAAG GGAPATGGTT G~ ATG 1920 TTTGACAAAG ACAGCTTTGT Gr-~r-~rATTT GAAGGATGGG CAAAAACAGT GGTTACTGGC 1980 (2) INFORMATION FOR SEQ ID NO: 2:
(i) ~U~N~'~ CHARACTERISTICS
(A) LENGTH 258 base pairs (B) TYPE nucleic acid (C) STR~N~ )N~C single (D) TOPOLOGY linear (ii) MOLECULE TYPE: CDNA
(iii) ~Y~U1~1CAL: NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYPE
(Vi) ORIGINAL SOURCE
(Xi) ~UU~NL~ DESCRIPTION SEQ ID NO:2:
L-L1CLL~CAA ACA11~1~G AC~1~11~1 ATTAC Q~AC CTCTGGACCC TCCAGA Q GA 60 CL1~11~L1 ACA1CCC1~A GAACA QTGC GATC Q CGTG QGCTATCTG L~1~1AGAT 120 GA Q GC QAG GGA~ATGGTT G~1~1ATG TTTGA Q~AG A QG~111~1 GGAGACATTT 180 GAAGGATGGG CAA~AACAGT GGTTACTGGC AGAG QPAGC TTGGAGGAAT 1CL1~1~C 240 (2) INFORMATION FOR SEQ ID NO:3:

(i) ~UU~NL~ CHARACTERISTICS:
RECTIFIED SHEET (RUEE 91) ISA/EP

W O 96/31609 . . PC~rrUS96/0 tA) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STR~N~ N~:qC: single (D~ TOPOLOGY: linear (ii) MOT~T~T7T~T~- TYPE: peptide (iii) ~Y~UL~LlCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: i n t ~n~ 1 (vi) ~RTGTN~T~ SOURCE:
(Xi) ~:yU~N~ DESCRIPTION: SEQ ID NO:3:
Val Met Lys Met (2) INFORMATION FOR SEQ ID NO:4:
(i) ~;yu~;N~ RZ~ ~RT~cTIcs (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STR~NIJ~:~N~:cS: single (D) TOPOLOGY: linear (ii) MOT~R~T~T~ TYPE: cDNA
( iii ) ~Y~l~h L lCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SO~RCE:
(Xi ) ~UU~N - ~ DESCRIPTION: SEQ ID NO:4:
GCCAGATTCC ~r~A~.CAT ATATCC 26 (2) INFORMATION FOR SEQ ID NO:5:
(i) ~UU~N~ ~R~rTT~-RT.~TICS:
(A) LENGTH: 7470 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~:~S: Ringle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~U~ lCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ~RTr.TN~T. SOURCE:
(Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:5:
AT 'l~lV~'l'Vl~l GGGCCACGGA ACGACAATGT CACAGCTTGG ATTAGCCGCA 60 G~lvC~-L~AA AGGCCTTGCC ACTACTCCCT AATCGCCAGA GAAGTTCAGC TGGGACTACA 120 TTCTCATCAT CTTCATTATC GAGGCCCTTA AACAGAAGGA A~AGCCATAC lvvll~ACTC 180 CGTGATGGCG GAGATGGGGT ATCAGATGCC A~AAAGCACA GCCAGTCTGT TCGTCAAGGT 240 CTTGCTGGCA TTATCGACCT CCCAAGTGAG GCA~cl~vCv AAGTGGATAT TTCACATGGA 300 TCTGAGGATC CTAGGGGGCC AA~TTCT TATCA~ATGA ATGGGATTAT CAATGA~ACA 360 CATAATGGAA GACATGCCTC AGTGTCCAAG ~l~vllvAAT L 1 lVlVC~C ACTAGGTGGC 420 AA~ r~A TTCACAGTAT ATTAGTGGCC AACAATGGAA TGGCAGCAGC A~AATTTATG 480 TTCGTAGAGG Lv~l~lGG A~AT AATAACTACG CCAATGTTCA ACTCATAGTG 660 GGGATGGCAC A~AAACTAGG lVl 1 lV'LVCT ~1 ~.vG~lv vll~G~l~A L~ll.~vAG 720 RECTIFIE~ SHEET (RULE 91) ISA/EP

CA 022l7367 l997-lO-03 W O~6131609 . 79 PCTrUS96/04625 AATC ~ GAAC TGCCAGATGC ATTGACCGCA AAA~,-~TCG ... ~..................... ~ CCCACCTGCA 780 TCATCAATGA A~...~G AGATAAGGTC GGCTCAGCTC TCA--~-~A AGCAGCCGGG 840 GTCCCAACTC ..~..~AG TGGATCACAT GTTGAAGTTC CATTAGAGTG ~-~-AGAC 900 GCr~AT~CCTG AGGAGATGTA TAGA~AAG ~ ~C.~l~ACTA CCACAGAGGA AGCAGTTGCA 960 A~i~AAG .~..~.~A TCCTGCCATG ATTAArGrAT ~-~GG~AGG .~.~AAA 1020 GGAATAAGAA AGGTTCATAA TGATGATGAG GTT-Ar~Ar~CGC TGTTTAAGCA AGTACAAGGT 1080 GAA~-CC~ G~CCC~AAT A...~. ATG A~..~ AT CCCAGAGTCG GCATCTTGA~A 1140 GTTCAGTTGC LLL~ATCA ATATGGTAAT GTAGCAGCAC TTCA QGTCG TGATTGCAGT 1200 ~ GTGCAACGGC r-ArArrAr-~A GATTATTGAA GAAGGTC QG TTA~.~.. ~C ~C'~-~.~AG 1260 ACAGTTA~AG CACTTGAGCA GGCAGCAAGG AGG~..~A A~G~.~.~GG TTA.~.-~1 1320 AAL~CC~jA~ TACAGGTTGA GCATCCAGTC ACTGAGTGGA TAGCTGAAGT GAA~-GC~- 1440 GCAGCTCAAG 1~C.~.~G AATGGGCATA C~.. 1~GC AGATTCCAGA AATCAGACGT 1500 TTCTATGGAA TGGACTATGG AGGAGGGTAT GACATTTGGA GGAAAACAGC AG~.~..~1 1560 AGAATTACTA GTr~r~--ArCC AGATGATGGT TTCAAACCTA ~.~G~AA AGTr~Ar~A~. 1680 ATAAGTTTTA AAAGCAAGCC TAA~ GCCTACTTCT C~AGTAAAGTC .~AGGC 1740 ATTCATGAAT ~-~-~ATTC TCA~-..~A CA~.G~...-~ CATATGGACT CTCTAGACCA 1800 GCAGCTATAA CAAACATGTC TCTTGCATTA AAAGAGATTC AGA..~-~G AGAAATTCAT 1860 TCAAATGTTG ATTACACAGT TGAC~ A AACGCTTCAG ACTTCAGAGA AAACAAGATC 1920 CACACTGGTT GGCTGGATAC A~GAATAGCT A~GC~1LC AAGCTGAGAG GCCCC~ATGG 1980 TATATCTCAG ~~.-~AGG TGCTTTATAT A~AACAGTAA CrArr~ATGC AGCCACTGTT 2040 TCTGAATATG TTAGTTATCT CACCAAGGGC CATATTCCAC CAAAGCATAT A~CC~.~.C 2100 GGACATGGTA GCTACAGGTT GAGAATGA~T GATTCAACAG TTGAAGCGAA TGTACAATCT 2220 TTATGTGATG ~-~GC~-~1- AATGCAGTTG GATGGA~ACA GCCATGTAAT TTATGCAGAA 2280 CATGATCCAT CGAAGTTATT AGCTGAGACA CCCTGCAAAC ~..~..~... ~1-~G-1 2400 GA1~1G~1C ATGTTGATGC GGATGTACCA TACGCGGAAG TTGAGGTTAT GAAGATGTGC 2460 A.~..1~1 TGTCACCTGC ~-~L~l~-C ATTCATTGTA TGA~1~.~A GGGCCAGGCA 2520 TTGCAGGCTG GTGATCTTAT -A~-rA;Ar-GTTG GATCTTGATG ACC~-.~GC' TGTGAAAAGA 2580 GTACA Q ~A GATATG ~ GC AAGTTTGAAT G~1~-~AA .~1C~LGC AGGATATGAG 2700 QCAATATTA ATGAAGTCGT T QAGATTTG GTATGCTGCC TGGACAACCC TGAG~.C-1 2760 TTCCTACAGT GGGATGAACT TA.~.~.~1. CTAG QACGA GG~AAG AAATCT QAG 2820 GACTTTCCAT CCAAGTTGCT AA~.~r-ArATC ATTGAGGAAA A1~L1~ A ~ L~AGAG 2940 AAGGAAAAGG CTA QAATGA GAGG~1~ GAGC~-.--A TGAACCTACT GAAGTCATAT 3000 GA w ~l~G~A GAGAGAGCCA TGCACATTTT ~ll~-~AAGT ~l~lL.l~A w AGTATCTT 3060 ACAGT w AAG AACTTTTTAG TGAT w CATT CAGTCTGACG TGATTGAAAC ATTGC w QT 3120 CAGCACAGTA AAGACCTGCA GAA~-A GACATTGTGT l~l~l~ACCA w ~l~l~AGG 3180 AACAAAGCTA AGCTTGTAAC w CACTTATG GA,AAAGCTGG TTTATCCAAA 'L~'l'W~'l' 3240 TACAGGGATC TGTTAGTTCG ~.I..~...C CTCAATCATA AAAGATATTA TAAGTTGGCC 3300 CTTAAAGCAA GTGAACTTCT TGAACAAACC AAACTAAGTG AA ~C~lGC AAGCGTTGCA 3360 AGAAGCCTTT CGGATCT w G GATGCATAAG Gr--AriAAATGA GTATTAAGGA TAACATGGAA 3420 GATTTAGTCT ~CC~ATT AC~ ~AA GATGCTCTGA ...~..~.. TGATTACAGT 3480 GATCGAACTG TT QGCAGAA AGTGATTGAG ArATArATAT CACGATTGTA CCAGCCTCAT 3540 CTTGTAAAGG ATAGCATCCA AATGAAATTC AAGGAATCTG GTGCTATTAC'...Ll w ~AA ,3600 TTTTATGAAG GGCATGTTGA TACTAGAAAT w ACAT w GG CTATTATT w TGGeAAGCGA 3660 lG W ~l~CCA ~~l'~'l-~- CAAATCACTT GAA~ L CAACAGCCAT TGTGGCTGCA 3720 ATGGAAAAGC TTAGCAAGAT ACTGAA w AT ACTAGCGTTG CAAGTGATCT CCAAGCTGCT 3900 G~lll~AAGG TTATAAGTTG CA .~.. AA AGAGATGAAG CTCGCATGCC AATGCGCCAC 3960 - ACAl~C~l~l G~.. ~ATGA CAAGAGTTGT TATGAAGAAG AGCAGATTCT CCGGCATGTG 4020 GAGC~'lCCCC TCTCTACACT TCTTGAATTG GATAAGTTGA A w TGAAA w ATACAATGAA 4080 ATGAAGTATA ~.~'l--~CG TGACCGCCAA TGGCATATCT ACACACTAAG AAATACTGAA 4140 AACCCCAAAA TGTTGCATAG ~.~...l.C CGAACTATTG TCA w CAACC CAATGCAGGC 4200 AACAAGTTTA GA~CGG~. A GATCAGCGAC GCTGA w TAG GA~ C~A AGAATCTCTT 4260 CTTGACCTCA TTCCATTTTC A w GAGTACA A~.~--~ATG l~GC~AAGA TGAAGCTACC 4440 RECTIFIED StlEET (RULE 91) ISA/EP

