CA2218139A1 - Acetyl-coa carboxylase compositions and methods of use - Google Patents

Abstract

The present invention provides isolated and purified polynucleotides that encode plant and cyanobacterial polypeptides that participate in the carboxylation of acetyl-CoA. Isolated cyanobacterial and plant polypeptides that catalyze acetyl-CoA carboxylation are also provided. Processes for altering acetyl-CoA carboxylation, increasing herbicide resistance of plants and identifying herbicide resistant variants of acetyl-CoA carboxylase are also provided.

Description

WC>96~32484 PCTJUSSli~'65a~5 DESCRIPIION

ACETYL-CoA CARBOXYLASE COMPOSITIONS AND MET~IODS OF USE

S 1. BACKGROUND OF THE INVENIION
The present application is a continll~tion-in-part of U. S. Serial Number 08/422,560, filed April 14, 1995, which is a contin~l~tion-in-part of U. S. Serial Number 07/956,700, filed October 2, 1992; the entire texts and figures of which disclosures are specifically incorporated herein by reference without disclaimer. The 10 United States government has certain rights in the present invention pursuant to Grant #90-34190-5207 from the United States Department of Agriculture.

1.1 Field of the Invention The present invention relates to the field of molecular biology. More 15 specifically, it concerns nucleic acid compositions comprising cyanobacterial and plant acetyl-CoA carboxylases (ACC), methods for making and using native and recombinant ACC polypeptides, and methods for making and using polynucleotides encoding ACC polypeptides.

20 1.2 Des~ lionofthe R~ te-l Art 1.2.1 Acetyl-CoA Carboxylase Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the first committ~-l step in de novo fatty acid biosynthesis, the addition of CO2 to acetyl-CoA to yield malonyl-CoA. It belongs to a 25 group of carboxylases that use biotin as cofactor and bicarbonate as a source of the carboxyl group. ACC catalyzes the addition of CO2 to acetyl-CoA to yield malonyl-CoA in two steps as shown below.
BCCP + ATP + HCO3 ~ BCCP-CO2 +ADP +Pi(l) BCCP-CO2 + Acetyl-CoA ~ BCCP +malonyl-CoA (2)~0 CA 02218139 1997- lo- 14 wo 96/32484 PCT/US96/05095 First, biotin becomes carboxylated at the expense of ATP. The carboxyl group is then transferred to Ac-CoA (Knowles, 1989). This irreversible reaction is theco,l",.i~ d step in fatty acid synthesis and is a target for multiple regulatorymech~nicms. Reaction (1) is catalyzed by biotin carboxylase (BC); reaction (2) by 5 transcarboxylase (TC); BCCP = biotin carboxyl carrier protein.
There are two types of ACC: prokaryotic ACC in which the three functional domains: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and carboxyltransferase (CT) are located on separable subunits (e.g., E. coli, P.
aeruginosa, Anabaena, Synechococcus and probably pea chloroplast) and eukaryotic10 ACC in which all the domains are located on one large polypeptide (e.g., rat, chicken, yeast, diatom and wheat).
E. coli ACC consists of a dirner of 49-kDa BC monomers, a dimer of 17-kDa BCCP monomers and a CT tetramer cont~inin~ two each of 33-kDa and 35-kDa subunits. The primary structures of all of the E. coli ACC subunits (Alix, 1989;Muramatsu and Mizuno, 1989; Kondo etal., 1991; Li and Cronan, 1992; Li and Cronan, 1992) as well as the structure of the BC and BCCP of Anabaena 7120 (Gornicki et al., 1993), and P. aeruginosa (Best and Knauf, 1993) are known, based on the gene sequences. The genes encoding the subunits of E. coli ACC are called:
accA (CT oc subunit), accB (BCCP), accC (BC) and accD (CT 13 subunit). accC and 20 accB form one operon, while accA and accD are not linked to each other or to accCB
(Li and Cronan, 1992). In cyanobacteria, accC and accB are l-nlink~d as well (Gornicki et al., 1993).
Yeast, rat, chicken and human ACCs are cytoplasmic enzymes consisting of 250- to 280-kDa subunits while diatom ACC is most likely a chloroplast enzyme 25 consisting of 230-kDa subunits. Their primary structure has been deduced fromcDNA sequences (Al-feel et al., 1992; Lopez-Casillas et al., 1988; Takai et al., 1988;
Roessler and Ohlrogge, 1993; Ha et al., 1994). In eukaryotes, homologs of the four bacterial genes are fused in the following order: accC, accB, accD and accA. Animal ACC activity varies with the rate of fatty acid synthesis or energy requirements in 30 different nutritional, hormonal and developmental states. In the rat, ACC mRNA is WO 961324~4 PCTlUS9~'C,511!~, transcribed using dirrer~llt promoters in different tissues and can be regulated by ~lt~ tive splicing. The rat enz~he activity is also allosterically regulated by a number of metabolites and by reversible phosphorylation (Ha et al., 1994 and references therein). The expression of the yeast gene was shown to be coordinated S with phospholipid metabolism (Chirala, 1992; Haslacher et al., 1993).
Much less is known relating to plant ACC. Early attempts at characterization of plant ACC led to the suggestion that it consisted of low molecular weight subunits similar to those of bacteria (Harwood, 1988). More recent efforts indic~te that at least one plant isozyme is composed of >200-kDa subunits, sim~ilar to the enzyme from other eukaryotes (Egin-Buhler and Ebel, 1983; Slabas and Hellyer, 1985; Gornickiand Haselkorn, 1993; Egli et al., 1993; Betty et al., 1992).
While strong evolutionary conservation exists among biotin carboxylases and biotin carboxylase domains of all biotin-dependent carboxylases, BCCP dom~in~
show very little conservation outside the conserved sequence E(A/V)MKM (lysine residue is biotinylated) (Knowles, 1989; Samols et al., 1988). Although the three functional domains of the E. coli ACC are located on separate polypeptides, plant ACC is quite ~lirfelellt, having all 3 domains on a single polypeptide.
At least one form of plant ACC is located in plastids, the primary site of fattyacid synthesis. The gene encoding it, however, must be nuclear because no corresponding sequence has been seen in the complete chloroplast DNA sequences of tobacco, liverwort or rice. The idea that in some plants plastid ACC consisted of several smaller subunits was revived by the discovery of an accD homolog in somechloroplast genomes (Li and Cronan, 1992). Indeed, it has been shown that the product of this gene in pea binds two other peptides, one of which is biotinylated. The complex may be a chloroplast isoform of ACC in pea and some other plants (Sasakietal., 1993).
It has been shown recently that plants have indeed more than one form of ACCase (reviewed in Sasaki et al., 1995). The one located in plastids, the primary site of plant fatty acid synthesis, can be either a eukaryotic-type high molecular weight multi-functional enzyme (e.g., in wheat and maize) or a prokaryotic-type WO 96132484 PCT/U~;3.1~u!~i multi-subunit enzyme (e.g., in pea, soybean, tobacco and Arabidopsis). The otherplant ACCase, located in the cytoplasm, is of the eukaryotic type.
In Gr~min~, genes for both cytosolic and plastid eukaryotic-type ACCase are nuclear. No ACCase coding sequence can be found in the complete sequence of rice5 chloroplast DNA.
In other plants, subunits of ACCase other than the carboxyltransferase subunit encoded by a homolog of the E. coli accD gene, present in the chloroplast genome(Sasaki et al., 1995; Li and Cronan, 1992), must be also encoded in the nuclear DNA.
Like the vast majority of plastid proteins, plastid ACCases are synthesi7e-l in the 10 cytoplasm and then transported into the plastid. The amino acid sequence of the cytosolic and some subunits of the plastid ACCases from several plants have beendeduced from genomic or cDNA sequences (Egli et al., 1995; Li and Cronan, 1992;
Gornicki etal., 1994; Schulte etal., 1994; Shorrosh etal., 1994; Shorrosh etal.,1995; Roesler et al., 1994; Anderson et al., 1995).
There is experimPnt~l evidence suggesting that, in plants, ACCase activity controls carbon flow through the fatty acid pathway and therefore may serve as an important regulation point of plant metabolism (Page et al., 1994; Post-Beitenmiller et al., 1992; Shintani and Ohlrogge, 1995).
The possibility of different ACC isoforms, one present in plastids and another 20 in the cytoplasm, is now accepted. The rationale behind the search for a cytoplasmic ACC isoform is the requirement for malonyl-CoA in this cellular compartment, where it is used in fatty acid elongation and synthesis of secondary metabolites. Indeed, two isoforms were found in maize, both consisting of >200-kDa subunits but differing in size, herbicide sensitivity and immunological ~lu~llies. The major form was found 25 to be located in mesophyll chloroplasts. It is also the major ACC in the endosperm and in embryos (Egli et al., 1993).

1.2.2 Cyanobacteria Unlike monocot plants, members of the cyanobacteria are resistant to these 30 herbicide f~milit-s Cyanobacteria are prokaryotes that carry out green plant WO 96J32484 rC~US96~'~5l~95 photosynthesis, evolving ~2 in the light. They are believed to be the evolutionary aneestors of ehloroplasts. Virtually nothing is known about fatty aeid biosynthesis in cyanobacteria.
Synechococcus is a llnicell~ r obligate phototroph with an efficient DNA
5 transformation system. Replieating veetors based on endogenous plasmids are available, and selectable markers include resistance to kanamycin, chloramphenicol, streptomyein and the PSII inhibitors diuron and atrazine. Inaetivation and/or deletion of Synechococcus genes by transformation with suitable cloned material interrupted by rçcict~nee c~CC,e,tt~s is well known in the art. Genes may also be replaced by 10 specifiç~lly mut~tecl versions using seleetion for elosely linked resistanee eassettes.
Anabaena differentiates specialized cells for nitrogen fixation when the cultureis deprived of a souree of eombined nitrogen. The differentiated eells have a unique glycolipid envelope cont~ining C26 and C28 fatty acids (Murata and Nishida, 1987), whose synthesis must start with the reaetion eatalyzed by ACC. Therefore ACC must 15 be developmentally regulated in Anabaena. Powerful systems of genetie analysis exist forAnabaena as well (Golden etal.,1987).
That eyanobaeteria and plants are evolutionarily-related make the former useful sources of cloned genes for the isolation of plant cDNAs. This method is well known to those of skill in the art. For example, the cloned gene for the enzyme 20 phytoene desaturase, whieh functions in the synthesis of carotenoids, isolated from cyanobacteria was used as a probe to isolate the cDNA for that gene from tomato (Pecker et al.,l992).

1.2.3 Herbicide R~ ..r~
Although the mech~nicmc of inhibition and recic~nee are unknown (~ iehtenth~ler, 1990), it has been shown that aryloxyphenoxypropionates and cyclohexane- 1 ,3-dione derivatives, powerful herbicides effective against monocot weeds, inhibit fatty acid biosynthesis in sensitive plants.
The aryloxyphenoxypropionate class comprises derivatives of aryloxyphenoxy-propionic acid such as diclofop, fenoxaprop, fluazifop, haloxyfop, CA 02218139 lgg7- lo- l4 wo 96/32484 PCT/US96/OSo95 propaquizafop and quizalofop. Several derivatives of cyclohexane-1,3-dione are also important post-emergence herbicides which also selectively inhibit monocot plants.
This group comprises such compounds as oxydim, cycloxydim, clethodim, sethoxydim, and tralkoxydim.
Recently it has been determine-l that ACC is the target enzyme for both of these classes of herbicide at least in monocots. Dicotyledonous plants, on the other hand, such as soybean rape, sunflower, tobacco, canola, bean, tomato, potato, lettuce, spinach, carrot, alfalfa and cotton are resistant to these compounds, as are other eukaryotes and prokaryotes.
Important grain crops, such as wheat, rice, maize, barley, rye, and oats, however, are monocotyledonous plants, and are therefore sensitive to these herbicides.
Thus herbicides of the aryloxyphenoxypropionate and cyclohexane-1,3-dione groupsare not useful in the agriculture of these important grain crops owing to the inactivation of monocot ACC by such chemicals.
1.2.4 Derlciencies in the Prior Art The genetic transformation of important commercial monocotyledonous agriculture crops with DNA segments encoding herbicide-resistant ACC enzymes would be a revolution in the farming of such grains as wheat, rice, maize, barley, rye, and oats. Moreover the availability for modnl~ting the herbicide resistance of plants through the alteration of ACC-encoding DNA segments and the polypeptides themselves would be highly desirable. Methods of identifying and assaying the levels of ACC activity in these plants would also be important in genetically en~ineering grain crops and the like with desirable herbicide-resistant qualities. Likewise the availability of DNA segments encoding dicotyledonous ACC and nucleic acid segm~nt.~ derived th~l~;rl~lll would provide a much-needed means of genetically altering the activity of ACC in vivo and in vitro. ~, What is lacking in the prior art, therefore, is the identific~tion of DNA
segments encoding plant and cyanobacterial ACC enzymes, and the development of methods and processes for their use in creation of modified, transgenic plants which WO 96132484 PCTJlJS9f 'C~S~gS

have altered herbicide rç~ict~n~e. Moreover, novel methods providing transgenic plants using DNA segmPntc encoding ACC polypeptides to modulate ACC activity, fatty acid biosynthesis in general, and oil content of plant cells in specific, are greatly needed to provide transformed plants altered in such activity. Methods for S cletPrmining ACC activity in vivo and qll~nti~ herbicide rçci~t~nçe in plants would also represent major i~ vvt;lllenl~, over the current state of the art.