W O 96/31609 PC~rrUS96/~4625 G~1~ AC TTTTA~AATC AA,~C,,,~ AAr.ATAt~ATG AG~1~1~G TGrAAr.r.ATG 4soo U ~ U~, TArCr.At7.AAr. TGGAGGAAAT AGAATCACAG AAGTTAGTGT ACCATTCAGC CA~ A 4680 GCTGt.At~rAT TGCATGGTGT TGCACTGAAT AATCCATATC AACCTTTGAG TGTGATTGAT 4740 CTAAAGCGCT G~L~,~LAG t'.AAt'AAt'At'A At~AAt~ATATT GCTATGATTT ~l~CC 4800 TTTGAAACTG CACTGCAGAA GTCATGGCAG TCCA~ATGGCT CTA~,~.,,,C TGAAGGCAAT 4860 GA~AATAGTA AATCCTACGT GAAG~CAACT GAGCTAGTGT ,,~,~aAAA ACA1~C 4920 TGGGGCACTC CTATAATTCC GATGGAACGC CCTG ~ GGGC TCAACGACAT TGGTATGGTC 4980 GCTTGGATCA TGGAGATGTC AAt'At'CTGAA ,,,~C~ATG GCAGGCAGAT TA~ A 5040 GCAA~ATGATA TCACTTTCAG AGCTGGATCA ~GC~ AA GGr.AAt~.ATGC A~1111~AA 5100 ACTGTCACTA AC~1~G~11G t~rAAAt.t.AAA ~11~1~11A TATACTTGGC At~rAAAt~CT 5160 G~lG~lAGGA TTGG QTAGC TGATGAAGTA AAATCTTGCT lC~l~l~aG A~L~AC 5220 GAAGGCAGTC CTr.AA~7t7.At7.r, GTTTCAGTAC ATCTATCTGA ~r-AA~~A~~A CTATGCTCGC 5280ATTAGCTCTT CTGTTATAGC ArATAAt~.CTG GAG~'TAt.ATA GTGGTGAAAT TAGGTGGATT 5340 ATTGACTCTG '~1 W aCAA ~,rAt.r.ATGGG ~ .1~1a~ AGAACATACA TGGAAGTGCT 5400 GCTATTGCCA GTGCTTATTC TAGGGCATAT GAGGAGACAT TTACACTTAC Al~ ~ACT 5460 W GCGGACTG TAGGAATAGG AGCTTATCTT GCTCGACTTG GTATACGGTG t7ATAt7At7~cGT 5520 CTTGACCAGC CTATTATTTT AAt'At'.r~GTTT TCTGCCCTGA ACAAGCTCCT TG W CGGGAA 5580 GTGTACAGCT CCCACATGCA G~LLW ,~. CCTAAt~.ATCA TGGC-7.Art7AA ~ C 5640 CACCTCACTG TTCCAGATGT CCTTGAAGGT GTTTCCAATA TATTGA W TG GCTCAG ~AT 5700 ~1L~1~CAA ACA,,~.1~G AC~1~LLC~ ATTACCAAAC CTCTW ACCC TCCAGACAGA 5760 C~.1L~CTT ACALCC~A GAACACATGC GATCCACGTG CAGCTATCTG ~L~-lAGAT 5820 GACAGCCAAG GGAAATGGTT WW L~71ATG TTTGACAAAG ACAGCTTTGT GGAGACATTT 5880 GAA W ATGGG CAAAAACAGT GW TTACTGGC AGAGCAA-AGC TTGrAr~AAT l~l~-L~WC 5940 GATTCCCATG AGCGATCTGT CC~l~l~ WACA~AGTGT Wl l~CL-AGA TTCTGC-AACC 6060 AAr.ArrGCTC AGGCATTATT AGACTTCAAC CGTGAAGGAT TGC~L~l~ll CAlC~L 6120 AATT W AGAG G~1~1~LW TGGACAAAGA GA~ L~ AA W AATTCT TCAGW CTGGG 6180 TCAACAATTG TCGAGAACCT TArr.ArATAT AATCAGCCTG ~1111~1~.1A CATTCCTATG 6240 GCTGW AGAGC ~1~.1 W AGG AG~1~ 7L~ATA GCAAAATAA~A TCCAGACCGC 6300 GAAATCAAGT TCAGGTCAGA GGAACTCCAA GA ~ GTATGG GTAGGCTTGA ccrAr~ArTTG 6420 ATAAATCTGA AAGCAAAACT CrAArATGTA AATCATGGAA ATGGAAGTCT ArrArAr~T~ 6480 ATTGCAATAC GGTTTGCTGA ATTGCATGAT A~11~1AA GAAT W CAGC TAAAGGTGTG 6600 ATTAAGAAAG TTGTAGACTG GGAAGAATCA CG~1~.11~1 TCTATA~AAG GCTACGGAGG 6660 AGGATCGCAG AAGATGTTCT TGCA~AAGAA ATAAGGCAGA TA~1~ W 1~A TAAATTTACG 6720 AGCACTGW AT GW GATGACGA TGATGCTTTT GTTGCCTGGA A W ACAGTCC TGA~AACTAC 6840 GACTCCAGTT CAGATCTGCA AGCATTCTCG CA~.1~111 CTACGCTATT AGATAAGATG 6960 GA1CC~L~LC AGAGAGCGAA ~111~1~AG GAAGTCAAGA AG~. lC~ ~L~A TTGATGATAC 7020 CAACACATCC AACACAATGT GTGCATGTCA CA1~1LG TTCTAGTACA TACATAGA~AG 7080 GATATTGCTT W ~1L~ATT GATCATGTCT GATTTAAGTC GACTATTATT -~l-~AATT 7140 L1~L1LL~A ~1G~.1~1A '1~LL~ATGG ATGTATATTG GATATGTGCG L1~1~C~AGG 7200 TGTAAGCACA AAGGTTTAGA C~RAYMRAR~ ~rAAr~Ar~CGA GTGAACCTGT.~ .--LL,a 7260 CA~.1~.11~A GTAAGGCAGA AAGTTGTTAA ACCGTAGTTC TGAGATGTAT TACC,~AGTGNC 7320 AAAGTTTGAA CTCAAATAAC A'L~.1L~1~ TAAGCATATG TACCGTACCT CTACG?GAAA 7440 (2~ INFORMATION FOR SEQ ID NO:6:
~i) S~U~NC~ CHARACTERISTICS: .' (A) LENGTH: 2325 amino acids (B) TYPE: amino acid ( C ) STRAN I ) I~ N I':,~S: single (D) TOPOLOGY: linear (ii) MOLEC~'LE TYPE: protein (iii) ~Y~ul~LlCAL: NO
RECTIFIED SHEET ~RULE 91 ISA/EP

W O96/31609 . 81 PCTrU59-'01'~

( iv) ANTISENSE: NO
(v) ERAGMENT TYPE: N-t~inAl ~d C-to- nAl (full length protein) (vi ) ORIGINAL SOURCE:
(Xi) ~ U~;N~:~i DESCRIPTION: SEQ ID NO:6:
Met Ser Gln Leu Gly Leu Ala Ala Ala Ala Ser Lys Ala Leu Pro Leu ~ 5 10 15 Leu Pro Asn Arg Gln Arg Ser Ser Ala Gly Thr Thr Phe Ser Ser Ser Ser Leu Ser Arg Pro Leu Asn Arg Arg Lys Ser His Thr Arg Ser Leu Arg Asp Gly Gly Asp Gly Val Ser Asp Ala Lys Lys His Ser Gln Ser Val Arg Gln Gly Leu Ala Gly Ile Ile Asp Leu Pro Ser Glu Ala Pro Ser Glu Val Asp Ile Ser Hi9 Gly Ser Glu Asp Pro Arg Gly Pro Thr Asp Ser Tyr Gln Met Asn Gly Ile Ile Asn Glu Thr His Asn Gly Arg His Ala Ser Val Ser Lys Val Val Glu Phe Cys Ala Ala Leu Gly Gly Lys Thr Pro Ile His Ser Ile Leu Val Ala Asn Asn Gly Met Ala Ala Ala Lys Phe Met Arg Ser Val Arg Thr Trp Ala Asn Asp Thr Phe Gly Ser Glu Lys Ala Ile Gln Leu Ile Ala Met Ala Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Gly Met Ala Gln Lys Leu Gly Val Ser Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Thr Ala Lys Gly Ile Val Phe Leu Gly Pro Pro Ala Ser Ser Met Asn Ala Leu Gly Asp Lys Val Gly Ser Ala Leu Ile Ala Gln Ala Ala Gly Val Pro Thr Leu Ala Trp Ser Gly Ser His Val Glu Val Pro Leu Glu Cys Cys Leu Asp Ala Ile Pro Glu Glu Met Tyr Arg Lys Ala Cys Val Thr Thr Thr Glu Glu Ala Val Ala Ser Cys Gln Val Val Gly Tyr Pro Ala Met Ile Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile Arg Lys Val Hi3 Asn Asp ~ 325 330 335 Asp Glu Val Arg Ala Leu Phe Lys Gln Val Gln Gly Glu Val Pro Gly Ser Pro Ile Phe Val Met Arg Leu Ala Ser Gln Ser Arg His Leu Glu Val Gln Leu Leu Cys Asp Gln Tyr Gly Asn Val Ala Ala Leu His Ser ~ ~ Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Gly Pro Val Thr Val Ala Pro Arg Glu Thr Val Lys Ala Leu Glu Gln Ala Ala Arg Arg Leu Ala Lys Ala Val Gly Tyr Val Gly Ala Ala Thr Val Glu Tyr Leu Tyr Ser Met Glu Thr Gly Asp Tyr Tyr Phe Leu Glu I-eu RECTIFIED SHEET (RULE 91 ISA/EP