2. SUMl\IARY OF THE INVENTION
The present invention seeks to overcome these and other inherent deficiencies in the prior art by providing compositions comprising novel ACC polypeptides from plant and cyanob~ctPri~l species. The invention also provides novel DNA segmentsencoding eukaryotic and prokaryotic ACCs, and methods and processes for their use in regulating the oil content of plant tissues, for conferring and mod~ ting resi~t~n~e to particular herbicides in a variety of plant species, and for altering the activity of ACC in plant cells in vivo. Also disclosed are methods for tlptermining herbicide resistance and kits for identifying the presence of plant ACC polypeptides and DNA
segmPnt.~.

2.1 ACC Genes and Polynucleotides The present invention provides polynucleotides and polypeptides relating to a whole or a portion of acetyl-CoA carboxylase (ACC) of cyanobacteria and plants as well as processes using those polynucleotides and polypeptides.
As used herein the term "polynucleotide" means a sequence of nucleotides connçct~d by phosphodiester linkages. A polynucleotide of the present invention can comprise from about 2 to about several hundred thousand base pairs. Preferably, a polynucleotide comprises from about 5 to about 150,000 base pairs. Preferred lengths ~, of particular polynucleotides are set forth hereinafter.
A polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. Where a polynucleotide is aDNA molecule, that molecule can be a gene or a cDNA molecule. Nucleotide bases are inrlic ~ted herein by a single letter code: ~rlenin~ (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
In one embodiment, the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding polypeptides which have the 5 ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium. Preferably, the cyanobacterium is Anabaena or Synechococcus. A
plGr~llcd Anabaena is Anabaena 7120. A ~lert;llcd Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).
Preferably, a polypeptide is a biotin carboxylase enzyme of a cyanob~ctel ;1~lll10 This enzyme is a subunit of cyanobacterial acetyl-CoA carboxylase and par~icir~tes in the carboxylation of acetyl-CoA. In a ~lcr~ cd embodiment, a BC polypeptide is encoded by a polynucleotide compri~ing an accC gene which has the nucleic acid sequence of SEQ ID NO:5 (Anabaena accC) or SEQ ID NO:7 (Synechococcus accC), or functional equivalents thereof. The BC polypeptide preferably comprises the 15 amino acid sequence of SEQ ID NO:6 (Anabaena BC) or SEQ ID NO:8 (Synechococcus BC), or functional equivalents thereof.
~ a second embo~lim~nt the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding a biotin carboxyl carrier protein of a cyano~r-trril-m Preferably, the cyanob~rterillm is Anabaena or 20 Synechococcus. A l.,ert;"cd Anabaena is Anabaena 7120. A ~lGr~;llcd Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).
Preferably, a polypeptide is a biotin carboxyl carrier protein of a cyanob~cteril-m This polypeptide is a subunit of cyanob~r,ten~l acetyl-CoA
carboxylase and participates in the carboxylation of acetyl-CoA. In a ~,cÇe"~d 25 embodiment, a BCCP polypeptide is encoded by a polynucleotide comprising an accB
gene which has the nucleic acid sequence of SEQ ID NO: 1 (Anabaena accB) or SEQ
ID NO:3 (Synechococcus accB), or functional equivalents thereof. The BCCP
polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2 (Anabaena BCCP) or SEQ ID NO:4 (Synechococcus BCCP), or functional 30 equivalents thereof.

_9_ In a third embodiment, the present invention co.l~ lates isolated and purified polynucleotides comprising DNA segments encoding a carbo~ylL,dllsferaseprotein of a cyanobacterium. Preferably, the cyanobart~rillm is Anabaena or > Synechococcus. A plGrellGd Anabaena is Anabaena 7120. A preferred 5 Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).
Preferably, a polypeptide is a ca,lJo~yll,dl~sferase a or ~ subunit protein of acyanobacterium. These polypeptides are subunits of cyanobacterial acetyl-CoA
carboxylase and participate in the carboxylation of acetyl-CoA. In a preferred embodiment, a CTa polypeptide is encoded by a polynucleotide comprising an accA
10 gene which has the nucleic acid sequence of SEQ ID NO: 11 (Synechococcus accA), or a functional equivalent thereof. The CTa polypeptide preferably comprises the amino acid sequence of SEQ ID NO:12 (Synechococcus CTa), or a functional equivalent thereof.
In a fourth embo-lim~nt, the present invention co~ llpl~tes isolated and 15 purified polynucleotides compri~ing DNA segments encoding an acetyl-CoA
carboxylase protein of a plant. Preferably, the plant is a monocotyledonous or adicotyledonous plant. An exemplary and l"~er~,ll.,d monocotyledonous plant is wheat, rice, maize, barley, rye, oats or timothy grass. An exempl~ry and ~,cfe".,d dicotyledonous plant is soybean, rape, sunflower, tobacco, Arabidopsis, petunia, pea, 20 canola, bean, tomato, potato, lettuce, spinach, alfalfa, cotton or carrot. A ~,GfGl,~d monocotyledonous plant is wheat, and a preferred dicotyledonous plant is canola.Preferably, a polypeptide is an acetyl-CoA carboxylase (ACC) protein of a plant. This polypeptide participates in the carboxylation of acetyl-CoA. In a plGrGlr~d embodiment, an ACC polypeptide is encoded by a polynucleotide comprising an ACC
25 cDNA which has the nucleic acid sequence of SEQ ID NO:9 (wheat ACC) or SEQ IDNO:19 (canola ACC), or functional equivalents thereof. The ACC polypeptide preferably comprises the amino acid sequence of SEQ lD NO:10 or SEQ ID NO:31 (wheat ACC) or SEQ ID NO:20 (canola ACC), or functional equivalents thereof.
In yet another aspect, the present invention provides an isolated and purified 30 DNA molecule comprising a promoter opeldlively linked to a coding region that WO 96/32484 PCT/U' _5'~5C95 encodes (1) a polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium, (2) a biotin carboxyl carrier protein of a cyanobacterium or (3) a plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a S transcription-tennin~ting region, whereby said promoter drives the transcription of said coding region.
In another aspect, the present invention provides an isolated polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium such as Synechococcus. Preferably a biotin carboxyl carrier protein 10 gene includes the nucleic acid sequence of SEQ ID NO:2 and the polypeptide has the amino acid residue sequence of SEQ ID NO:6.

2.2 ACC roly~Jtides and Anti-ACC Antibodies The present invention also provides ( 1 ) an isolated and purified biotin 15 carboxyl carrier protein of a cyanobacterium such as Anabaena or Synechococcus, which protein includes the arnino acid residue sequence of SEQ ID NO:2 or SEQ IDNO:4, respectively; (2) an isolated and purified biotin carboxylase of a cyanobacterium such as Anabaena or Synechococcus, which protein includes the amino acid residue sequence of SEQ ID NO:6 or SEQ ID NO:8, respectively; (3) an isolated and purified carboxyltransferase a subunit protein of a cyanobacterium such as Synechococcus, which protein incl~cles the amino acid residue sequence of SEQ ID
NO:12; (4) an isolated and purified monocotyledonous plant polypeptide from wheat having a molecular weight of about 220 kDa, dimers of which have the ability to catalyze the carboxylation of acetyl-CoA, which protein includes the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:31; and (5) an isolated and purified dicotyledonous plant polypeptide from canola having the ability to catalyze the carboxylation of acetyl-CoA, which protein includes the arnino acid sequence of SEQ
ID NO:20.
Another aspect of the invention concerns methods and compositions for the use of the novel peptides of the invention in the production of anti-ACC antibodies.

WO 96/32484 PCT/US!~C~ 5J~;

The present invention also provides methods for identifying ACC and ACC-related polypeptides, which methods comprise contacting a sample suspected of cont~ininpsuch polypeptides with an immunologically effective amount of a composition comprising one or more specific anti-ACC antibodies disclosed herein. Peptides that S include the amino acid sequence of any of SEQ ID NO:4 through SEQ ID NO:8 and their d~livaliv~s will be L~l-,r~ d for use in generating such anti-ACC antibodies.
Samples which may be tested or assayed for the presence of such ACC and ACC-related polypeptides include whole cells, cell extracts, cell homogenates, cell-free sup~rn~t~nt.s, and the like. Such cells may be either eukaryotic (such as plant cells) or 10 prokaryotic (such as cyanobacterial and bacterial cells).
In certain aspects, diagnostic reagents comprising the novel peptides of the present invention and/or DNA segments which encode them have proven useful as test reagents for the detection of ACC and ACC-related polypeptides.

15 2.3 ACC Traneformation and T~lPntification of Herbicide-R ~;et:~nt Variante In yet another aspect, the present invention provides a process of mocl~ ting the herbicide resistance of a plant cell by a process of transforming the plant cell with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a herbicide resistant polypeptide having the ability to catalyze the 20 carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-t~rmin~ting region, whereby the promoter is capable of driving thetranscription of the coding region in a monocotyledonous plant.
Preferably, a polypeptide is an acetyl-CoA carboxylase enzyme and, more preferably, a plant acetyl-CoA carboxylase. In a preferred embodiment, a coding 25 region includes the DNA sequence of SEQ ID NO:9 or SEQ ID NO: 19 and a promoter is CaMV35.
In a preferred embodiment, a cell is a cyanobacterium or a plant cell and a plant polypeptide is a monocotyledonous plant acetyl-CoA carboxylase enzyme suchas wheat acetyl-CoA carboxylase enzyme. The present invention also provides a 30 transformed cyanobacterium produced in accordance with such a process.

WO 96/32484 PCT/US9G~'~5035 The present invention still further provides a process for determining the inh~rit~n~e of plant resist~n~e to herbicides of the aryloxyphenl~y~l~,pionate or cyclohexane-1,3-dione classes, which generally involves measuring resistance to these herbicides in a parental plant line and in the progeny of the parental plant line, 5 ~l~tecting the presence of complexes between DNA restriction fragments and the ACC
gene, and then correlating the herbicide recict~n~e of the parental and progeny plants with the presence of particular sizes of ACC gene-cont~ining DNA fr~gm~nts as anindication of the inhlq~. it~nce of resi~t~n- e to herbicides of these classes.
Preferably, the acetyl-CoA carboxylase is a dicotyledonous plant acetyl-CoA
10 carboxylase enzyme or a mllt:~t~l monocotyledonous plant acetyl-CoA carboxylase that confers herbicide resistance or a hybrid acetyl-CoA carboxylase comprising a portion of a dicotyledonous plant acetyl-CoA carboxylase, a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA carboxylase.
Where a cyanobacterium is transformed with a plant ACC DNA molecule, that cyanobacterium can be used to identify herbicide resistant mutations in the geneencoding ACC. In accordance with such a use, the present invention provides a process for identifying herbicide resistant variants of a plant acetyl-CoA carboxylase comprising the steps of:
(a) transforming cyanobacteria with a DNA molecule that encodes a monocotyledonous plant acetyl-CoA carboxylase enzyme to form transformed or transfected cyanobz~tPri~;
(b) inactivating cyanob~t~ri~l acetyl-CoA carboxylase;
(c) exposing the transformed cyanobacteria to an effective herbicidal amount of a herbicide that inhibits acetyl-CoA carboxylase activity;
(d) identifying transformed cyanobacteria that are resistant to the herbicide; and (e) characterizing DNA that encodes acetyl-CoA carboxylase from the cyanobacteria of step (d).

WO 961324~4 PCTJIJS~ ;0!9S

Means for transforming cyanobacteria as well as expression vectors used for such transformation are preferably the same as set forth above. In a preferred embodiment, cyanobacteria are transformed or transfected with an expression vector comprising a coding region that encodes wheat ACC. Cyanobacteria resistant to the 5 herbicide are identified. Identifying comprises growing or c-llt-lrin~ transformed cells in the presence of the herbicide and recovering those cells that survive herbicide exposure. Transformed, herbicide-resistant cells are then grown in culture, collected and total DNA extracted using standard techniques. ACC DNA is isolated, amplified if needed and then characterized by comparing that DNA with DNA from ACC
10 known to be inhibited by that herbicide.
In still yet another aspect, the present invention provides a process for identifying herbicide resistant variants of a plant acetyl-CoA carboxylase. Suchmethods generally involve transforming a cyanobacterium or a bacterium or a yeast cell with a DNA molecule that encodes a plant acetyl-CoA carboxylase enzyme, 15 inactivating the host-cell acetyl-CoA carboxylase, and exposing the cells to a herbicide that inhibits monocotyledonous plant acetyl-CoA carboxylase activity.
Transformed cells may be identified which are resistant to the herbicide; and the DNA
that encodes resistant acetyl-CoA carboxylase in these transformed cells may be examined and characterized.
2.4 ACC Transgenes and Transgenic Plants In yet another aspect, the present invention provides a process of altering the carboxylation of acetyl-CoA in a cell comprising transforrning the cell with a DNA
molecule comprising a promoter operatively linked to a coding region that encodes a 25 plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is ~c~dliv~ly linked to a transcription-terrnin~ting region,whereby the promoter is capable of driving the transcription of the coding region in the cell. The invention also provides a means of reducing the amount of ACC in plants by expression of ACC antisense mRNA.

WO 96/32484 PCT/US~6/U50~5 Another aspect of the invention relates generally to transgenie plants which express genes or gene segments eneoding the novel polypeptide compositions diselosed herein. As used herein, the term "transgenic plants" is inte.n(1e-l to refer to plants that have incorporated DNA sequenees, ineluding but not limited to genes 5 whieh are perhaps not normally present, DNA sequenees not normally transeribed into RNA or tr~ncl~t~cl into a protein ("expressed"), or any other genes or DNA sequenees whieh one desires to introduee into the non-transformed plant, sueh as genes whieh may normally be present in the non-transformed plant but whieh one desires to either genetieally engineer or to have altered expression. It is eontemplated that in some 10 instances the genome of transgenic plants of the present invention will have been ~ gme.nte-l through the stable introduetion of the transgene. However, in other instanees, the introduced gene will replaee an endogenous sequence.
A preferred gene which may be introduced includes, for example, the ACC
DNA sequences from eyanobaeterial or plant origin, partieularly those deseribed 15 herein which are obtained from the cyanobaeterial speeies Synechococcus or Anabaena, or from plant speeies sueh as wheat or eanola, of any of those sequenees which have been genetically engineered to decrease or inerease the activity of the ACC in such transgenic speeies.
Veetors, pl~cmicls, eosmids, YACs (yeast artificial ehromosomes) and DNA
20 ~e.gment.c for use in transforming sueh eells will, of eourse, generally eomprise either the eDNA, gene or gene sequenees of the present invention, and partieularly those eneoding ACC. These DNA eonstruets ean further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired. The DNA
segment or gene may encode either a native or modified ACC, which will be 25 expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant.
Such transgenic plants may be desirable for increasing the herbicide resistance of a monocotyledonous plant, by incorporating into such a plant, a transgenic DNA
segment encoding a plant acetyl-CoA carboxylase enzyme which is resistant to 30 herbicide inactivation, e.g., a dieotyledonous ACC gene. Alternatively a WO 96/32484 PcTJus96~ 5 cyanobacterial ACC polypeptide-encoding DNA segment could also be used to prepare a transgenic plant with increased resi.ct~nce to herbicide inactivation.~ ltern~tively transgenic plants may be desirable having an decreased herbicide reci~t~n-~e This would be particularly desirable in creating transgenic plants which 5 are more sensitive to such herbicides. Such a herbicide-sensitive plant could be prepared by incorporating into such a plant, a transgenic DNA segment encoding aplant acetyl-CoA carboxylase enzyme which is sensitive to herbicide inactivation, e.g., a monocotyledonous ACC gene, or a m~lt~t~d dicotyledonous or cyanob~-~teri~l ACC-encoding gene.
In other aspects of the present invention, the invention concerns processes of modifying the oil content of a plant cell. Such modifications generally involve expressing in such plant cells transgenic DNA segments encoding a plant or cyanobacterial acetyl-CoA carboxylase composition of the present invention. Suchprocesses would generally result in increased expression of ACC and hence, increased 15 oil production in such cells. ~ltenl~tively, when it is desirable to decrease the oil production of such cells, ACC-encoding transgenic DNA segments or ~nti~n~e (complementary) DNA segments to genomic ACC-encoding DNA sequences may be used to transform cells.
Either process may be facilitated by introducing into such cells DNA segments 20 encoding a plant or cyanobacterial acetyl-CoA carboxylase polypeptide, as long as the resulting transgenic plant expresses the acetyl-CoA carboxylase-encoding transgene.
The present invention also provides a transformed plant produced in accordance with the above process as well as a transgenic plant and a transgenic plant ~ seed having incorporated into its genome a transgene that encodes a herbicide resistant 25 polypeptide having the ability to catalyze the carboxylation of acetyl-CoA. All such transgenic plants having incorporated into their genome transgenic DNA segments encoding plant or cyanobacterial acetyl-CoA carboxylase polypeptides are aspects of this invention.

30 2.5 ACC Screening and Tmmllno-l~tec~ n Kits WO 96/32484 PcTlus!~ u~ 5 The present invention contemplates methods and kits for screening samples suspected of c-)nt~ining ACC polypeptides or ACC-related polypeptides, or cells producing such polypeptides. Said kit can contain a nucleic acid segmP.nt or an antibody of the present invention. The kit can contain reagents for ~iPtecting an 5 interaction between a sample and a nucleic acid or antibody of the present invention.
The provided reagent can be radio-, fluorescently- or enzym~tic~lly-labeled. The kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.
The reagent of the kit can be provided as a liquid solution, attached to a solid10 support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconctitl-t~.-l by the addition of a suitable 15 solvent, that may be provided.
In still further embo-liment.~7 the present invention concerns immunodetection methods and associated kits. It is proposed that the ACC peptides of the presentinvention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be 20 employed to detect ACC or ACC-related epitope-cont~ining peptides. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then ~ietecting the presence of the 25 immunocomplex.
In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like.
30 Generally, immunocomplex formation will be detected through the use of a label, W~ 96132484 PC'r~JSg~5~'GSa35 such as a radiolabel or an enzyme tag (such as ~lk:~line phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
For assaying purposes, it is proposed that virtually any sample suspected of comprisin~ either an ACC peptide or an ACC-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the pl~aldlion of kits that may be employed to detec~ thepresence of ACC or ACC-related proteins or peptides and/or antibodies in a sample.
Samples may include cells, cell supern~t~nts, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing ACC peptides. Generally speaking, kits in accordance with the present invention will include a suitable ACC peptide or an antibody directed against such a protein orpeptide, together with an immunodetection reagent and a means for cont~ining theantibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for cont~ining the antibody, , antigen, and reagent containers in close confinem~nt for commercial sale. Such containers may include injection or blow-molded plastic cont~inerS into which the desired vials are retained.

WO 96/32484 PCT/US~C~'~50!JS

2.6 ELISAs and Tmml-no~ ;latiu .
ET.TSAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incorporating ACC antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of 5 a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the10 background caused by nonspecific binding of antisera onto the surface.
After binding of antigenic m~teri.ql to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
15 Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween~). These added agents also tend to assist in the reduction of nonspecific background. Thelayered antisera is then allowed to incubate for from about 2 to about 4 hours, at temperatures preferably on the order of about 25~ to about 27~C. Following 20 incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween(~), or borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of25 immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an a~plv~liate chromogenic substrate. Thus, forexample, one will desire to contact and incubate the antisera-bound surface with a 30 urease or peroxidase-conjugated anti-human IgG for a period of time and under WC~ 96132484 PCTlUSg~J'~ 3S

conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room tG~ GlalulG in a PBS-cont~inin~ solution such ac PBS
~ Tween(~9).
After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-b~n7thi~701ine)-6-sulfonic acid (ABTS) and H2O2, in the case of peroxidase as the enzyme label. Qu~ntific~tion is then achieved by measuring the degree of color generation, e.g., using a visible spectra spG~;l-ophotometer.
The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to ~ii.ccrimin~te: or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Nonionic salts are ~Gfclled, since other agents 15 such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g. enzyme-substrate pairs.

20 2.7 WesternBlots The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-peptide antibodies may be used as high-affinity primary reagents for the iclentific~tion of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with 25 immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the WO 96132484 PCT/US9Gi(~35 detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
Tmmlln~11Ogically-based detection methods for use in conjunction with Western blotting include enzym~tir~lly-, radiolabel-, or fluorescently-tagged secondary 5 antibodies against the toxin moiety are considered to be of particular use in this regard.

2.8 Epitopic Core Sequences The present invention is also directed to protein or peptide compositions, free 10 from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more anti-ACC antibodies.
As used herein, the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-ACC antibodies" is intended to refer to a peptide 15 or protein antigen which inchlcles a primary, secondary or tertiary structure similar to an epitope located within an ACC polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the ACC
polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in 20 conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
The identificzltion of ACC immunodomin~nt epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554,101,25 incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988;
Wolf et al., 1988; U.S. Patent Number 4,554,101). The amino acid sequence of these WO 96/32484 PC'r)US~ il~S

"epitopic core sequences" may then be readily incorporated into peptides, eitherthrough the application of peptide synthesis or recombinant technology.
~ lerell~,d peptides for use in accordance with the present invention willgenerally be on the order of 8 to 20 amino acids in length, and more preferably about 5 8 to about 15 amino acids in length. It is proposed that shorter antigenic ACC-derived peptides will provide advantages in certain circ~lm~t~nces, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantagesinclude the ease of plGpalalion and puri~c~tion, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide directed to ACC and ACC-related sequences. These epitopic core sequencesare identified herein in particular aspects as hydrophilic regions of the ACC
polypeptide antigen. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimlll~tion, and, hence, elicit specific antibody production.
An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on transferrin-binding protein antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term "complementary"
refers to amino acids or peptides that exhibit an attractive force towards each other.
Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly s crucial, so long as it is at least large enough to carry the i(l~ntified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure WO 96/32484 PCT/U3~J'~50~!1., would generally be on the order of about 8 amino acids in length, with sequences on the order of 10 to 20 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic 5 epitopic core sequence.
The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Patent 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer 10 programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysis programs (e.g., DNAStar~) software, DNAStar, Inc., Madison, WI) may also be useful in decigning synthetic peptides in accordance with the present disclosure.
Syntheses of epitopic sequences, or peptides which include an antigenic 15 epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commerciallyavailable peptide synthPci7er such as an Applied Biosystems Model 430A Peptide Synth~ci7er). Peptide antigens synth.-.ci7~d in this manner may then be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous 20 solutions or, even more preferably, in a powder or lyophilized state pending use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will 25 generally be desirable to include agents inclll~ing buffers such as Tris or phosphate buffers to mz~int~in a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4~C, or more preferably, frozen. Of course, where the peptides are stored 30 in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in W~ 961~2484 PCTJUS!~6~1~51~!i metered aliquots that may be rehydrated with a precleterrnint-(l amount of water (preferably distilled) or buffer prior to use.
..
2.9 DNA Se~ nt.c The present invention also concerns DNA segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode the novel peptides disclosed herein. DNA segments encoding these peptide species may proveto encode proteins, polypeptides, subunits, functional domains, and the like of ACC-related or other non-related gene products. In addition these DNA segments may be synthe~i7ed entirely in vitro using methods that are well-known to those of skill in the art.
As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA
segment encoding an ACC peptide refers to a DNA segment that contains ACC
coding sequences yet is isolated away from, or purified free from, total genomic DNA
of the species from which the DNA segment is obtained. Included within the term "DNA segment", are DNA segments and smaller fr~gment.c of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified ACC gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other ~lement.c such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional terrn includes both genomic sequences, cDNA sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.
'' "Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding ACC, forms the significant part of the coding WO 96/32484 PCT/U:,r . '051 region of the DNAsegm~rlt, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fr~gm~ntc or other functional genes or cDN~ coding regions. Of course, this refers to the DNAsegment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular emborliments, the invention concerns isolated DNA segm~nt~ and recombinant vectors incorporating DNA sequences that encode an ACC peptide species that includes within its arnino acid sequence an amino acid sequence essentially as set forth in any of SEQ IDNO:2, SEQ IDNO:4, SEQ IDNO:6, SEQ ID
NO:8, SEQ IDNO:10, SEQ ~ NO:12, SEQ ~ NO:20, and SEQ ~ NO:31.
The term "a sequence essenti~lly as set forth in any of SEQ IDNO:2, SEQ ID
NO:4, SEQ ~ NO:6, SEQ ~ NO:8, SEQ ~ NO:10, SEQ ~ NO:12, SEQ ~ NO:20 and SEQ IDNO:31" means that the sequence substantially corresponds to a portion of the sequence of either SEQ ~ NO:2, SEQ ~ NO:4, SEQ ~ NO:6, SEQ ~ NO:8, SEQ ID NO:10, SEQ l:DNO:12, SEQ IDNO:20 or SEQ IDNO:31, and has relatively few arnino acids that are not i~l~ntic~l to, or a biologically functional equivalent of, the amino acids of any of these sequences. The term "biologically functional equivalent"
is well understood in the art and is further defined in detail herein (for example, see Preferred Embodiments). Accordingly, sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% amino acid sequence identity or functional equivalence to the amino acids of any of SEQ ID NO:2, SEQ ID NO:4, SEQ ~ NO:6, SEQ ~ NO:8, SEQ ~ NO:10, SEQ ~ NO:12, SEQ ~ NO:20, and SEQ IDNO:31will be sequences that are "essentially as set forth in any of SEQ IDNO:2, SEQ ~ NO:4, SEQ ~ NO:6, SEQ ~ NO:8, SEQ ~ NO:10, SEQ ~ NO:12, SEQ ~ NO:20, and SEQ ~ NO:31."
It will also be understood that arnino acid and nucleic acid sequences may include additional residues, such as additional N- or C-termin~l arnino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the .