W O 96/31609 . PCTAUS96/04625 Asn Pro Arg Leu Gln Val Glu His Pro Val Thr Glu Trp Ile Ala Glu Val Asn Leu Pro Ala Ala Gln Val Ala Val Gly Met Gly Ile Pro Leu 465 470 475 480~rp Gln Ile Pro Glu Ile Arg Arg Phe Tyr Gly Met Asp Tyr Gly Gly 485 490 495~ly Tyr Asp Ile Trp Arg Lys Thr Ala Ala Leu Ala Thr Pro Phe Asn Phe Asp Glu Val Asp Ser Gln Trp Pro Lys Gly His Cy3 Val Ala Val Arg Ile Thr Ser Glu Asp Pro Asp Asp Gly Phe Lys Pro Thr Gly Gly Lys Val Lys Glu Ile Ser Phe Lys Ser Lys Pro Asn Val Trp Ala Tyr 545 550 555 560~he Ser Val Lys Ser Gly Gly~Gly Ile His Glu Phe Ala Asp Ser Gln 565 570 575~he Gly His Ala Phe Ala Tyr Gly Leu Ser Arg Pro Ala Ala Ile Thr Asn Met Ser Leu Ala Leu Lys Glu Ile Gln Ile Arg Gly Glu Ile His Ser Asn Val Asp Tyr Thr Val Asp Leu Leu Asn Ala Ser Asp Phe Arg Glu Asn Lys Ile His Thr Gly Trp Leu Asp Thr Arg Ile Ala Met Arg 625 630 635 640~al Gln Ala Glu Arg Pro Pro Trp Tyr Ile Ser Val Val Gly Gly Ala 645 650 655~eu Tyr Lys Thr Val Thr Thr Asn Ala Ala Thr Val Ser Glu Tyr Val Ser Tyr Leu Thr Lys Gly His Ile Pro Pro Lys His Ile Ser Leu Val Asn Ser Thr Val Asn Leu Asn Ile Glu Gly Ser Lys Tyr Thr Ile Glu Thr Val Arg Thr Gly His Gly Ser Tyr Arg Leu Arg Met Asn Asp Ser 705 710 715 720~hr Val Glu Ala Asn Val Gln Ser Leu Cys Asp Gly Gly Leu Leu Met 725 730 735~ln Leu Asp Gly Asn Ser His Val Ile Tyr Ala Glu Glu Glu Ala Gly Gly Thr Arg Leu Gln Ile Asp Gly Lys Thr Cys Leu Leu Gln Asn Asp His Asp Pro Ser Lys Leu Leu Ala Glu Thr Pro Cys Lys Leu Leu Arg Phe Leu Val Ala Asp Gly Ala His Val Asp Ala Asp Val Pro Tyr Ala 785 790 795 800~lu Val Glu Val Met Lys Met Cys Met Pro Leu Leu Ser Pro Ala Ser 805 810 815~ly Val Ile His Cys Met Met Ser Glu Gly Gln Ala Leu Gln Ala Gly Asp Leu Ile Ala Arg Leu Asp Leu Asp Asp Pro Ser Ala Val Lys Arg Ala Glu Pro Phe Asp Gly Ile Phe Pro Gln Met Glu Leu Pro Val Ala Val Ser Ser Gln Val His Lys Arg Tyr Ala Ala Ser Leu Asn Ala Ala 865 870 875 880~rg Met Val Leu Ala Gly Tyr Glu His Asn Ile Asn Glu Val Val Gln 885 890 895~sp Leu Val Cys Cys Leu Asp Asn Pro Glu Leu Pro Phe Leu Gln Trp Asp Glu Leu Met Ser Val Leu Ala Thr Arg Leu Pro Arg Asn Leu Lys Ser Glu Leu Glu Asp Lys Tyr Lys Glu Tyr Lys Leu Asn Phe Tyr His RECTIFIED S~IEET (RULE 91) ISA/EP

W O 96/31609 . 83 PCTrUS96/04625 Gly Lys Asn Glu Asp Phe Pro Ser Lys Leu Leu Arg Asp Ile Ile Glu Glu Asn Leu Ser Tyr Gly Ser Glu Ly8 Glu Lys Ala Thr Asn Glu Arg Leu Val Glu Pro Leu Met Asn Leu Leu Lys Ser Tyr Glu Gly Gly Arg Glu Ser His Ala His Phe Val Val Lys Ser Leu Phe Glu Glu Tyr Leu 995 ~ 1000 1005 Thr Val Glu Glu Leu Phe Ser Asp Gly Ile Gln Ser Asp Val Ile Glu Thr Leu Arg His Gln His Ser Lys Asp Leu Gln Lys Val Val Asp Ile Val Leu Ser His Gln Gly Val Arg Asn Lys Ala Lys Leu Val Thr Ala Leu Met Glu Lys Leu Val Tyr Pro Asn Pro Gly Gly Tyr Arg Asp Leu Leu Val Arg Phe Ser Ser Leu Asn His Lys Arg Tyr Tyr Lys Leu Ala Leu Lys Ala Ser Glu Leu Leu Glu Gln Thr Lys Leu Ser Glu Leu Arg 1090 1095 . 1100 Ala Ser Val Ala Arg Ser Leu Ser Asp Leu Gly Met His Lys Gly Glu Met Ser Ile Lys Asp Asn Met Glu Asp Leu Val Ser Ala Pro Leu Pro Val Glu Asp Ala Leu Ile Ser Leu Phe Asp Tyr Ser Asp Arg Thr Val Gln Gln Lys Val Ile Glu Thr Tyr Ile Ser Arg Leu Tyr Gln Pro His Leu Val Lys Asp Ser Ile Gln Met Lys Phe Lys Glu Ser Gly Ala Ile Thr Phe Trp Glu Phe Tyr Glu Gly His Val Asp Thr Arg Asn Gly His 185 ll90 1195 1200 Gly Ala Ile Ile Gly Gly Lys Arg Trp Gly Ala Met Val Val Leu Lys Ser Leu Glu Ser Ala Ser Thr Ala Ile Val Ala Ala Leu Lys Asp Ser Ala Gln Phe Asn Ser Ser Glu Gly Asn Met Met His Ile Ala Leu Leu Ser Ala Glu Asn Glu Ser Asn Ile Ser Gly Ile Ser Ser Asp Asp Gln Ala Gln His Lys Met Glu Lys Leu Ser Lys Ile Leu Lys Asp Thr Ser Val Ala Ser Asp Leu Gln Ala Ala Gly Leu Lys Val Ile Ser Cys Ile Val Gln Arg Asp Glu Ala Arg Met Pro Met Arg His Thr Phe Leu Trp Leu Asp Asp Lys Ser Cys Tyr Glu Glu Glu Gln Ile Leu Arg His Val 1315 . 1320 1325 Glu Pro Pro Leu Ser Thr Leu Leu Glu Leu Asp Lys Leu Lys Val Lys Gly Tyr Asn Glu Met Lys Tyr Thr Pro Ser Arg Asp Arg Gln Trp His Ile Tyr Thr Leu Arg Asn Thr Glu Asn Pro Lys Met Leu His Arg Val Phe Phe Arg Thr Ile Val Arg Gln Pro Asn Ala Gly Asn Lys Phe Arg Ser Ala Gln Ile Ser Asp Ala Glu Val Gly Cys Pro Glu Glu Ser Leu Ser Phe Thr Ser Asn Ser Ile Leu Arg Ser Leu Met Thr Ala Ile Glu Glu Leu Glu Leu His Ala Ile Arg Thr Gly His Ser His Met Tyr Leu RECTIFIED StJEET (RULE 91 ISA/EP