Wo 96132484 PCT/US~9GJ'~5(~5 maintenance of biological protein activity where protein expression is concerned. The addition of termin~l sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences fl~nkin~ either of the 5' or 3' portions of the coding region or may include various int~rn~l sequences, i.e., introns, which are known to occur within genes.
The nucleic acid segm~nt~ of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of p~ dlion and use in the int~n(lt~l recombinant DNA protocol. For example, nucleic acid fr~gment~ may be prepared that include a short contiguous stretch encoding either of the peptide sequences disclosed in any of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:20 and SEQ ID NO:31, or that are iclenti~l to or complementary to DNA sequences which encode any of the peptides disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:20, and SEQ ID
NO:31, and particularly those DNA segments disclosed in SEQ ID NO:l, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:l9, or SEQ ID NO:30. For example, DNA sequences such as about 14 nucleotides, and that are up to about 13,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 14 base pairs in length (including all intermediate lengths) are also co,.lel-,plated to be useful.
It will be readily understood that "interrnt~ te lengths", in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.;
21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000;
* 2,000-3,000; 3,000-5,000; 5,000-10,000, 10,000-12,000, 12,000-13,000 and up to and including sequences of about 13,000, 13,001, 13,002, or 13,003 nucleotides etc. and the like.
It will also be understood that this invention is not limited to the particular }
nucleic acid sequences which encode peptides of the present invention, or which S encode the amino acid sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:20, and SEQ ID NO:31, including those DNA sequences which are particularly disclosed in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID
NO:l9, and SEQ ID NO:30. Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
The DNA segments of the present invention encompass biologically-functional equivalent peptides. Such sequences may arise as a consequence of codon re(llln~l~ncy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the ~l~,p~lLies of the amino acids being exchanged. Changesclçsignçfl by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mllt~ntc in order to examine activity at the molecular level.If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for pllrific~tion or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively). J

WO 96132484 PCT)US9CJO!~O~S

Recombinant vectors form further aspects of the present invention.
Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA ~e~m~.nt, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the 5 form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
In other embo~limt-ntc, it is contempl~t~d that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is int~nded to refer to a promoter that is not normally associated with a DNA ~egmt-nt encoding an ACC peptide in its natural environment. Such promoters may include 15 promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or plant cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of 20 molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the a~plopliate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression in~ de, but are not 25 limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
In connection with expression emborlimentc to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred.
However, it will be appreciated that the use of shorter DNA segments to direct the 30 expression of ACC peptides or epitopic core regions, such as may be used to generate WO 96t324X4 PCT/US~6J1~5G1~5 anti-ACC antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly 5 useful. Such peptide e~ilu~es may be amino acid sequences which comprise contiguous amino acid sequences from any of SEQID NO:2, SEQID NO:4, SEQID
NO:6, SEQID NO:8, SEQID NO:10, SEQID NO:12, SEQID NO:20, or SEQID
NO:31.
In addition to their use in directing the expression of ACC peptides of the 10 present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embo~lim~ontc. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 15 nucleotide long contiguous DNA segment any of SEQID NO: 1, SEQID NO:3, SEQ
ID NO:5, SEQID NO:7, SEQID NO:9, SEQID NO: 11, SEQID NO: 19, and SEQID
NO:30 will find particular utility. Longer contiguous identical or complementarysequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1,000, 2,000, 5,000, 8,000, 10,000, 12,000, 13,000 etc. (including all interm~ te lengths and up to and 20 including full-length sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to ACC-encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the ~le~alation of mutant species 25 primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions con~i~ting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to DNA sequences of any of SEQ ID NO:l, SEQ ID
NO:3, SEQID NO:5, SEQID NO:7, SEQID NO:9, SEQID NO: 11, SEQID NO: 19, 30 and SEQID NO:30 are particularly contemplated as hybridization probes for use in, wo s6J32484 PCT~US~0~35 e.g., Southern and Northern blotting. Smaller fr~gm~nt~ will generally find use in hybridization embo-lim~-nt~, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and about 100 or 200 nucleotides, but larger contiguous complemPnt~rity stretches may be used, according to the length 5 complementary sequences one wishes to detect.
The use of a hyhritli7~tion probe of about 14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally plcrelled, though, in order to increase stability and selectivity of the ~ 10 hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 contiguous nucleotides, or even longer wheredesired.
Of course, fr~gment~ may also be obtained by other techniques such as, e.g., by mechanical ~h~ring or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthe~i7ing the fragment by ch~mic~l means, as is commonly practiced using an automated oligonucleotide synth~i7~r. Also, fr~gm.ont~ may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patents 4,683,195 and 4,683,202 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAfr~gment~. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to forrn the hybrids, e.g., one will select relatively low salt and/or high telllpel~lulc; conditions, such as provided by WO 96/32484 PCTNS~G~ 50!~5 about 0.02 M to about 0.15 M NaCl at te~ ,.dLul~;s of about 50~C to about 70~C.
Such selective conditions tolerate little, if any, mism~tch between the probe and the template or target strand, and would be particularly suitable for isolating ACC-encoding DNA segments. Detection of DNA segments via hyhril1i7~tion is well-known to those of skill in the art, and the teachings of U.S. Patents 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al 1993; Segal 1976; Proskop, 1991; and Kuby, 1991, are particularly relevant.
Of course, for some applications, for example, where one desires to prepare mlltPlnt~ employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate ACC-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these ~;hcu~ ces, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20~C to about 55~C. Cross-hybridizing species can thereby be readily identified as positively hybri-li7ing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of form~mide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus,hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiment~, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an a~plupl;ate means, such as a label, for determining hybridization. A wide variety of app~ iate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human CA 022l8l39 l997- lO- l4 WO 9G132484 PCTlUS96)0S095 eye or spe~ ophotometrically~ to identify specific hybridization with complementary nucleic acid-cont~ining samples.
In general, it is envisioned that the hyhri(li7~tion probes described herein will be useful both as reagents in solution hybridization as well as in embo~lim.o.nt~
S employing a solid phase. In emborlimt-.nt~ involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for 10 exarnple, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even qll~ntit~t~.-l, by means of the label.

15 2.10 Biological Functional Equivalents Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon ch~nging the amino acids of a protein to create 20 an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by ch~nging the codons of the DNA sequence, according tothe codons listed in Table 1.

Amino Acids Codons Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F WC WU

WO 96/32484 PCT/U~ 'OrJ55 Amino Acids Codons Glycine Gly G GGA GGC GGG GGU
E~icti~line His H CAC CAU
Isoleucine Ile I AUA AUC AW
Lysine Lys K AAA AAG
T ~u~in.o Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Ghlt~mine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GW
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with S structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like ~lopellies. It is thus 10 contemplated by the inventors that various changes may be made in the peptidesequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring 15 interactive biologic function on a protein is generally understood in the art (Kyte and WO 96132484 I'CT/lJSg6~0~ J5 Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, 5 and the like.
Each arnino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenyl71l71nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); hicti~lin~ (-3.2); ghlt7lm7lte (-3.5); ghlt7lminP (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginlne (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of arnino acids whose hyd~ul~alhic indices are within _2 is preferred, those which are within + 1 are particularly preferred, and those within _0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to aTnino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ~
l); gl~lt7lm7lte (+3.0 + 1); serine (+0.3); asparagine (+0.2); gl~lt7lmine (+0.2); glycine ~- (0); threonine (-0.4); proline (-0.5 _ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).

WO 96132484 PCT/US5''~503S

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hy~philicity values are within i2 is preferred, those which are 5 within i 1 are particularly preferred, and those within iO.5 are even more particularly t;r~ d.
As outlined above, amino acid substitutions are generally therefore based on the relative .cimil~rity of the amino acid side-chain substituent~, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions 10 which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glut~m~t~ and aspartate;
serine and threonine; glut~min~ and asparagine; and valine, leucine and isoleucine.

2.11 Site-Specific Mutagenesis Site-specific mutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of ml-t~ntc through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art,as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors W~ 96132484 PCTJU5~ !35 such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which elimin~tes the step of transferring the gene of interest from a plasmid to a phage.
~ general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mllt~ted sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymP.ri7ing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-ml-t~ted sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform ap~lu~l;ate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mllt~d sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA
segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequencemay be treated with mutagenic agents, such as hydroxylamine, to obtain sequence v~ri~ntc ~ 12 Monoclonal Antibody Generation Means for plel~alhlg and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immllni7.ing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immllni7e-1 animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a h~m.~ter, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a plefell~;d choice for production ofpolyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often neces.s~ry therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysllccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stim~ tors of the imml~ne response, known as adjuvants. Exemplary and p-erelled adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants andalllminnm hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immllni7.~tion. A variety of routes can be used to ~-lmini~ter the immunogen (subcutaneous, intr~mll~c~ r~ intra(lerm~l, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immnni7t-.d animal at various points following immnni7~tion. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immnni7e.~1 animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.

W 096132484 PCTAUS9610~095 mAbs may be readily prepared through use of well-known techniques, such as those exemplifi~d in U.S. Patent 4,196,265, incorporated herein by reference.
Typically, this technique involves immllni7.ing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified ACC protein, S polypeptide or peptide. The immllni7.ing composition is ~mini~tered in a manner effective to stim~ te antibody producing cells. Rodents such as mice and rats are preferred ~nim~l~, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most ~lc;fell~,d as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immnni7~tion, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are plefe,.ed, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of ~nim~l~ will have been immlmi7ed and thespleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immlmi7e.d mouse contains approximately 5 x 107 to 2 x 108 lymphocytes.
The antibody-producing B lymphocytes from the immnni7e-1 animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immnni7e-l Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83~ 1984). For example, where the immllni7ed animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-ll, MPCll-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210;
and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One ~rertilled murine myeloma cell is the NS-l myeloma cell line (also termed P3-NS-l-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-a_aguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the ratio may vary from about 20: 1 to about 1: 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically in(l~lced fusion methods is also a~,v~.iate (Goding, 1986, pp. 71-74).
Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10-6 to 1 x 10-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective metlillm The selective me~ m is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas a_aserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypox~nthine and thymidine as a source of nucleotides (HAT medium). Where ~7~çrine is used, the media is supplemented withhypox~nthine~

wo96/32484 PC'rllJS~6J~

The ~lcrelled selection m~ lm is HAT. Only cells capable of operdLillg nucleotide salvage palllw~y~ are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage ~lhwa5/, e.g., hypox~nthin~
phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate 5 this palllw~, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This c-llt--ring provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing 10 the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supern~t~nt~ (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radiohl"llulloassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic 20 and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture m.Q(lillm from which they can 25 be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various .. chromatographic methods such as HPLC or affinity chromatography.

WO 96132484 PCT/USg~/O~O~.S

3. BRIEF DESCRIPTION OF THE DRAWINGS
The drawings form part of the present specific~tion and are in~ cle.cl to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with thedetailed description of specific embodillle~ . presented herein.
FIG.l. Structure of the cytosolic ACCase gene from wheat. Arrows indicate fragments of the genomic clones analyzed in more detail. Sequenced fr~gm~nt~ aremarked in black. The loc~li7~tion of the ACCase functional ~lom~in~ was established by amino acid sequence comparison with other biotin-dependent carboxylases (Gornicki et al., 1994). BC, biotin carboxylase; BCC, biotin carboxyl carrier; CT, carboxyltransferase .
EIG. 2. ~lignm~nt of cDNA sequences corresponding to the 3'-end of the mRNA encoding wheat cytosolic ACCase. Only the sequence of the 3'-end of the RACE clones is shown. The putative polyadenylation signals are nnc~erlined.
~.~teri~k~ indicate identical nucleotides. Sixteen additional 3'-RACE clones were sequenced, these matched one or another of the four sequences shown.
FIG. 3. DNA sequence of the wheat genomic ACC clone. The entire sequence is given in SEQ ID NO:30.
FIG. 4. Deduced amino acid sequence of the wheat genomic ACC clone shown in FIG. 3. The sequence is presented in SEQ ID NO:31.
FIG. 5. Shown is the 5' fl~nking sequence of the ACCase 1 gene (about 3 kb u~sllealll of the translation initiation codon, of clone 71L. The sequence isshown in SEQ ID NO:32.
FIG. 6. Shown is the 5' fl~nking sequence of the ACCase 2 gene ~lecign~tP~l 153. The sequence is shown in SEQ ID NO:33.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
4.1 Definitions The following words and phrases have the m~ning.c set forth below:

WO 96132484 l'CT~S~IC'J5~5 ~1-Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to ~~ produce a polypeptide.
Promoter: A recognition site on a DNA sequence or group of DNA sequences S that provide an ex.~lession control element for a structural gene and to which RNA
polymerase specifically binds and initi~t~s RNA synthesis (transcription) of that gene.
Regeneration: The process of growing a plant from a plant cell (e.g., plant protoplast or explant).
Structural gene: A gene that is expressed to produce a polypeptide.
Transfonnation: A process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.
Transformed cell: A cell whose DNA has been altered by the introduction of an exogenous DNA molecule into that cell.
Transgenic cell: Any cell derived or regenerated from a transformed cell or ~ derived from a transgenic cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.
Transgenic plant: A plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous DNA
molecule not originally present in a native, non-transgenic plant of the same strain.
The terms "transgenic plant" and "transformed plant" have sometimes been used in the art as synonymous terms to define a plant whose DNA contains an exogenous DNA
molecule. However, it is thought more scientifically correct to refer to a regenerated plant or callus obtained from a transformed plant cell or protoplast as being a transgenic plant, and that usage will be followed herein.
Vector: A DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the ~tt~ch~d segment. A plasmid is an exemplary vector.

WO96/32484 PCT/US~ 050g5 4.2 Polynurl~oti ' Amino acid sequences of biotin carboxylase (BC) from Anabaena and Synechococcus show great ~imil~rity with amino acid residue sequences from other5 ACC enzymes as well as with the amino acid residue sequences of other biotin-cont~ining enzymes. Based on that homology, specific nucleotide sequences were chosen for the construction of primers for polymerase chain reaction amplification of a corresponding region of the gene for ACC from wheat. Those primers have the nucleotide sequences shown below:
Primer 1 5'-TCGAATTCGTNATNATHAARGC-3' (SEQ ID NO: 13);
Primer 2 5'-GCTCTAGAGKRTGYTCNACYTG-3' (SEQ ID NO: 14);
whereNisA,C,GorT;HisA,CorT;RisAorG;YisTorCandKisG
or T. Primers 1 and 2 comprise a 14-nucleotide specific sequence based on a conserved amino acid sequence and an 8-nucleotide extension at the 5'-end of theprimer to provide anchors for rounds of amplification after the first round and to provide convenient restriction sites for analysis and cloning.
In eukaryotic ACCs, a BCCP domain is located about 300 amino acids away from the end of the BC domain, on the C-terminal side. Therefore, it is possible to amplify the cDNA covering the interval between the BC and BCCP domains using primers from the C-te.rmin~l end of the BC domain and the conserved MKM region of the BCCP. The BC primer was based on the wheat cDNA sequence obtained as described above. Those primers, each with 6- or 8-base 5'-extensions, are shown below:
Primer 3 5'-GCTCTAGAATACTATTTCCTG-3' (SEQ ID NO:15) Primer 4 5'-TCGAATTCWNCATYTTCATNRC-3' (SEQ ID NO: 16) where N, R and Y are as defined above. W is A or T. The BC primer (primer 3) was based on the wheat cDNA sequence obtained as described above. The MKM
primer (primer 4) was first checked by determining whether it would amplify thefabE
gene coding BCCP from Anabaena DNA. This PCRTM was primed at the other end by using a primer based on the N-terminal amino acid residue sequence as determined on WO 96/32484 PCTIUS9-~'05 protein purified from Anabaena extracts by affinity chromatography. Those primers are shown below:
Primer 5 5'-GCTCTAGAYTTYAAYGARATHMG-3' (SEQ ID NO:17) Primer 4 5 ' -TCGAATTCWNCATYTTCATNRC-3 ' (SEQ ID NO: 18) where H, N, R, T, Y and W are as defined above. M is A or C. This amplification (using the conditions described above) yielded the correct fragment of the AnabaenafabE gene, which was used to identify cosmids that contained the entire fabE gene and fl~nking DNA. An about 4-kb XbaI fragment cont~ining the gene was cloned into the vector pBluescriptKS(~) for sequencing. Primers 3 and 4 were then used to amplify the illte~ve;llillg sequence in wheat cDNA. Again, the product of the first PCRTM was eluted and reamplified by another round of PCRTM, then cloned into the Invitrogen vector pCRlI(~).
The amino acid sequence of the polypeptide predicted from the cDNA
sequence for this entire fragment of wheat cDNA (1473 nucleotides) was compared with the amino acid sequences of other ACC enzymes and related enzymes from various sources. Rat, chicken and yeast are more closely related to each other than to the BC subunits of bacteria, and the BC domains of other enzymes such as pyruvate carboxylase of yeast and propionyl CoA carboxylase of rat. The amino acid identities between wheat ACC and other biotin-dependent enzymes, within the BC domain are no higher than 60%, and shown below in Table 2.

% identity # identity with wheat ACC with rat ACC
rat ACC 5~ (100) chicken ACC 57 yeast ACC 56 Synechococcus ACC 32 A~abaena ACC 30 E.coli ACC 33 rat propionyl CoA carboxylase 32 31 yeast pyruvate carboxylase 31 4.3 Probes and Primers In another aspect, DNA sequence information provided by the invention 5 allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences of the selected polynucleotides disclosed herein. In these aspects, nucleic acid probes of an a~lop.iate length are prepared based on a consideration of a selected ACC gene sequence, e.g., a sequence such as that shown in SEQ ID NO:9 or SEQ ID NO:19, or a selected gene sequence 10 encoding a subunit of a cyanobacterial ACC, e.g., a sequence as that shown in SEQ ID
NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO: 11. The ability of such nucleic acid probes to specifically hybridize to an ACC gene sequence lend them particular utility in a variety of embo~lim~nt~. Most importantly, the probes can be used in a variety of assays for cletecting the presence of complementary sequences in a 15 given sample.
In certain embodiments, it is advantageous to use oligonucleotide primers.
The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mllt~ting a defined segment of an ACC
gene from a cyanob~rt~rillm or a plant using PCRTM technology. Segm.-ntc of ACC
20 genes from other org~ni~m~ may also be amplified by PCRTM using such primers.

WO 96/32484 PCT~US~6~'SC5S

To provide certain of the advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 14 to 30 or so long nucleotide stretch of an ACC-encoding or ACC subunit-encoding sequence, such as that shown S in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ
ID NO:ll, or SEQ ID NO:l9. A size of at least 14 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complement~ry sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase 10 stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 14 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synth~ ~i7.ing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patents 4, 683,195, and 4,683,202, herein incorporated by reference, or by excising selected DNA
fragments from recombinant pl~cmi~ls cont~inin~ a~rop.iate inserts and suitable restriction sites.
Accordingly, a nucleotide sequence of the invention can be used for its ability 20 to selectively form duplex molecules with complementary stretches of the gene.
Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degree of selectivity of the probe toward the target sequence. For applications requiring a high degree of selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, 25 for example, one will select relatively low salt and/or high te~l.peldLu.c; conditions, such as provided by about 0.02 M to about 0.15 M NaCl at tel-lpeldlu-~s of about50~C to about 70~C. These conditions are particularly selective, and tolerate little, if any, mi~m~tch between the probe and the template or target strand.
Of course, for some applications, for example, where one desires to prepare 30 mllt~nt~ employing a mutant primer strand hybridized to an underlying template or WO96/32484 PCT/U5~ 5~35 where one seeks to isolate an ACC coding sequences for related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circum~t~nces, one maydesire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures S ranging from about 20~C to about 55~C. Cross-hybridizing species can thereby be readily i~lentified as positively hybridizing signals with respect to control hybri~li7~tions. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of ff)rm~mi~le7 which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus,10 hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it is advantageous to employ a polynucleotide of the present invention in combination with an ~ iate label for clett~.cting hybrid formation. A wide variety of ~pl~l~.iate labels are known in the art, including 15 radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
In general, it is envisioned that a hybridization probe described herein is useful both as a reagent in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is 20 adsorbed or otherwise affixed to a selected matrix or surface. This fixed nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions depend as is well known in the art on the particular circumstances and criteria required (e.g., on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe). Following washing 25 of the matrix to remove nonspecifically bound probe molecules, specific hybridization is detected, or even qll~ntit~te~l, by means of the label.

4.4 Expression Vectors The present invention contemplates an expression vector comprising a 30 polynucleotide of the present invention. Thus, in one embodiment an expression Wo 96132~4 PCTnJS~G,'15~95 ~7-vector is an isolated and purified DNA molecule comprising a promoter op~;ldli~/ely linked to an coding region that encodes a polypeptide having the ability to catalyze the t carboxylation of a biotin carboxyl carrier protein of a cyanobacterium, which coding region is operatively linked to a transcription-t~rmin~ting region, whereby the promoter drives the transcription of the coding region.
As used herein, the term "operatively linked" means that a promoter is connect~-1 to an coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art.
Where an expression vector of the present invention is to be used to transform a cyanobacterium, a promoter is selected that has the ability to drive and regulate expression in cyanobacteria. Promoters that function in bacteria are well known in the art. An exemplary and preferred promoter for the cyanobacterium Anabaena is the glnA gene promoter. An exemplary and ~refi;;lled promoter for the cyanob~-~terillm Synechococcus is the psbAI gene promoter. ~ltern~tively, the cyanobacterial acc gene promoters themselves can be used.
Where an expression vector of the present invention is to be used to transform a plant, a promoter is selected that has the ability to drive expression in plants.
Promoters that function in plants are also well known in the art. Useful in expressing the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A promoter is also selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be near-constitutive, such as the CaMV 35S promoter, or tissue-specific or developmentally specific promoters affecting dicots or monocots.
Where the promoter is a near-con~ ulivt; promoter such as CaMV 35S, .~ increases in polypeptide expression are found in a variety of transformed plant tissues 30 (e.g., callus, leaf, seed and root). ~lt~.rnzltively, the effects of transformation can be CA 02218139 1997- lO- 14 W096/32484 PCTrUS96/05095 directed to specific plant tissues by using plant integrating vectors cont~inin~ a tissue-specific promoter.
An exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The Lectin protein in soybean seeds is encoded by a single gene (~) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA. The lectin gene and seed-specific promoter have been fully char~(~teri7e~1 and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983; Lindstrom et al., 1990.) An expression vector cont:~ining a coding region that encodes a polypeptide of interest is engineered to be under control of the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method (Dhir et al., 1991). The expression of the polypeptide is directed specifically to the seeds of the transgenic plant.
A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows t_e effects of transformation in more than one specific tissue.
Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea smallsubunit RuBP Carboxylase (Poulsen et al., 1986; ~chmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), CaMV 35S transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989). Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunit RuBP carboxylase promoter.
The choice of which expression vector and l~ltim~tely to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, e.g., the location and timing of protein expression, and the host cell W096132484 PCTJUS9~JD3D~5 to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is opelaliv~ly linked.
Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et aL, 1987). However, several otherplant integrating vector systems are known to function in plants including pCaMVCN
transfer control vector described (Fromm et al., 1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S
promoter.
In preferred embodiments, the vector used to express the polypeptide inc~lndes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. One preferred drug re~i~t~nce marker is the gene whose expression lS results in kanamycin resistance; i.e., the chimeric gene contAining the nopaline synthase promoter, TnS neomycin phosphotransferase II and nopaline synthase 3' nontr~n~lAted region described (Rogers et al., 1988).
RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to t~rminAte transcription. Those DNA
sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are described in United States Patent Nos. 4,971,908, 4,940,835, 4,769,061 and 4,757,011, the disclosures of which are incorporated herein by reference. Those vectors can be modified to include a coding sequence in accordance with the present invention.
A variety of methods has been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
A coding region that encodes a polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanob~terillm is preferably a biotin carboxylase enzyme of a cyanobacterium, which enzyme is a subunit of acetyl-CoA carboxylase and participates in the carboxylation of acetyl-CoA. In a preferred embodiment, such a polypeptide has the amino acid residue sequence of SEQ ID
NO:6 or SEQ ID NO:8, or a functional equivalent of those sequences. In accordance 10 with such an embodiment, a coding region comprises the entire DNA sequence ofSEQ ID NO:5 or the DNA sequence of SEQ ID NO:5 comprising the Anabaena accC
gene. Alternatively, a coding region comprises the entire DNA sequence of SEQ IDNO:7 or the DNA sequence of SEQ ID NO:7 comprising the Synechococcus accC
gene.
In another embodiment, an expression vector comprises a DNA segment that encodes a biotin carboxyl carrier protein of a cyanobacterium. That biotin carboxyl carrier protein preferably includes the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4, or functional equivalents thereof. In accordance with such an embodiment, a coding region comprises the entire DNA sequence of SEQ ID NO: 1 or20 the DNA sequence of SEQ ID NO: 1 comprising the Anabaena accB gene.
.Altern~tively, a coding region comprises the entire DNA sequence of SEQ ID NO:3 or the DNA sequence of SEQ ID NO:3 comprising the Synechococcus accB gene.
In another embodiment, an expression vector comprises a DNA segment that encodes a carboxyltransferase protein of a cyanobacterium. That carboxyltransferase 2~ protein preferably includes a CTa or CT,~ subunit, and preferably includes the amino acid residue sequence of SEQ ID NO:12, or a functional equivalent thereof. In accordance with such an embodiment, a coding region comprises the entire DNA
sequence of SEQ ID NO:11 or the DNA sequence of SEQ ID NO:11 compricing the SynechococcusaccA gene.