W O96~1609 . PCTrUS96/04625 Cys Ile Leu Lys Glu Gln Lys Leu Leu Asp Leu Ile Pro Phe Ser Gly 144S 1450 1455~er Thr Ile Val Asp Val Gly Gln Asp Glu Ala Thr Ala Cy8 Ser Leu 1460 1465 1470 ,~eu Lys Ser Met Ala Leu Lys Ile His Glu Leu Val Gly Ala Arg Met His His Leu Ser Val Cys Gln Trp Glu Val Lys Leu Lys Leu Asp Cy8 Asp Gly Pro Ala Ser Gly Thr Trp Arg Val Val Thr Thr Asn Val Thr 505 1510 1515 1520~ly His Thr Cys Thr Ile Asp Ile Tyr Arg Glu Val Glu Glu Ile Glu 1525 -1530 1535~er Gln Lys Leu Val Tyr His Ser Ala Ser Ser Ser Ala Gly Pro Leu 1540 1545 1550~is Gly Val Ala Leu Asn Asn~Pro Tyr Gln Pro Leu Ser Val Ile Asp Leu Lys Arg Cys Ser Ala Arg Asn Asn Arg Thr Thr Tyr Cys Tyr Asp Phe Pro Leu Ala Phe Glu Thr Ala Leu Gln Lys Ser Trp Gln Ser Asn 585 1590 1595 1600~ly Ser Thr Val Ser Glu Gly Asn Glu Asn Ser Lys Ser Tyr Val Lys 1605 1610 1615~la Thr Glu Leu Val Phe Ala Glu Lys His Gly Ser Trp Gly Thr Pro 1620 1625 1630~le Ile Pro Met Glu Arg Pro Ala Gly Leu Asn Asp Ile Gly Met Val Ala Trp Ile Met Glu Met Ser Thr Pro Glu Phe Pro Asn Gly Arg Gln Ile Ile Val Val Ala Asn Asp Ile Thr Phe Arg Ala Gly Ser Phe Gly 665 1670 1675 1680~ro Arg Glu Asp Ala Phe Phe Glu Thr Val Thr Asn Leu Ala Cys Glu 1685 1690 1695~rg Lys Leu Pro Leu Ile Tyr Leu Ala Ala Asn Ser Gly Ala Arg Ile 1700 1705 1710~ly Ile Ala Asp Glu Val Lys Ser Cys Phe Arg Val Gly Trp Ser Asp Glu Gly Ser Pro Glu Arg Gly Phe Gln Tyr Ile Tyr Leu Thr Glu Glu Asp Tyr Ala Arg Ile Ser Ser Ser Val Ile Ala His Lys Leu Glu Leu 745 1750 1755 1760~sp Ser Gly Glu Ile Arg Trp Ile Ile Asp Ser Val Val Gly Lys Glu 1765 1770 1775~sp Gly Leu Gly Val Glu Asn Ile His Gly Ser Ala Ala Ile Ala Ser 1780 1785 1790~la Tyr Ser Arg Ala Tyr Glu Glu Thr Phe Thr Leu Thr Phe Val Thr Gly Arg Thr Val Gly Ile Gly Ala Tyr Leu Ala Arg Leu Gly Ile Arg Cys Ile Gln Arg Leu Asp Gln Pro Ile Ile Leu Thr Gly Phe Ser Ala 825 1830 1835 1840~eu Asn Lys Leu Leu Gly Arg Glu Val Tyr Ser Ser His Met Gln Leu 1845 1850 1855~ly Gly Pro Lys Ile Met Ala Thr Asn Gly Val Val His Leu Thr Val 1860 1865 1870~ro Asp Val Leu Glu Gly Val Ser Asn Ile Leu Arg Trp Leu Ser Tyr Val Pro Ala Asn Ile Gly Gly Pro Leu Pro Ile Thr Lys Pro Leu Asp Pro Pro Asp Arg Pro Val Ala Tyr Ile Pro Glu Asn Thr Cys Asp Pro ~rg Ala Ala Ile Cys Gly Val Asp Asp Ser Gln Gly Lys Trp Leu Gly RECTIFIED SHEET (RULE 91) ISA/EP

CA 022l7367 l997-l0-03 W O 96131609 , PC~rrUS96/04625 Gly Met Phe Asp Lys Asp Ser Phe Val Glu Thr Phe Glu Gly Trp Ala Lys Thr Val Val Thr Gly Arg Ala Lys Leu Gly Gly Ile Pro Val Gly Val Ile Ala Val Glu Thr Gln Thr Met Met Gln Ile Ile Pro Ala Asp . 1970 1975 1980 Pro Gly Gln Leu Asp Ser His Glu Arg Ser Val Pro Arg Ala Gly Gln Val Trp Phe Pro Asp Ser Ala Thr Lys Thr Ala Gln Ala Leu Leu ASp Phe Asn Arg Glu Gly Leu Pro Leu Phe Ile Leu Ala Asn Trp Arg Gly Phe Ser Gly Gly Gln Arg Asp Leu Phe Glu Gly Ile Leu Gln Ala Gly Ser Thr Ile Val Glu Asn Leu Arg Thr Tyr Asn Gln Pro Ala Phe Val Tyr Ile Pro Met Ala Gly Glu Leu Arg Gly Gly Ala Trp Val Val Val Asp Ser Lys Ile Asn Pro Asp Arg Ile Glu Cys Tyr Ala Glu Arg Thr Ala Lys Gly Asn Val Leu Glu Pro Gln Gly Leu Ile Glu Ile Lys Phe Arg Ser Glu Glu Leu Gln Asp Cys Met Gly Arg Leu Asp Pro Glu Leu Ile Asn Leu Lys Ala Lys Leu Gln Asp Val Asn His Gly Asn Gly Ser Leu Pro Asp Ile Glu Gly Ile Arg Lys Ser Ile Glu Ala Arg Thr Lys Gln Leu Leu Pro Leu Tyr Thr Gln Ile Ala Ile Arg Phe Ala Glu Leu ~i~ Asp ~hr Se~ Lêu A~g MeL Ald Ala Lys ~ly val Ile Lys Lys Val Val Asp Trp Glu Glu Ser Arg Ser Phe Phe Tyr Lys Arg Leu Arg Arg Arg Ile Ala Glu Asp Val Leu Ala Lys Glu Ile Arg Gln Ile Val Gly Asp Lys Phe Thr His Gln Leu Ala Met Glu Leu Ile Lys Glu Trp Tyr Leu Ala Ser Gln Ala Thr Thr Gly Ser Thr Gly Trp Asp Asp Asp Asp Ala Phe Val Ala Trp Lys Asp Ser Pro Glu Asn Tyr Lys Gly His Ile Gln Lys Leu Arg Ala Gln Lys Val Ser His Ser Leu Ser Asp Leu Ala Asp Ser Ser Ser Asp Leu Gln Ala Phe Ser Gln Gly Leu Ser Thr Leu Leu ASp Lys Met Asp Pro Ser Gln Arg Ala Lys Phe Val Gln Glu Val Lys Lys Val Leu Asp (2) INFORMATION FOR SEQ ID NO:7:
(i) S~yU~N~ CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) sT~NnRnNR.~.~ single ~ (D) TOPOLOGY: linear (ii) M~T~R~J~R TYPE: cDNA
(iii) ~Y~u~ CAL: NO
(iv) ANTISENSR: NO
RECTIFIED SHEET (RULE 91) ISA/EP

W O 96/31609 . PC~rrUS9~'0 (v) FRAGMENT TYPE:
~vi) ORIGINAL SOURCE:
(Xi ) ~UU~N~ DESCRIPTION: SEQ ID NO:7:
G~~ AAT TGTGCTGTCT GG 22 (2) INFORMATION FOR SEQ ID NO:8:
U~N~ ~P~TT'~T~TICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRZ~N~ )N~ S: ging1e (D) TOPOLOGY: linear (ii) M~T~CTTT~T' TYPE: cDNA
(iii) ~Y~Ol~LlCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(i) ~i~!;~Ul!;N~I': CH~rTT~'~T-~:TICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STI7PNI)~CI)NI~:CS: sinyle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~ul~LlCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CACAGCCAGT ~ C~l~A AGG 23 (2) INFORMATION FOR SEQ ID NO:l0:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STR~Nn~N~c~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(Xi) ~ U~N~ DESCRIPTION: SEQ ID NO:l0:

(2) INFORMATION FOR SEQ ID NO:ll:
RECTlFtED SHEET (RULE 91?
ISA/EP

CA 022l7367 l997-l0-03 WO 96/31609 , PC~rrUS9G/~2 (i) ~Uu N~ r~ARArT~RT~TICS U ~ U~, ~B
(A) LENGTH 20 base pairs (B) TYPE nucleic acid (C) ST~ANl~l)N~Cs single (D) TOPOLOGY linear (ii) MOT~T'r~TT~ TYPE cDNA
(iii) ~Y~l~llCAL NO
(iv) ANTISENSE NO
(v) FRAGMENT TYPE
(vi) ORIGINAL SOURCE
(xi) _~U~NL~ DESCRIPTION SEQ ID NO 11 CATAGCTATG GCAACTCCGG _ - 20 (2) lN~ ATION FOR SEQ ID NO 12 (i) ~i~;yU~;NLlS CHARArT~!RTcTIcs (A) LENGT~ 3488 base pairs (B) TYPE nucleic acid (C) STRAN~ N~S single (D) TOPOLOGY linear (ii) MOLECULE TYPE Genomic DNA
(iii) ~Y~u~ lCAL NO
(iv) ANTISENSE NO
(v) FRAGMEN-T TYPE
(vi) ORIGINAL SOURCE
(xi) x~yU~NL~ DESCRIPTION SEQ ID NO: 7 2:
AAGCTTGGTA TGGATTCGTC AGCGCCAAGC C~ll~lG CAl~LGCCL~ AcTGr~AA~rc 60 GAAllCC~l~ AGCC~uLAC RRCAATGGCA ArCCrA~rGT TAL~ ~lu GCTGAATGGT 120 LlC~GLllAC GCAATTGTTT GTGGCAGCWG CGTGGGCTAA ATGTARGTTG lL~ll~ll~ 180 CACTGCARGA TGGATGGGTA G--Ll-l~LGC L~CL1LLGe1 A~l~l~ARC ~ll~GLluAC 240 lulG~lllAT TrAr~;rATGc CCATGCCCAT GCTAGATTGA TA~l~CLAT TCTAATGGTA 300 GGTGGCGGTA AGGTTTATTA AGCTGYAGYA TCAGTAGGTA ACCTCATGAA TCAG~lllA 360 AGCACACCTT l~LLlll~lG TGGGTGCATA AGGAATGCAC ll~LllC~l lCCLl~ATAG 420 l'~lll'~S'l'LA l~l~l ATTC TACCAAGTGG GTTACTGTAA CATTGCACTC TATGATGGTT 480 G~1~L1~1~ CAl~lllYlG Ll~CLLLl~G ~lulLlAATA CCTGCATGTA ACTGATGACC 540 YYLYLllATG TATCATATAG ATTACATCCT lll~ll~lAC ATCTCAATTC TGAAAAAACA 600 Al~Llll~CA TTLTTAGCGC ~l~luLACA AGGA~AAGGA ~ll~lACCT GCAACTTTTT 660 'llllL~AGAA AAAACAAACC ~ ~luAAAG G QGTGATCA TTTAGTATAA AGAAAATTTG 720 ATTTACTTTC TTCAGAGAGA ATATKCCAAR rAAA-rAATTT TCTTACTGTC Tr~prcrArr~A ' 780 AARcrAAAT~ GGTGATTCAT AGAATGAGAR AAArAArCTG TTGCCATTTG GGGACCTTGT 900 TGTGTACTCA TTAlCCLCCC TGCTCAGGTT GA~lllLLl TGCCACTGCC ACCLL11~C 960 CLLllLllAT ArPArrATCT CCATTGAAAA AGAlllluLA CTACATTTGG GLllL~lATG 1020 ACAP~AAGG APAATAAA~AC TAAArPr~rpr~ AAACATAGTA TAATTATAGG TAAAAr~GTTc 1080 TGG Q AGTTT GAGTGGAAGA GACLll~lA TATTTGGACA TATTTCACTA GTAAPTArTT 1140 TTATAATGTT GL~ ATTT CTTTGATATT rAAAArTTCC TAAGAGTATT CTGCTAGAGC 1320 , TCTGATGGTG 'l'Lllll~CLl CTGTCAGATT TTCrPr~r~pr~T lll~ll.'CLl TTTTATGGCA 1380 Ll~L~C~lll rA~AAr~TCT TCA~ATTGTGC l~l~lGGGCL ACGGAACGAC AATGT QCAG 1440 CTTGGATTAG CCGCAGCTGC CTCAAAGGCC TTGCCACTAC TrccTAATcG CCAGAGAAGT 1500 CATACTCGTT CALlCL~luA l~GC~LAGAT GGGGTATCAG ATGCQ~AAA GCACAGCCAG 1620 l~l~l~lC AAGGTACTGT GAATATCTTT TGATACAAGC TAAAATTTTG rTArArAATA 1680 TATATTTAAA GA~ll-lllC TTGGCTGGTG ll~lllATTT GTTTAACATS Cr-AAAr~GCC 1740 TCTAGTTGGA ll~llAGGT GGSCTGAATA CCACTCCTTA AG~L~ll~AG 'l'll~Lll~lL 1800 RECTIFIED SHEET tRlll F 91) ISA/EP