WO 96132484 PCTJUS9~ 5~5 In still yet another embodiment, an expression vector comprises a coding region that encodes a plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA. Such a plant polypeptide is preferably a monocotyledonous or a dicotyledonous plarlt acetyl-CoA carboxylase enzyme. A preferred S monocotyledonous plant polypeptide encoded by such a coding region is preferably wheat ACC, which ACC in~ es the amino acid residue sequence of SEQ ID NO: 10 or SEQ ID NO:31 or functional equivalents thereof. A preferred coding region includes the DNA sequence of SE~Q ID NO:9 or SEQ ID NO:30. ~l~rn,.tjvely, a preferred dicotyledonous plant ACC, such as canola ACC, is also preferred. Such an 10 ACC enzyme is encoded by the DNA segment of SEQ ID NO:l9 and has the amino acid sequence of SEQ ID NO:20.

4.5 Polypeptides The present invention provides novel polypeptides that define a whole or a 15 portion of an ACC of a cyanob~ct~rium or a plant. In one embodiment, thus, the present invention provides an isolated polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl ca~~Tier protein of a cyanobacterium such as Anabaena or Synechococcus. Preferably, a biotin carboxyl carrier protein from Anabaena includes the amino acid sequence of SEQ ID NO:2, with such amino acid 20 sequence listing encoded by the DNA segment of SEQ ID NO: 1. Preferably, a biotin carboxyl carrier protein from Synechococcus includes the amino acid sequence of SEQ ID NO:4, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:2.
In another embodiment, the present invention provides an isolated polypeptide 25 comprising a biotin carboxylase protein of a cyanobacterium such as Anabaena or Synechococcus. Preferably, a biotin carboxylase protein from Anabaena includes the amino acid sequence of SEQ ID NO:6, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:5. Preferably, a biotin carboxylase protein fromSynechococcusinch~ s the amino acid sequence of SEQ ID NO:8, with such amino 30 acid sequence listing encoded by the DNA segment of SEQ ID NO:7.

WO 96/32484 PCT/US9G~'O~ S

In another embodiment, the present invention provides an isolated polypeptide comprising a carboxyltransferase protein of a cyanobacterium such as Synechococcus Preferably, a carboxyltransferase protein comprises a CTa or CT~ subunit and includes the amino acid sequence of SEQ ID NO:12, with such amino acid sequence 5 listing encoded by the DNA se.gm~.nt of SEQ ID NO: 11.
In another embodiment, the present invention contemplates an isolated and purified plant polypeptide having a molecular weight of about 220 kDa, dimers ofwhich have the ability to catalyze the carboxylation of acetyl-CoA. Such a polypeptide preferably in~ fles the amino acid residue sequence of SEQ ID NO:10 or 10 SEQ ID NO:31, with such arnino acid sequence listing encoded by the DNA segment of SEQ ID NO:9 or SEQ ID NO:30. Alternatively the present invention provides an isolated and purified plant polypeptide from canola which has the ability to catalyze the carboxylation of acetyl-CoA. Such a polypeptide preferably includes the arnino acid residue sequence of SEQ ID NO:20, with such amino acid sequence listing 15 encoded by the DNA segment of SEQ ID NO: 19.

4.6 Transformed or Transgenic Cells or Plants A cyanobacterium, a yeast cell, or a plant cell or a plant transformed with an ekpLession vector of the present invention is also contemplated. A transgenic 20 cyanobacterium, yeast cell, plant cell or plant derived from such a transformed or transgenic cell is also contemplated. Means for transforming cyanobacteria and yeast cells are well known in the art. Typically, means of transformation are sirnilar to those well known means used to transform other bacteria or yeast such as E. coli or Saccharomyces cerevisiae. Synechococcus can be transformed simply by incubation 25 of log-phase cells with DNA. (Golden et al., 1987) Methods for DNA transformation of plant cells include Agrobacterium-me~ t~d plant transforrnation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into imm~fllre embryos and particle bombardment. Each of these methods has distinct advantages and disadvantages.
30 Thus, one particular method of introducing genes into a particular plant strain may not wo 96132484 ~CT~USS5'~5C)!75 . .

necç~.c~nly be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain.
~. There are many methods for introducing transforrning DNA segments into cells, but not all are suitable for delivering DNA to plant cells. Suitable methods are 5 believed to include virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-m~.r1i~t~d transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-me~ t~.rl DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc. In certain10 embo-lim-~nt.c, acceleration methods are preferred and inchlr1e, for example, microprojectile bombardment and the like.
Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described:
(1) ch~mi~l methods (Graham and van der Eb, 1973; Zatloukal etal., 1992); (2) 15 physical methods such as rnicroinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm etal., 1985) and the gene gun (Johnston and Tang, 1994;
Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a; 1988b); and (4) receptor-mediated mech~ni.sm~ (Curiel et al., 1991;
1992; Wagner et al., 1992).
4.6.1 Ele~lro~ol ~lion The application of brief, high-voltage electric pulses to a variety of animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane.
DNA is taken directly into the cell cytoplasm either through these pores or as a25 consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation can be extremely efficient and can be used both for transient expression of clones genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-m~ t~.~l transfection and protoplast fusion, frequently gives rise to cell 30 lines that carry one, or at most a few, integrated copies of the foreign DNA.
.

WO 96132484 PCTIUS~/0~35 The introduction of DNA by means of ele~ upola~ion, is well-krrown to those of skill in the aTt. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation, by mech~nic~l wounding. To effect ttransformation by ele~;l~opoldtion one may employ either friable tissues such as a suspension culture of cells, or embryogenic callus, or ~It~rn~tively, one may transform imm~tllre embryos or other or~ni7~1 tissues directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mech~nic~lly wounding in a controlled manner. Such cells would then be recipient to DNA transfer by ele~;llol,oldlion, which may be carried out at this stage, and transformed cells then icl~ntifi~d by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.

4.6.2 Microprojectile Bomba~ "~l,t A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, pl~tinllm, and the like.
An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforrning monocots, is that neither the isolation of protoplasts (~ristou et al., 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNAinto maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured insuspension. The screen disperses the particles so that they are not delivered to the ,.
recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles Wo 96/32484 PCT/US96/0~09S

aggregate and may contribute to a higher frequency of transformation by redl~cing damage inflicted on the recipient cells by projectiles that are too large.
For the bombardment, cells in suspension are preferably concentrated on filters or solid culture m~ m ~lt~ tively, imm~tllre embryos or other target cells may S be arranged on solid culture m~ lm The cells to be bombarded are positioned at an a~plol,liate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells tr;~n~iently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 1 to 3.
In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the m~ximllm numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in rnanipulation of cells before and imm~ te~ly after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as line~ri7t~cl DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immsltl-re embryos.
Accordingly, it is contemplated that one may wish to adjust various of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flightdistance, tissue distance, and helium pressure. One may also minimi~:e the trauma * reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum CA 02218139 1997- lO- 14 W096/32484 PCT/U~r J, ~5~35 transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.
Agrobacterium-m~ t~-l transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, 5 thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-m~ t~1 plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described (Fraley et al., 1985; Rogers et al., 1987). Further, the intt-~r:~tion of the Ti-DNA is a relatively precise process res-llting in few rearr~ngem~ont~ The region of DNA to be transferred 10 is defined by the border se~uences, and intervening DNA is usually inserted into the plant genome as described (Spielmann et al., 1986; Jorgensen et al., 1987).
Modern Agrobacterium transformation vectors are capable of replication in E.
coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors for 15 Agrobacterium-m~ t.od gene transfer have improved the arrangement of genes and restriction sites in the vectors to f~cilit~te construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are~0 suitable for present purposes. In addition, Agrobacterium Cont?~ining both armed and rmt~d Ti genes can be used for the transformations. In those plant strains whereAgrobacterium-mP~ tPrl transformation is efficient it is the method of choice because of the facile and defined nature of the gene transfer.
Agrobacterium-mediated transformation of leaf disks and other tissues such as 25 cotyledons and hypocotyls appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-m~cli~t~d transformation is most efficient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterillm vectors as described (Bytebier et al., 1987). Therefore, commercially important cereal 30 grains such as rice, corn, and wheat must usually be transformed using alternative Wo 96/32484 PCT~U5~5~50gS

methods. However, as mentioned above, the transformation of asparagus using Agrobacterium can also be achieved (see, for e~mple, Bytebier et al., 1987).
A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such tr~n~ge~ic plants can be S referred to as being h~le~ yg~us for the added gene. However, in~cmllch as use of the word "helel~zy~ ous" usually implies the presence of a complementary gene at the sarne locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant cont~inin~ one added gene as here, it is believed that a more accurate name for such a plant is an independent segregant, because the added, exogenous gene 10 segregates independently during mitosis and meiosis.
More plere-led is a tr~.n.~genic plant that is homozygous for the added structural gene; i.e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant 15 transgenic plant that contains a single added gene, gerrnin~ting some of the seed produced and analyzing the resulting plants produced for enh,.nced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
It is to be understood that two different transgenic plants can also be mated to20 produce offspring that contain two independently segregating added, exogenousgenes. Selfing of a~lupliate progeny can produce plants that are homozygous for both added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.
Transformation of plant protoplasts can be achieved using methods based on 25 calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, for example, Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).
Application of these systems to different plant strains depends upon the ability30 to regenerate that particular plant strain from protoplasts. Illustrative methods for the W096132484 PCTrUS~G~'~50~5 -~8-regeneration of cereals from protoplasts are described (Fujhllul~ et al., 1985;
Toriyama et al., 1986; Yamada et al., 1986; Abdu~ah et al., 1986).
To transform plant strains that cannot be sllrces~fully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utili7~cl S For example, regeneration of cereals from imm~hlre embryos or explants can be effected as described (Vasil, i988). II1 addition, "particle gun" or high-velocity microprojectile technology can be l~tili7Pd (Vasil, 1992) Using that latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al., 1987;
Klein et al., 1988; McCabe et al., 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.
Thus, the amount of a gene coding for a polypeptide of interest (i.e., a polypeptide having carboxylation activity) can be increased in monocotyledonous plants such as corn by transforming those plants using particle bombardment methods 15 (Maddock et al., 1991). By way of example, an expression vector cont~ining ancoding region for a dicotyledonous ACC and an a~,upLiate sçl~ct~hle marker is transformed into a suspension of embryonic maize (corn) cells using a particle gun to deliver the DNA coated on microprojectiles. Transgenic plants are regenerated from transformed embryonic calli that express ACC. Particle bombardment has been used20 to succescfully transform wheat (Vasil et al., 1992).
DNA can also be introduced into plants by direct DNA transfer into pollen as described (Zhou et al., 1983; Hess, 1987; Luo et al., 1988). Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described (Pena et al., 1987). DNA can also be injected directly 25 into the cells of imm~tllre embryos and the rehydration of desiccated embryos as described (Neuhaus et al., 1987; Benbrook et al., 1986).
The development or regeneration of plants from either single plant protoplasts or various explants is well known in the art (Weissbach and Weissbach, 1988). This regeneration and growth process typically includes the steps of selection of 30 transformed cells, culturing those individualized cells through the usual stages of wo 96132484 ~CTJV~ '0~~5S

embryonic development through the rooted plantlet stage. Tr~nsgçnic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter ~ planted in an a~ liate plant growth medium such as soil.
The development or regeneration of plants contairling the foreign, exogenous 5 gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described (Horsch et al., 1985). In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transforrned as described (Fraley e~ al., 1983).
This procedure typically produces shoots within two to four months and those shoots are then transferred to an a~plopliate root-inducing m~ m cont~ining the selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the15 particular plant strain employed, such variations being well known in the art.
Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, as discussed before. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is 20 used to pollinate regenerated plants.
A transgenic plant of the present invention cont~ining a desired polypeptide is cultivated using methods well known to one skilled in the art. Any of the transgenic plants of the present invention can be cultivated to isolate the desired ACC or fatty acids which are the products of the series of reactions of which that catalyzed by ACC
25 is the first.
A transgenic plant of this invention thus has an increased amount of an coding region (e.g., gene) that encodes a polypeptide of interest. A ~rcrcllcd transgenic plant is an independent segregant and can transrnit that gene and its activity to its progeny.
A more preferred transgenic plant is homozygous for that gene, and transmits that 30 gene to all of its off.~pring on sexual mating.

W 096/32484 PCTnUS96/05095 Seed from a transgenic plant is grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants.
The progeny from these plants become true breeding lines that are evaluated for, by way of example, herbicide re~i.et~nce, preferably in the field, under a range of5 environmental conditions.
The commercial value of a transgenic plant with increased herbicide resi~t~nce or with altered fatty acid production is enh~nce-l if many different hybrid combinations are available for sale. The user typically grows more than one kind of hybrid based on such differences as time to maturity, standability or other agronomic 10 traits. Additionally, hybrids adapted to one part of a country are not necessarily adapted to another part because of differences in such traits as maturity, disease and herbicide rçsi~t~nt~e. Because of this, herbicide resistance is preferably bred into a large number of parental lines so that many hybrid combinations can be produced.
15 4.7 Process of Increasing Herbicide R~c;et~n-~e Herbicides such as aryloxyphenoxypropionates and cyclohexane- 1 ,3-dione derivatives inhibit the growth of monocotyledonous weeds by interfering with fatty acid biosynthesis of herbicide sensitive plants. ACC is the target enzyme for those herbicides. Dicotyledonous plants, other eukaryotic org~ni~m~ and prokaryotic 20 organisms are resistant to those compounds.
Thus, the resistance of sensitive monocotyledonous plants to herbicides can be increased by providing those plants with ACC that is not sensitive to herbicide inhibition. The present invention therefore provides a process of increasing theherbicide resistance of a monocotyledonous plant comprising transforming the plant 25 with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a herbicide resistant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-termin~ting region, whereby the promoter is capable of driving thetranscription of the coding region in a monocotyledonous plant.

CA 022l8l39 l997- lO- l4 WC~ 96132.484 PCT)USS~'~3l~g5 Preferably, a herbicide resistant polypeptide, a dicotyledonous plant polypeptide such as an acetyl-CoA carboxylase enzyme from soybean, rape, sunflower, tobacco, Arabidopsis, petunia, canola, pea, bean, tomato, potato, lettuce, spinach, alfalfa, cotton or carrot, or functional equivalent thereof. A promoter and a S tranSCription-termin~tin~ region are preferably the same as set forth above.
Transformed monocotyledonous plants can be i~entifi~cl using herbicide resistance. A process for identifying a transformed monocotyledonous plant cell involves transforrning the monocotyledonous plant cell with a DNA molecule that encodes a dicotyledonous acetyl-CoA carboxylase enzyme, and det~.~ining the 10 resi.~t~nçe of the plant cell to a herbicide and thereby the identification oi the transformed monocotyledonous plant cell. Means for transforrning a monocotyledonous plant cell are the same as set forth above.
The re.~i~t~n- e. of a transformed plant cell to a herbicide is preferably c~eterrnined by exposing such a cell to an effective herbicidal dose of a preselected 15 herbicide and m~int~ining that cell for a period of time and under culture conditions sufficient for the herbicide to inhibit ACC, alter fatty acid biosynthesis or retard growth. The effects of the herbicide can be studied by measuring plant cell ACC
activity, fatty acid synthesis or growth.
An effective herbicidal dose of a given herbicide is that amount of the 20 herbicide that retards growth or kills plant cells not cont~ining herbicide-resistant ACC or that amount of a herbicide known to inhibit plant growth. Means for determining an effective herbicidal dose of a given herbicide are well known in the art. Preferably, a herbicide used in such a process is an aryloxyphenoxypropionate or cyclohexanedione herbicide.

4.8 Process of Altering ACC Activity ACC catalyzes the carboxylation of acetyl-CoA. Thus, the carboxylation of acetyl-CoA in a cyanob~ct~rillm or a plant can be altered by, for example, increasing an ACC gene copy number or ch~n~ing the composition (e.g., nucleotide sequence) of 30 an ACC gene. Changes in ACC gene composition may alter gene expression at either the transcriptional or translational level. ~lt~rn~tively, changes in gene composition can alter ACC function (e.g., activity, binding) by çh~nging primary, secondary or tertiary structure of the enzyme. By way of example, certain changes in ACC
structure are associated with changes in the resiet~n- e of that altered ACC to herbicides. The copy number of such a gene can be increased by transforming a cyanobacterium or a, plant cell with an a~plu~l;ate expression vector comprising a DNA molecule that encodes ACC.
In one embodiment, therefore, the present invention contemplates a process of altering the carboxylation of acetyl-CoA in a cell comprising transforming the cell with a DNA molecule comprising a promoter operatively linked to a coding region that encodes a polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is o~ aliv~ly linked to a transcription-t~rmin~ting region, whereby the promoter is capable of driving the transcription of the coding region in the cyanobacterium.
In a preferred embodiment, a cell is a cyanobacterium or a plant cell, a polypeptide is a cyanobacterial ACC or a plant ACC. Exemplary and preferred expression vectors for use in such a process are the same as set forth above.

4.9 Determining Herbicide R~ei.et~nce Inheritability In yet another aspect, the present invention provides a process for det~rmining the inheritance of plant resistance to herbicides of the aryloxyphenoxypropionate or cyclohexanedione class. That process involves measuring resistance to herbicides of the aryloxyphenocypropionate or cycloh~x~n~lione class in a parental plant line and in progeny of the parental plant line and rl~tP.cting the presence of a DNA segment encoding ACC in such plants.
The inheritability of phenotypic traits such as herbicide resistance can be determined using RFLP analysis. Restriction fragment length polymorphisms (RFLPs) are due to sequence differences detectable by lengths of DNA fragments generated by digestion with restriction enzymes and typically revealed by agarose gel 30 electrophoresis. There are large numbers of restriction endonucleases available, WO 96/32484 PCTIUS96JD51~9r, char~cttori7~1 by their recognition sequences and source. From these studies, it is possible to correlate herbicide re~i.ct~nre with a particular DNA fragment and analyze the inht~rit~nce of such r~ t~n~e in progeny plants.
In a preferred embodiment, the herbicide resistant variant of acetyl-CoA
carboxylase is a dicotyledonous plant acetyl-CoA carboxylase enzyme or a portionthereof. In another plerell~d embodiment, the herbicide resistant variant of acetyl-CoA carboxylase is a m~lt~ter1 monocotyledonous plant acetyl-CoA carboxylase that confers herbicide re~ict~nce or a hybrid acetyl-CoA carboxylase comprising a portion of a dicotyledonous plant acetyl-CoA carboxylase, a portion of a monocotyledonous plant acetyl-CoA carboxylase or one or more domains of a cyanobacterial acetyl-CoA
carboxylase.
Restriction fragment length polymorphism analyses are conducted, for example, by Native Plants Illc~olaled (NPI). This service is available to the public on a contractual basis. For this analysis, the genetic marker profile of the parental inbred lines is ~lrtçrrnin~d. If parental lines are çc~çnti~lly homozygous at all relevant loci (i.e., they should have only one allele at each locus), the diploid genetic marker profile of the hybrid offspring of the inbred parents should be the sum of thoseparents, e.g., if one parent had the allele A at a particular locus, and the other parent had B, the hybrid AB is by inference.
Probes capable of hybridizing to specific DNA segments under ~plo~liate conditions are prepared using standard techniques well known to those skilled in the art. The probes are labelled with radioactive isotopes or fluorescent dyes for ease of detection. After restriction fragments are separated by size, they are identified by hybridization to the probe. Hybridization with a unique cloned sequence permits the identification of a specific chromosomal region (locus). Because all alleles at a locus are detectable, RFLP's are co-dominant alleles. They differ from some other types of markers, e.g., from isozymes, in that they reflect the primary DNA sequence, they are not products of transcription or translation.
.~

WO 96/32484 I'CT/U~Gl'~50~5 4.10 Oil Content of Seed~c Manipulation of the oil content and quality of seeds may benefit from knowledge of this gene's structure and regulation. Underst~ncling the basis of resict~n~e to herbicides, on the other hand, will be useful for future attempts to construct transgenic grasses and to provide crop plants such as wheat with selective reci.ct~nre Genes of the present invention may be introduced into plants, particularly monocotyledonous plants, particularly commercially important grains. A wide range of novel transgenic plants produced in this manner may be envisioned depending on the particular constructs introduced into the transgenic plants. The largest use of grain is for feed or food. Introduction of genes that alter the composition of the grain may greatly enhance the feed or food value.
The introduction of genes encoding ACC may alter the oil content of the grain, and thus may be of cigni~lc~nt value. Increases in oil content may result in increases in metabolizable-energy-content and -density of the seeds for uses in feed and food.
The introduction of genes such as ACC which encode rate-limiting enzymes in fatty acid biosynthesis, or replacement of these genes through gene disruption or deletion mutagenesis could have significant impact on the quality and quantity of oil in such transgenic plants.
Likewise, the introduction of the ACC genes of the present invention may also alter the balance of fatty acids present in the oil providing a more healthful or nutritive feedstuff. ~lt(-rn~tively, oil properties may also be altered to improve its perforrnance in the production and use of cooking oil, shortenings, lubricants or other oil-derived products or improvement of its health attributes when used in the food-related applications. Such changes in oil ~ Jp~lLies may be achieved by altering the type, level, or lipid arrangement of the fatty acids present in the oil. This in turn may be accomplished by the addition of genes that encode enzymes that catalyze the synthesis of novel fatty acids and the lipids pocceccing them or by increasing levels of native fatty acids while possibly reducing levels of precursors.

WO 96132484 PCT)US9'~ iO9~;

.~ltern~tively, introduction of DNA segmP.nt~ which are complem~nt~ry to the DNA segments disclosed herein into plant cells may bring about a decrease in ACCactivity in vivo and lower the level of fatty acid biosynthesis in such transformed cells.
Therefore, transgenic plants co~ ing such novel constructs may be hllpo~ l due to their decreased oil content in such cells. Introduction of specific mutations in either the DNA se~m~nt~ disclosed, or in their compl~m~-nts, may result in transformed plants having interm-odi~tt~ ACC activity.
The following examples are included to demonstrate ~,r~;fe.led embo~liment~
of the invention. It should be appreciated by those of skill in the art that thetechniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embo-lim~nts which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

5. EXAMPLES
5.1 EXAM[PLE 1 -- Cloning and Sequencing of the Anabaena acc Genes 5.1.1 Biotin Carboxylase (accC) The gene for the BC subunit was cloned with a fragment of the E. colifabG
gene as a heterologous hybridization probe. Southern analysis of Anabaena sp. strain PCC 7120 DNA digested with various restriction enzymes, carried out at low stringency (57~C, 1 M NaCl, GeneScreen Plus~ membrane [DuPont]) in accordance with the m~nnfa~t~lrer's protocol, with an ssm-pstI fragment consisting of ~90% of the coding region of thefabG gene from E. coli as a probe revealed, in each case, only one strongly hybri~li7ing restriction fr~gmPnt The 3.1-kb Hindm fragment identified by this probe in the Anabaena sp. strain PCC 7120 DNA digest was purified by gelelectrophoresis and then was digested with NheI, yielding a 1.6-kb NheI-Hindm fragment that hybridized with the samefabG probe. The 1.6-kb fragment was purified .

by gel electrophoresis and cloned into XbaI-Hindm-~ligeste~1 pUC18. The ends of the insert were sequenced.
A fragment of an open reading frame coding for a polypeptide with very high similarity to an internal sequence of E. coli BC was found at the NheI end of the 5 insert. This result in~ te-1 that the 3.1-kb Hindm fragment contained the entire Anabaena sp. strain PCC 7120 BC gene. The 1.6-kb Anabaena sp strain PCC 7120 DNA fragment was then used as a probe to screen, at high stringency (65'C, 1 M
NaCl), a cosrnid library of Anabaena sp. strain PCC 7120 DNA in the cosmid vector pWB79 (Charng etal., 1992), constructed by W.J. Buikema (University of Chicago) 10 with a sized partial Hindm digest of chromosomal DNA. Five cosmids containingoverlapping fragments of Anabaena sp. strain PCC 7120 DNA were found in the 1,920-member bank, all of which contained the sarne size Hindm and NheI fr~gm~ntc as those identified by the E. coli probe previously. From one of the cosmids, the 3.1-kb Hindm fragment was subcloned into pUC 18 and sequenced.
Nucleotide sequences of both strands were ~l~terrnin~l on double-stranded templates by the dideoxy chain terrnin~tion method with Sequenase (United StatesBiochernicals). Sets of nested deletions generated with an Erase-a-Base kit (Promega) as well as specific primers were used for sequencing. The 3065-nucleotide DNA
segment comprising the Anabaena accC gene is given in SEQ ID NO:5. The 477-20 arnino acid translation of the accC gene encoding the Anabaena BC protein is given in SEQ ID NO:6.