W O96/31609 . PCTnUS96/04625 CC~N~AGC GAATTTTAGG CTAGGGTTAC CrCCCrArrC CrArCrrAAT CTG QCAGYC 1860 ~ W Y~Y~Y C~Y~ATA TAGGCTACGA TGTCATTGTG TA1~ ~G Cr~X~,... 1920 AAGAGTTTTC TTGACCTTTG TT~r~ArATC TTAAT~ATAr AATGTCCAAG GG~U1~A 1980 CCCTGTAGGT CGA~ L ~ ~ L ~A ~ ~ ~ ~ ~ ~ ~ ~AA CATGGTAATG TTTGAAGCCT CATTCTAGGT 20 40 Rrr~TATAr ATATGCTCAC TGCTCAGTTT CAAATGTTTG TCTGCATGTA ~lC~ ~ 2100 GCATTATCGA CCTCCCAAGT rAr~Gr~rCTT CCGAAGTGGA TATTTCACAG T~ArrArT~C 2160 A~TATTTTGC GTA~...~. TTTGGA~AAA rAAAATATTC TCAGCTTATT T~T~rTArCT 2220 TCGCTAATAC TGA~TGCTG TCTTAATGTC ~.~.~. ATGCTCAATC TTTCATAGTA 2280 AATGCTGCAA AATATGTGAT GTAACTGTTG rAArAr~rCC Ar~G~ArCTGT TATTTAGAGC 2340 ATGGTGAATG ~ ~A GTTATATGAT GTAGTTATAG CTCATGTTGA AGAATTAGTT 2400GCA~1~L..~ CTGrArAATG GTCACTTATT ATA~ATCATA TCTGrATAr~ CA~ ~AC 2460 '~L~L~G~ GTAAATGCCC GCA..... ~ ArAAAAATTT AAA.~.. ~ rCT~AATTGG 2520 ACATATATGA TArArAAArC TGATTTGAAC ~L~ATT TTTGACATCC ATGCATATTG 2580 TCA~1~,,~, rAAAArAATA CTAATCCTTT ~~ ~L~ .L..~AGTG GATCTGAGGA 2640 TCCTAGGG W CCAACAGATT CTTATCAAAT GAAT W GATT ATCAATGAAA rArATAATGG 2700 AAGACATGCC TCA~LC~A A W ..~..~a A....~.~ Gr~rTArGTG GrAAAAr~rC 2760 CCGGACAT W GCTAATGATA ~---.~ATC TrArAAr~GCA ATTCAACTCA TAGCTATGGC 2880 AACTCCGGAA GACATGAGGA TAAATGCAGA ACACATTAGA A--~-~ACC AATTACGTAG 2940 A W TGCCTGG TGrAArAAAr AATAATAACT ACGCCAATGT TCAACTCATA GTGGAGGTTA 3000 GCCTTGCTAA ~ AGTT TACTACT W T ~-G~.~...C CTTTATTTGT TGTATAATGA 3060 TTrArATATT TAAGTAGAGA AATTTATATT .~.U~.~.~C ~L~AA GTCCAATTGT 3120 CATCATTAAC TGTGAAATAT TGCAGATGGC ACAAAAACTA ~l~...~.~ ~l~l..~CC 3180 ~ ~W~ CA.~....~ AGAATCCTGA ACTGCCAGAT GCATTGACCG r~AAAr~rAT 3240 C~ L ~~~ GGccrArcTG SATCATCAAT GAA1~ rrArAT~Ar~ TCGGCTCAGC 3300 TCTCATTGCT rAArrArCCG W ~N~C~AAC ~ ~ AGTGGATCAC ATGTGAGTCT 3360 CA~ ~A TTACTATCCG C~ CAT ~~ ~1 TTCATATTCT AATrArArTA 3420 (2) INFORMATION FOR SEQ ID NO:13 (1) ~UU~N~ r~ARA~T~RT~TICS
(A) LENGTH 1328 bage Pair8 (8) TYPE: nUC1eiC aCid (C) STR~N"~,~N~qS: Sing1e (D) TOPOLOGY: 1 inear (ii) MOT~rUT~ TYPE GenOmiC DNA
(iii) ~Y~U1~11CAL: NO
(iV) ANTISENSE: NO
(V) ERAGMENT TYPE
(Vi) ORIGINAL SOURCE
(Xi ) ~UU N~ DESCRIPTION: SEQ ID N0:13:
GAA~1~ AGCC~ AC GGCAATGGCA ArCrrAr~GT TA~-~W L~ GCTGAATGGT 60 CTCGGCTTAC GCAATTGTTT GT W CAGCTG ~L~1~A ATGTAGGTTG ~C1C1~ G 120 CACTGCA W A TGGATGGGTA GC-.~L~GC CGC---~L AGTGTCTAGC ~Ll~Ul~ACT 180 ~1~111ATT rArGrATGCC CATGCCCATG CTAGATTGAT AGGTCATAGG TGCCATTCTA 240 GGTTTAAGCC CAC~Llc-iC '~l~lWW TGr~TAArr~ ATGCACTTGG ~11~11CUC 360 TGCTAGTCTT TGCTCATGTG TCATTCTACC AA~1~A CTGTAACATT GCACTCTATG 420 ALW ~ L~ ~ ~aTC ~----G~--C ~.~..~. CTAATACCTG CATGTAACTG 480 ATGACCTTCT TTTATGTATC ATATAr-ATTA CA.~L...~L TGTACATCTC AATTCTGAAA 540 AACAATGTTT TGCATTCTTA G~L~L~1~ CACAAGGAAA AGGAGGTTTT ACCTGCAACT 600 '1L1~ ; AGAAAAAACA AAC~...~.~ AAArGrAr~TG ATCATTTAGT AT~AAriAAAA 660 TTTGATTTAC 11~C1LCAGA rAr~AATATTC rAAArAAACA AL~ -AC AGTCTGAGCC 720 AAAAAAACCA AATAGW TGAT TCATAGAATG AGAA~AAGAA C..~L.~CCA 1~G~;ArC 840 ~1L~1~1A CTCATTATCC CCC~L~.~a GGTTGAGGTT L~UAC TGCr~rCrCT 900 TGG~C~ TTATACAACC ATCTCCATTG AAAAArATTT TG QCTACAT ~GGU11CG 960 RECTIFIED StJEET (Rl)LE 91) ISA/EP

CA 022l7367 1997-10-03 WO 96/31609 , PC~rrUS9~'01'2 T~T~Ar~AAA ~Arr.~AA~T~ AAArTAA~rA Gr~r~A~r~T AGTATAATTA TAGGTAAAAG 1020 vllL~lvGC~A GTTTGAGTGG T~r~r-~rCTT TGr~TATTTG r-~rAT~TTTC ACTAGTAAAT 1080 AVLLLL~LAA AATGTTCATG AALGvLvGCC A~AACTTG ATAAGATCTC AACATGGCAG 1140 V~L~L~A AATr-~r-~r-r-~ AAACTGGA~A CATr~r~AAT A~LLLLLAGC GAVLVVC~LA 1200 TAAATTATAA lvLlv~LC ALLL~LLL~A TATTCA~AAC TTCCTA~GAG TA'LL~V~LA 1260 GAG~L~1VAT GVLV1~LL L, GC~-~,~L - A GA~lL L L~AG GAv- LLL~L' C~L lLLLAT 1320 (2) INFORMATION FOR SEQ ID NO:14:
(i) ~UU~N~ CHP~rTR~TSTICS:
(A) LENGTH 1565 base pairs (B) TYPE nucleic acid (C) STR~ N~qC: single (D) TOPOLOGY: linear~
(ii) II~T~T'rl~ TYPE Genomic DNA
(iii) ~Y~u~ lCAL: NO
(iv) AWTISENSE: NO
(v) FRAGMENT TYPE
(vi) ORIGINAL SOURCE:
(xi) ~Uu~ DESCRIPTION SEQ ID NO 14:
AGCATCCCTT GGGATTGTGA TNACTCACAT A~ATTCTTGC GAAL~LVLLvA CATTCTAGTG 60 ATTTGAGTTC CGTTCTAGTG TGCTAGTCAN TTGAGCTCAA VL~lLVVLLl TAlvLvLv~v 120 TATTCACTGT GALiLLLVlV LCV~V~VlVA VLLVLLVATC ~;l LCC~LlVC IC~ViVATTC 180 L, ~V1VA-A-AT CTTTTGAAAG GGCGAGAGGC TCCAAGCTGT GGAGATTCCT CGCAAGTGGG 240 ATTAAGAAAA GCAAAGCAAC AC'Cvlv~lAT TCAAGTTGGT CTTTGGACCG CTTrAr-~r~r 300 GTTGATTGCA ACC~1CVLCC vllvGvACGC CACAACGTGG AGTAGGCAAG ~VllVVl~ll 360 GGCrr-~rr~ CGGGATAACC AC~vlv~AT ~l~lvlvATT GATATCTCTT GGTTATTGTG 420 llvLvll~AG A~L~11~L~1 AGCCACTTGG CAAATTACTG TGCTAArA~T TAATCAAGTT 480 TTGTGGCTTA AGATTTTGAA GTATTACAGG ATCTGCATCA lVVl~lVlVl CTCCACAGCT 540 ATGACACCCA CAGGAATTCA ~VlVllC~l~ GGAGCCACTC TTGGATGACC T~r-r-~TT 600 ATTTCTAACC GG~llvLACA QTATGATGC ATCAAGAGAT GAvlvllllA CTATGCGAGG 660 GGCCATGCTT ATGACCATAA GTAAT ~ TCC l~vlllAQAA A~v~llG~ll CTCATATGGT 720 TCATGGGAAA TTCGCATGCC lC~llvlvvl GAAAATGTCT GGACAAAACA GCTGAAGAAT 780 G~lCvlAAAT ~lLv~ AT GGGAAATCGC CAATATATTG ATCTTGATCA TTCTTATTGC 840 TTGGATGCAG A~lC--~lllv ATGGAACGAT AGACTTCGAA CAAAACCTAA AACCTATTAT goo GAlc~lC~AA llll~vATGA AATCATCACA ~llGvlvATT TCAAGAACTC AAAAAYTTAC 960 TATTTTAGTT GACAATAAAG TAGATGGGCA TCATCCTGAG lll~.~lllGV CAL~vlvlcv 1080 TTGAAGCGAA TGTACAATCT TTATGTGATG vlvv~l~llA ATGCAGGTAA CTAvllllll 1200 TTTATGCTTT ATTATTAATT AGTTGGATAA A'1V~111~.~A lll.~l~ATTG TTA~ANTGCA 1260 Al~G~LC~AG TTGGATGGNA ACAGCCANGT AATTTATGCA GAAGNAGNAG NlvvlvvlAC 1320 A~GvNll~AG ATTGATGGAA AGANATGTTT ATTGCAGGTA AATAWTCCCT l~ll~lllA 1380 TAllllLVlL GTh-TGATTGT ATAAWTTTGN TAGATTATTT GTATAATTTA TTATTGCATT 1440 TCACCCCACT AANTTATTTT TAAAAGATGG vlllLVll~l ~ ~V~ 'AGC Nr~rr-~r~TC 1500 ACATAAGNAA ATTGTGATTA A~1~11V11L llllv~AGNA TGACCATGAT CCATCAAAGT 1560 (2) INFORMATION FOR SEQ ID NO:15:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 1168 base pairs (B) TYPE: nucleic acid (C) sT~Nn~nN~s: sing1e (D) TOPOLOGY: linear (ii) MnT~T~'CUT~ TYPE: Genomic DNA
(iii) ~YY~l~LlCAL: NO
RECTIFIED SHEET (RULE 91 ISA/EP

W O 96/31609 . PCTrUS96/~462 ~ 90 (iV) ANTISENSE: NO
(V) FRAGMENT TYPE:
(V1) ORTGTN~T~ SOUROE:
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:15:
ACATAAGCTG GGTTAGTAGT GGTGAATTAG TGGATTATTG A~ L~1G ~7~r~Arr~rN 60 ~ LW'~ TCGAGA~AAA ACATGGAAGT GTGTATTGCC AGTGCTTATT ~GG~ATAT 120 GAGGGAATTW AMATTACATT TGTGACTGGG CGGACTGTAG r~T~rr~rTT A.~ LCG 180 ATTGGTATAC GGTGCATACA G~KS--~ACC AGCTATTATT TTAACAGGGT ~L~e~ 240 GAACAAGTCC TTGGGCGGGA AGTGTACAGC TCCrAr~TGC AG~L-~ TCCTAAr~TC 300 ATGGCGACCA A. W .~--~. CCACCTCACT GTTCCAGATG ACCTTGAAGG L~LC~AAT 360 ATATTGAGGT GGCTCAGCTA ~~..C~-G~A AACATTGGTG GAC~.~.LCC TATT~rr~A~ 420 ~C'1'W ACC CTCr~r~r~r- A~..~. TACATCCCTG Ar~r~rATG CGATCCACGT 480 GCAGCTATCT ~1 W L~'1'AGA TGACAGCCAA r~Gr-~AATGGT L~LW LAT GTTTGACAAA 540 GA QGCTTTG Tr,r-~r-~r~TT TGAAGGATGG Gr~A~Ar~r~ TGGTTACTGG CAGAGCAAAG 600 CTTr,r-~r-r-~A TTCCATGCAT CTTAATA~AC ACA~LLGGCC CTT~A~r~r~ GTGAACTTCT 660 TGAACAAACC A~ACTAAGTG AA~.~.~C AAGCGTTGCA Ar-~r-CCTTT CGGATCTGGG 720 GATGCATAAG Gr-~r-~TGA GTATTAAGGA TAACATGGAA GATTTAGTCT ~L~CCC~ATT 780 AC~1~LL~AA GATGCTCTGA 'LLLCL~L~L~ TGATTACAGT GATCGAACTG TTCAGCAGAA 840 AGTGATTGAG ~C~T~r~T~T CACGATTGTA CCAGGTATTA TATr~rT~A CTTAATGTCT 900 TTCATTTGAC ~rA~r~AC ATTr-Ar~AAT GAGATGCTGA CGAL.W ~-~ A~ATTAACTG 1020 G~11~1~AGAA ATTGTGATCT CCr~ArTTGT TAATGCACAA 'L~LW ~L AACTTGCCAA 1080 TA11LLLLCA GCCTCATCTT GTTNAGGATA GCANCCAAAT GAAATCCAAG GAl~l~LGC 1140 (2) INFORMATION FOR SEQ ID NO 16 (i) ~UU N~ rr~RArTRRT~TICS:
(A) LENGTH: 63 8 ba8e PairS
(B) TYPE: nUC1eiC acid (C) STR~NI)~I)N~C~ ~ing1e (D) TOPOLOGY 11near (ii) MOr~r~CUT~r~ TYPE: GenOmiC DNA
(iii) ~Y~L~ CAL: NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYPE
(V1) ORIGINAL SOURCE:
(Xi) ~UU~N~ DESCRIPTION SEQ ID NO 16 CTCCCAATAT TGTCATGAGG ~TTG QTCCC AGGTTAGTTT L L - L ~ CTGAAATTTA 60 TTATTGACAA ATG Q CTAAT GGT QT Q TA TTTGGAGATT AArATATTTA TCTTAATTGA 180 TGGGAACTCT TGAAAATGAC AA~ ~AG QGATAATTA A Q~..L... AATAAAAAAA 240 TCCATTTGTG T QATTTA Q GTCGGTATCA TGr~AAAr~GTT GT QTAATGG cTGr~Ar~ANAA 360 ACAACA QTC L~ cA A Qe L~GG GAGAAGANGT TTTACCTTTT TTrrTAAAAT 420 TA~LLLLl~l ArT~AATTGT ATAATTTTTC CAATATTCTC QTGATTATT GAA~L~L~l 480 ~l~LL~AAAC AGC QAAA Q L~LLLC~ATA CTTTACACCT TTATTTTTTA GATGGAACCT 540 GAATCCG Q C TTGAATT QG 'L L~'l L~L~A TCAATATG 638 (2) INFORMATION FOR SEQ ID NO:17 ( i ) ~UU N~ CHARACTERISTICS
(A) LENGTH: 558 base pairs (B) TYPE nucleic acid (C) STR~ N~'~ single (D) TOPOLOGY linear RECTIFIED StlEET (RULE 91 ISA/EP

CA 022l7367 l997-l0-03 W O 96/31609 . PC~rrUS96/04625 (ii) M ~~T~-~ TYPE: Genomic DNA
(iii) nr~u~ CAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) n~TGTNAT~ SOURCE:
(xi) ~Uu~N~ DESCRIPTION: SEQ ID NO:17:
GGTAACCACC ArA~ ,~GG CG ~ TAATGG CCGTA~Ar~G ~lCC~ATT CGCCATTCAG 60 ~l~C~AACT ~ll~;~A~G GCGATCGGTG ~ ~ll CGNTATTACG C QG~l~C~ 120 AAAGGGGGAT GTGCTGCAAG GCGATTAAGT TGGGTAACGC CA~lll L C C QGTCACGA 180 CGTTGTAA~A CGACGGCCAG TGA~C~ TAATArr-ACT CACTATAGGG CGAATTGGGT 240 AC~GCCCC CC~.o~AGGT CGAC ~ GCAG GTCAACGGAT C~TA~GGGC ~AA~A~ATTC 300 TTATCAAATG AATGGGATTA TCAATGAAAC A~ATAATGGA AGACATGC ~ CA~l~l~AA 360 G~ll~l~A ~ L L ~lGCGG ~A~T~GTGG CAAAACACCA ATTCACAGTA TATTAGTGGC 420 CAACAATGGA ATGGCAGCAG CAAAATTTAT GAGGAGTGTC CG~-A~ATGGG CTAATGATAC 480 lll'~ATCT GAGAAGGCAA TTCAACTCAT AGCTATGGCA A~lC~AAG ACATGAGGTA 540 A~TGCAGAAC ACATTAGA 558 (2) lN~O.~IATION FOR SEQ ID NO:18:
(i) ~yU~N~- C~ARACTERISTICS:
(A) LENGTH: 975 base pairs (B) TYPE: nucleic acid (C) STRA~ : single (D) TOPOLOGY: linear (ii ) M~T.~.T' TYPE: Genomic DNA
(iii) nY~ llCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) O~TGTNAT~ SOURCE:
(xi) ~yU~NC~ DESCRIPTION: SEQ ID NO:18:
GAATAATCTG CCTGCAGCTC AA~lla~l~L TGGAATGGGC ATAC~lelll GGCAGATTCC 60 AGGTAATTAC Q ATTTACCA ACTTATTTAG l.C~LlATTG TTTTATTCTC TAAllLl~lA 120 CTTATGTAGA AATCAGACGT TT ~ATGGAA TGGACTATGG AGGAGGGTAT GACATTTGGA 180 GGAAA~ACAGC AG ~ CTTGCT ACACCATTTA ATTTTGATGA AGTAGATTCT CAATGGCCAA 240 AGGGCCATTG TGTAGCAGTT AGAATTA ~A GTGAGGACCC AGATGATGGT TTCAAACCTA 300 ~la~l~G~AA AGTGAAGGTA A~lLLl~LAG ATGACATGTA TTATATATCG TTCAAAGAGA 360 TTAAGTTTGG TTA~ATGA ~ A~l~ll~AT TTTTTATCTT TCAGGAGATA AGTTTTAAAA 420 GCAAGCCTAA L~lll~GGCC TA~ll~l~AG TAAAGGTAAC TTGTTAACTT TAGTACGCTG 480 TCACATTATT ~llc~ll~lG AAAATAATTT GAAC~llcl ~TTTGTATTT TAACCATCCA . 540 lc~l~l~ATT TASCAGAGCA CACAAATATT TGCACTGACC CCC~L~C'C~1 TAlcl~lll 600 CA~l~l~L~ GAGGCATTCA TGAATTTGCT GATTCTCAGT TCGGTATGTG TAAA~CAA~ 660 GTALLoLlLa TAATTTATAT l~lC~l~AA TTTTGA~ATA TTG~l~ll~ cGTTArA~A 720 CAW~LllllG CATATGGG ~ ~ CTAGATCA GCAGCAATAA CAAACATGAC TCTTGCATTA 780 AA~NAGATTC A~A~l.~l~G AGAAATTCAT TCAAATGTTT GATTACACAG TTGATCTCTT 840 AAATGTTAAG AAATATTAAC CAC~llllAA ATCACATTTT CCATTATGTT TGATTCCATA 900 TCATTAATTT TGAlllL~lA TTATGGCTAA A~-1~L~1~ CTAlllL~L ATTATCCCAG 960 (2~ lN~O~IATION FOR SEQ ID NO:l9:
(i) ::i~':Uul!;N~ !; CHpR~T~r~Tc:TIcs:
(A) LENGTH: 852 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~:-C-C: single (D) TOPOLOGY: linear (ii) ~nr~RcTJn~ TYPE: Genomic DNA
RECTI~tED SHEET (RULE ~t~
ISA/EP

WO 96131609 PCT/US9.''~S'~';

(iii) llY~ CAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) ~S~;yU~;N~:~; DESCRIPTION: SEQ ID NO:19:
GGATCCTAGG GGGCt~ArZ~r~ ATTCTTATCA AATGRAATGG GATTATCAAT rZ~ r~T~ 60 ATGGAAGACA LGC~:1~AGTG TCCAAGGTTG TTGAATTTTG TGCGGCACTA GGTGGr~A 120 rZ~rr~Z~TTCA CAGTATATTA ~ -GC'-~Ar~ ATGGAATGGC Arrz~r.r~z~A~ TTTATGAGGA 180 C~7AC AL~,G~:~AAT GATACTTTTG GATCTGAGAA GGCAATTCAA CTCATAGCTA 240 TGGCAACTCC GGAAGACATG Arr.I~TZ~ATG r~r~Ar2~r~T TAGAATTGCT r~7~rr~ATTAc 300 GTAGARGTGC C~ AAC AAACAATAAT AArTZ~rGrr~ ATGTTCAACT CATAGTGGAA 360 GTTAGCCTTG CTAATCTGTT AGTTTACTAC~L~71~ 7~.L~7 111~ AT 1~711~,1ATA 420 ATGATTGACA TATTTAAGTA GAGAAA3~TTATAi11c~ 7L1-71 GGAAGTCCAA 480 TTGTCACCAT TAACTGTGAA ATATTGCAGATGGr2~r~z~ ACTAGGTGTT ~ 7~ 7L11 540 ~7C~ 7~711~7 GG,GTCATGCT TCTGAGAATCCTGAACTGCC AGATGCATTG ACCGCAAAAG 600 GGA1c~1111 '1'~:11~7CC--A CCTGCATCAT CAATGAATGC lll~ r.~T AAGGTCGGCT 660 CAGCTCTCAT TGCTCAAGCA GCCGGG~,1CC CAA~ 7C TTGGAGTGGA TCACATGTGA 720 GTCTCACTCT TTGATTACTA 1CC~7C~ ,1C TCA11G~1~ ATA TTCTAATGAC 780 ACTAAATTTA GGTTGAAGTT CCATTAGAGT G~:1G~11AGA CGCGATACCT r.Ar~-~r-~TGT 840 (2) INFORMATION FOR SEQ ID NO:20:
(i) ::i~;Uu~;N~:~; C~ARz~rTRRTcTIcs:
(A) LENGTH: 231 base pairs (B) TYPE: nucleic acid (C) STRZ~N~ I)NI.:.qS: single (D) TOPOLOGY: linear (ii) MOT-RCTlT-R TYPE: Genomic DNA
(iii) ~Y~J1r~11CAL: NO
(iv) ANTISENSE: NO
(v) FRAG~NT TYPE:
(vi) ORIGINAL SOURCE:
(xi) ~i~;5,?u~;N~:~; DESCRIPTION: SEQ ID NO:20:
AA11Ce~ ~ GGTGTTATAG CTGTGGAGAC ACAGACCATG ATGCAGCTCA 1CC~:1G--1~,A 60 TCCAGGTCAA CTTGATTCCC ATGAGCGATG 1~711C~:1C~7 GCTGGACAAG 1~71G~711cCC 120 AGATh-CTGCA ACCAAGACAG CTCAGGCATT ATTAGACTTC AACCGTGAAG GATTGCCTCT l80 GTTCATCCTG GCTAACTGGA GAC;G~ 1C 1~,ACAG AGAGATCTCT T 231 (2) lN~ L_TION FOR SEQ ID NO:21:
(i) ~7~;UU~;N~'~; C}I~RZ~rTRRTqTICS:
(A) LENGTH: 207 base pairs (B) TYPE: nucleic acid (C) STRP-N~ )Nl~:~qlq single (D) TOPOLOGY: linear (ii) M~T.RrrTT.R TYPE: Genomic DNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(Xi) 51~ UI:;NCI:; DESCRIPTION: SEQ ID NO:21:

AATTCATGCA TCTTAATA~A CACAGTTGGC CCTTAAAGCA AGTGAACTTC TTGAACAAAC 60 CAAACTAAGT GAA~:1cL~7LL CCAGCATTGC AAGAAGCCTT TCAGATCTGG GGATGCATAA 120 RECTIFIED SHEET (RULE g1) ISA/EP

W O ~6/31609 . PC~rrUS96104625 GGrAr-~AATG ACTATTAAGG ATAGCATGGA AGATTTAGTC ,~l-;NC~AT ~C~1~11~A 180 AGA~ ~ll All '~L11~1 TTGATTA 207 (2) INFORMATION FOR SEQ ID NO:22:
;UUlSN~c r~pRArTRRTqTIcs (A) LENGTH: 180 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~ .q single (D) TOPOLOGY: linear ( ii ) MOT~RCUT~R TYPE: Genomic DNA
(iii) ~Y~ul~LLCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(Xi ) ~ ~:UU~N~ DESCRIPTION: SEQ ID NO:22:
ATAGACCTGT CGCATACATC CCTGAGAACA CATGCGATCC G~lG~AGCC A1CC~1 WN~ 60 TAGATGACAG rrAAr~r~AA ~llWWl~ GTA~lLl~A CAAAGACAGC ~ w AGA 120 CATTTGAAGG ATGGGCAAAA ACA~l w llA CTGGTAGAGC AAAGCTTGGA Gr~Arr-~ATT 180 (2) INFORMATION FOR SEQ ID NO:23:
(i) ~;UU~;N~-~; r~ARA~TRT~?TqTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRA~l)~ Nl~ s: single (D) TOPOLOGY: linear (ii) MOT~RCUT~R TYPE: cDNA
(iii) ~Y~l~llCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(Xi) ~hyU~N~ DESCRIPTION: SEQ ID NO:23:

(2) INFORMATION FOR SEQ ID NO:24:
(i) ~UU N~: CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~:-~S: single (D) TOPOLOGY: linear (ii) M~T~RCUT~R TYPE: cDNA
(iii) ~Y~LlCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
_ (vi) ORIGINAL SOURCE:
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:24:
CCTCTACGTA All~AGC 20 (2) INFORMATION FOR SEQ ID NO:25:
(i) ~U~N~ CHARACTERISTICS:
RECTlflED SHEET (RULE 91) ISA/EP

W O 96~1609 . PC~rrUS96/04625 (A) LENGTH: 20 base pairs (B) TypE nucleic acid (C) ST~N~ N~S: single (D) TOPOLOGY: linear (ii) M~T~T~1T~ TYPE CDNA
(iii) ~Y~Ul~ l 1CAL: NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYPE:
(Vi) ORIGINAL SOURCE:
(Xi) ~UU~N~ DESCRIPTION SEQ ID NO:25:
C~llL~TG GCA~1~GCG 20 (2) INFORMATION FOR SEQ ID NO:26:
(1) ~U~N~ ~T~T.~TICS
(A) LENGTH: 22 base pairs.
(B) TYPE: nucleic acid (C) STR~NII~I~N~.CS single (D) TOPOLOGY: 1 inear (ii) MOT~T~TT~ TYPE CDNA
(iii) ~Y~GI~11CAL: NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYPE:
(Vi) ORIGINAL SOURCE
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:26:

RECTIFIED SHEET (RULE 91) ISA/EP

Claims (50)

WHAT IS CLAIMED IS:

1. An isolated and purified DNA molecule comprising a DNA sequence encoding an amino terminal chloroplast transit peptide operably linked to a DNA sequence encoding a maize acetyl CoA carboxylase.

2. The DNA molecule of claim 1 which comprises a DNA sequence comprising SEQ ID NO:5.

3. The DNA molecule of claim 1 which encodes a maize acetyl CoA
carboxylase comprising SEQ ID NO:6.

4. An isolated and purified DNA molecule comprising a DNA sequence comprising SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:l9, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22.

5. An isolated and purified DNA molecule which is complementary to a DNA sequence comprising SEQ ID NO:5, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:l9, SEQ ID NO:20, SEQ ID
NO:21, or SEQ ID NO:22.

6. A method of imparting cyclohexanedione or aryloxyphenoxypropanoic acid herbicide tolerance to a plant tissue comprising:
(a) introducing an expression cassette comprising a DNA molecule encoding a maize acetyl CoA carboxylase, which comprises an amino terminal chloroplast transit peptide, operably linked to a promoter functional in a plant cell into cells of a susceptible plant so as to yield transformed plant cells;
(b) regenerating said transformed plant cells to provide a differentiated transforrned plant; and (c) expressing the DNA molecule encoding the maize acetyl CoA
carboxylase in the cells of the differentiated transformed plant in an amount effective to render the plant tolerant to levels of a cyclohexanedione or an aryloxyphenoxypropanoic acid herbicide which are toxic to a corresponding susceptible plant which does not comprise said expression cassette.

7. The method according to claim 6 wherein the DNA molecule encodes a maize acetyl CoA carboxylase, the activity of which is not inhibited by an amount of cyclohexanedione or aryloxyphenoxypropanoic acid herbicide that inhibits the activity of an acetyl CoA carboxylase which is sensitive to inhibition by a cyclohexanedione or aryloxyphenoxypropanoic acid herbicide.

8. The method according to claim 6, wherein DNA molecule encoding the acetyl CoA carboxylase is expressed in the cells at about a 2- to 20-fold amount over that of an acetyl CoA carboxylase which is present in the corresponding sensitive plant and which is sensitive to inhibition by a cyclohexanedione or aryloxyphenoxypropanoic acid herbicide.

9. The method according to claim 6, wherein the DNA molecule comprises the DNA sequence of SEQ ID NO:5.

10. The method according to claim 6, wherein the DNA molecule encodes a protein comprising SEQ ID NO:6.

11. The method according to claim 6, wherein the herbicide is selected from the group consisting of sethoxydim, haloxyfop, and mixtures thereof.

12. A transformed plant prepared by the method of claim 6.

13. A transformed seed of the transformed plant of claim 12.

14. A transformed monocot plant, which plant is tolerant to herbicides comprising:
(a) a native acetyl CoA carboxylase gene that encodes an acetyl CoA
carboxylase, the activity of which is inhibited by a cyclohexanedione or an aryloxyphenoxypropanoic acid herbicide, and (b) a recombinant DNA molecule encoding a maize acetyl CoA
carboxylase which comprises an amino terminal chloroplast peptide, wherein the DNA molecule is expressed in the plant in an amount effective to confer tolerance to the plant to levels of a cyclohexanedione herbicide or an aryloxyphenoxypropanoic acid herbicide which inhibit the activity of the native acetyl CoA carboxylase.

15. The plant of claim 14 wherein the recombinant DNA molecule encodes an acetyl CoA carboxylase comprising SEQ ID NO:6.

16. The plant of claim 14 wherein the recombinant DNA molecule comprises SEQ lD NO:5.

17. The plant of claim 14 wherein the recombinant DNA molecule encodes an acetyl CoA carboxylase, the activity of which is not inhibited by an amount of cyclohexanedione or aryloxyphenoxypropanoic acid herbicide that inhibits the activity of an acetyl CoA carboxylase which is sensitive to inhibition by a cyclohexanedione or aryloxyphenoxypropanoic acid herbicide.

18. A method for altering the oil content in a plant comprising:
(a) introducing an expression cassette comprising a recombinant DNA molecule encoding a maize acetyl CoA carboxylase, which comprises an amino terminal chloroplast peptide, operably linked to a promoter functional in a plant cell into the cells of a plant so as to yield transformed plant cells; and (b) regenerating said transformed plant cells to provide a differentiated transformed plant, wherein said plant expresses the DNA molecule encoding the maize acetyl CoA carboxylase in an amount of said acetyl CoA carboxylase effective to alter the oil content of the plant cells.

19. The method according to claim 18, wherein the acetyl CoA carboxylase encoded by the DNA molecule is expressed at about a 2- to 20-fold increase over the level of the native acetyl CoA carboxylase.

20. The method according to claim 19, wherein the oil content of the plant cells is increased about 1.2- to 20-fold.

21. The method according to claim 18, wherein the DNA molecule comprises SEQ ID NO:5.

22. The method according to claim 18, wherein the acetyl CoA carboxylase comprises SEQ ID NO:6.

23. The method according to claim 18, wherein the DNA molecule encoding the acetyl CoA carboxylase encodes a variant acetyl CoA carboxylase, wherein the variant acetyl CoA carboxylase has a specific activity which is different than the specific activity of the native acetyl CoA
carboxylase.

24. A transformed plant prepared by the method of claim 18.

99 A transformed seed of the transformed plant of claim 24.

A method for altering the oil content in a plant comprising:
(a) introducing an expression cassette comprising a recombinant DNA molecule which is complementary to SEQ ID NO:5, SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:l7, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, or SEQ ID NO:22 operably linked to a promoter functional in a plant cell into the cells of a plant so as to yield transformed plant cells; and (b) regenerating said transformed plant cells to provide a differentiated transformed plant, wherein said plant expresses the recombinant DNA molecule in all amount effective to alter the oil content of the plant cells.

27. A transformed plant having an altered oil content in the plant cells comprising: a recombinant DNA molecule encading a maize acetyl CoA
carboxylase which comprises an amino terminal chloroplast peptide, wherein the DNA molecule is expressed in the cells of the plant as an amount of said acetyl CoA carboxylase effective to alter the oil content of the plant cells relative to the oil content in the cells of a corresponding transformed plant.

28. The transformed plant of claim 27, wherein the transformed plant has an increase in oil content of abaut 1.2- to 20-fold in its leaves, seeds, or fruit above that present in a corresponding untransformed plant.

29. The transformed plant of claim 27, wherein the DNA molecule comprises SEQ ID NO:5.

30. The transformed plant of claim 27, wherein the acetyl CoA carboxylase comprises SEQ ID N0:6.

31. The transformed plant of claim 27, wherein the DNA molecule encodes a variant acetyl CoA carboxylase, wherein the variant acetyl CoA
carboxylase has a specific activity which is different than the specific activity of the native acetyl CoA carboxylase.

32. The transformed plant of claim 27, which is a dicot.

33. The transformed plant of claim 27, which is a monocot.

34. A transformed seed of the transformed plant of claim 27.

35. A transformed plant having an altered oil content in the plant cells comprising: a recombinant DNA molecule which is complementary to SEQ ID NO:5, SEQ ID NO:12, SEQ ID N0:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID N0:21, or SEQ ID NO:22 wherein the DNA molecule is expressed in the cells of the plant in an amount effective to alter the oil content of the plant cells relative to the oil content in the cells of a corresponding untransformed plant.

36. A method of producing a plant acetyl CoA carboxylase comprising:
(a) introducing a recombinant DNA molecule encoding a plant acetyl CoA carboxylase which comprises an amino acid sequence comprising SEQ ID NO:6 or a biologically active subunit thereof, into a population of host cells;
(b) expressing the recombinant DNA molecule in the host cells so as to yield plant acetyl CoA carboxylase or a biologically active subunit of plant acetyl CoA carboxylase, and (c) recovering said plant acetyl CoA carboxylase or the biologically active subunit of plant acetyl CoA carboxylase from said cells.

37. The method of claim 36, wherein the host cells are ~rokaryotic cells.

38. The method of claim 36, wherein the host cells are eukaryotic cells.

39. The method of claim 38, wherein the eukaryotic cells are yeast cells.

40. The method of claim 38, wherein the eukaryotic cells are plant cells.

41. The method of claim 36, wherein the DNA molecule comprises SEQ ID
NO:5.

42. The method according to claim 36, wherein the acetyl CoA carboxylase is expressed as crystalline acetyl CoA carboxylase.

43. An isolated, purified plant acetyl CoA carboxylase comprising an amino acid sequence comprising SEQ ID NO:6, or a biologically active subunit thereof.

44. A method of expressing an exogenous maize acetyl CoA carboxylase gene into a host cell comprising:
(a) introducing into a host cell in vitro an expression cassette comprising a DNA molecule encoding a maize acetyl CoA
carboxylase, which comprises an animo terminal chloroplast peptide, operably linked to a promoter functional in the host cell so as to yield a transformed host cell; and (b) identifying a transformed host cell which expresses the DNA
molecule.

45. The method of claim 44 wherein the DNA molecule encodes an acetyl CoA carboxylase comprising SEQ ID NO:6.

46. The method of claim 44 wherein the DNA molecule comprises SEQ ID
NO:5.

47. The method according to claim 44, wherein the host cell is a plant cell.

48. The method according to claim 47, wherein the plant cell can be regenerated into a differentiated plant.

49. The method according to claim 48, wherein the differentiated plant is a monocot.

50. The method according to claim 48, wherein the differentiated plant is a dicot.