5.1.2 Biotin Carboxyl Carrier Protein (accB) A different approach had to be used to clone the Anabaena sp. strain PCC
25 7120 BCCP gene. An earlier attempt to clone the gene with a fragment of E.coli DNA cont~ining the fabE gene as a heterologous hybridization probe failed.
Furthermore, analysis of the sequence (~1.3-kb) located upstream of the Anabaena sp strain PCC 7120 BC gene revealed no open reading frame corresponding to BCCP, incontrast to the E.coli gene org~ni~tion in which the BCCP gene is located WO 96132484 PCTIUSS~ 35 imm~ t~1y u~ ealll of the BC gene. The BCCP gene was cloned by PCRTM
amplification.
The N-tf rTnin~l amino acid sequence of BCCP was used to design an upstream ,~ PCRTM primer. The downstream primer was targeted to the conserved sequence 5 encoding the biotinylation site. The primers had the following structure:
Amino acid sequence: LDFNEIR (SEQ ID NO:22) Primer I 5'-GCTCTAGAYTTYAAYGARATHMG-3' (SEQ ID NO:23) Arnino acid sequence: NMKMX (SEQ ID NO:24) (N= V or A) Primer II 3'-CRNTACTTYTACNWCTTAAGCT-5' (SEQ ID NO:25) where Y= T or C; R= A or G; M= C or A; H= A, C, or T; W= A or T; N= T, C, A, or G.
PCRTM was carried out as described in the GeneAmp(~) kit manual (Perkin-Elmer Cetus). All components of the PCRTM except the Taq DNA polymerase 15 were incubated for 3 to 5 min at 95~C. The PCRTM was then initi~t~cl by the addition of polymerase. Amplification was for 45 cycles, each 1 rnin at 95~C, 1 min at 42 to 45~C, and 2 min at 72~C, with 0.5 to 1.0 ,ug of template DNA per ml and 50 ~Lg of each primer per ml. The PCRTM amplification yielded a product ~450 bp in size (i.e., the correct size for the ~nticir~t--rl fragrnent of the Anabaena sp. strain PCC 7120 20 BCCP gene ~lecluce~-l from the E. coli sequence and allowing for a 60- to 90-nucleotide addition due to the polypeptide length difference). The PCRTM product was clonedinto the Invitrogen vector pCR1000 with the A/T tail method and was sequenced toconfirm its identity.
The fragment of the Anabaenasp. strain PCC 7120 BCCP gene was then used 25 as a probe to identify cosmids that contain the entire gene and fl~nking DNA. Three such cosrnids were ~ietectecl in a 1,920-member library (same as described above). A
4.2-kb XbaI fragment cont:lining the BCCP gene was subcloned into pBluescriptII~, and its Hindm-NheI fragment was sequenced with specific primers as described above. The 1458-nucleotide DNA segment comprising the Anabaena accB gene is WO 96/32484 PCI~/US9' given in SEQ ID NO:1. The 182-amino acid translation of the accB gene encoding the Anabaena BCCP is given in SEQ ID NO:2.
The amino acid sequence deduced from the DNA sequence of the BCCP gene exactly m~tchPs the N-t~-rminzll sequence obtained for purified protein. Likely 5 tr~n~l~tion initiation codons were i~1entified by comparison with E. coli. For the BC
gene, the AUG start codon is not preceded by an obvious ribosome-binding site.
There is a stop codon in the same open reading frame one codon upstream from theAUG codon, excluding the possibility of additional amino acids at the N terminus.
The GUG start codon for BCCP imm~ tely precedes codons for the amino acids 10 identified by protein sequencing of the N terminlls of purif1ed BCCP. A putative 5-nucleotide ribosome-binding site, GAGGU, is located 11 nucleotides upstream ofthe GUG codon. The open reading frame extends further upstream of the GUG codon (for about 60 codons), but there are no AUG or GUG codons that could serve as start sties from translation. This excludes the possibility that the purified BCCP
15 polypeptide lacks more than one amino acid (Met) because of rapid proteolytic degradation.
Structural similarities ~ ce~l from the available amino acid sequences suggest strong evolutionary conservation among BCs (Al-Feel et al., 1992; Knowles, 1989; Lopez-Casillas et al., 1988; Samols et al., 1988; Takai et al., 1988).
20 Comparison of the amino acid sequence of the BC domain defined as the part of the sequence between amino acids Lys-5 and Phe-432 of Anabaena sp. strain PCC 7120 BC, the two outermost amino acids present in all or all but one of the compared sequences, revealed that all highly conserved amino acid residues identif1ed before are present in Anabaena sp. strain PCC 7120 BC, incl~l-ling the ATP binding site motif 2~ and the conserved sequence including Cys-230 as a part of the bicarbonate binding site. The identity between the amino acid sequence of the Anabaena sp. strain PCC
7120 BC domain (based on the best multiple alignment) and that of rat (Lopez-Casillas et al., 1988), chicken (Takai et al., 1988), yeast (Al-Feel et al., 1992), and wheat ACCs was no more than 32 to 37%. Mitochondrial enzymes, rat 30 propionyl-CoA carboxylase (Browner etal., 1989) and yeast pyruvate carboxylase W<) 96/32484 PCT/~JS96)1)5095 (Lim et al., 1988), are only 45 to 47% i~lPntic~l. Simil~riti~s with carbamoyl-phosphate synth~t~ces observed for other BCs (Knowles, 1989; Li and Cronan, 1992; Lopez-Casillas et al., 1988; Samols et al., 1988; Takai et al., 1988) are also evident forAnabaena sp. strain PCC 7120 BC.
Anabaena sp. strain PCC 7120 BCCP is unique with its biotinylation site, the result of a single A-to-C base change resulting in a Met-to-Leu substitution. This base change explains the highly variable yield of the PCRTM amplification with primer II.
The structure of this part of the BCCP gene was confirm~.~1 by sequencing the corresponding PCRTM-cloned fragment of Anabaena sp. strain PCC 7120 DNA. The result is not entirely surprising, because in vitro analysis of mllt~nt~ of the 1.3S
subunit of transcarboxylase from Propionibacterium shennanii, in which the same Met-to-Leu change was introduced, showed that this methionine residue is not essçnti~l for efficient biotinylation of the apo~l~,tein (Shenoy etal., 1992). Urea carboxylase contains Ala at this position. The conserved motif may be required for some other functions. Furthermore, it was suggested that the distance between the biotinylated lysine residue and the C t~rrninl-c and the structure of the last two amino acids (hydrophobic one followed by acidic one) are important detrnnin~nt~ for the modification of at least some BCCP apoproteins (Shenoy etal., 1992). Two amino acids with the same properties are also found at an analogous position (with respect to the distance from the biotinylation site) of large eukaryotic biotin-dependent carboxylases. Anabaena sp. strain PCC 7120 BCCP also contains those amino acids,but they are separated from the biotinylation site by two additional amino acids.
Anabaena sp. strain PCC 7120 BCCP is about 30 amino acids longer than the E. coli protein, including a 21-amino-acid insertion near the N terminus. The moderate conservation of the amino acid sequence is reflected by rather low conservation at the nucleotide level (Table 3), which explains why the E. coli BCCP specific probe failed to identify the Anabaena sp. strain PCC 7120 gene.
Comparison of the amino acid sequence encoded by the additional short open reading frame located upstream of the BCCP gene and transcribed in the same WO 96/32484 PCT/U~ 610503S

direction and sequences deposited in GçnR~nk (release 75) revealed no similar proteins.

5.1.3 Northern analysis oftheBCCP ...~ C
S The size of Anabaena sp. strain PCC 7120 BCCP mRNA was established by Northern (RNA) analysis with the PCRTM-amplified fragment of the gene as a probe.
The major hybri~li7.ing mRNA is 1.45-kb in size. The two minor species are 1.85 and 2.05-kb in size. All of these are long enough to include the BCCP coding region. The amount of all three mRNAs seems to be higher (about twofold) in cells grown in the absence of combined nitrogen. The 24-h induction time correlates with the onset of nitrogen fixation in heterocysts, differentiated cells that fix nitrogen and have a unique glycolipid envelope cont~ining C26 and C28 fatty acids (Murata and Nishida, 1987). If the increase of the level of the BCCP mRNA is heterocyst specific, it must be significant because heterocysts in Anabaena sp. strain PCC 7120 filaments are formed 1~ only at ~10-cell intervals. This result suggests that ACC may be developmentally regulated in Anabaena sp. strain PCC 7120. Results of some recent experiments indicate that, in bacteria, modulation of ACC activity may indeed play an important role in the overall regulation of the biosynthesis of the cell lipids. It has been demonstrated that the level of transcription of the ACC genes is correlated in E. coli with the rate of cellular growth and nutritional upshifts and downshifts (Li andCronan, 1993). Mutations in the E. colifabGE operon which decrease the rate of phospholipid biosynthesis suppress a null mutation in the htrB gene by restoring the balance between phospholipid biosynthesis and cell growth (Karow etal., 1992).
Northern analysis with the 1.6-kb NheI-Hindm fragment as a BC-specific probe repeatedly gave a smeared band pattern which could not be interpreted.
Unlike the BCCP and BC genes of E. coli where they are cotranscribed, the BCCP and BC genes of the present invention are separated by at least several kilobases (no overlapping cosmids were seen when the cosmid library was screenedwith probes specific for BCCP and BC).

WO 96/32484 PCTllJS96/O~iO9~;

5.2 EXAl\IPLE 2 -- Purifi~ti~ and Chara~le.i~tion of Anob~en~ BCCP
Western immunoblot analysis of Anabaena sp. strain PCC 7120 proteins with - 35S-streptavidin revealed one biotinylated polypeptide ~25 kDa in size. Although the presence of other, much less abundant biotinylated proteins cannot be strictly ruled 5 out, this result strongly suggests that ACC is the only biotin-dependent enzyme in Anabaena sp. strain PCC 7120, with the BCCP subunit of 19 kDa, the calculated size;
25 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The polypeptide shows a slightly lower mobility than E. coli BCCP (~22.5 kDa), suggesting that Anabaena sp. strain PCC 7120 BCCP is longer by 20 to 30 amino acids. However, the unusual electrophoretic ~lo~elLies of the E. coli protein (Li and Cronan, 1992) make an accurate prediction of the polypeptide length difficult.
Separation of Anabaena sp. strain PCC 7120 proteins for Western analysis or sequencing) was by SDS-PAGE with 12.5% separating gels (Sambrook etal., 1989) followed by transfer onto polyvinylidene difluoride membrane (Immobilon-P(~);
Millipore) in 10 mM sodium 3-(cyclohexylamino)-1-propane-sulfonate buffer (pH
11)-10% methanol. Western blots were blocked with 3% bovine serum albumin solution in 10 mM Tris-HCl (pH 7.5) and 0.9% NaCl and then were incubated for 3 to 16 h with 35S-streptavidin (Amersham). The blots were washed at room temperaturewith 0.5% Nonidet P 40TM in 10 mM Tris-HCl (pH 7.5) and 0.9% NaCl.

WO 96/32484 PCT/U59G~'~50!~5 COMPARISON OF BC AND BCCP SUBUNITS FROM
Anabaena AND E. coli ACC subunit No. of amino acids (mol wt)b Identity (.cimilzlrity) Anabaena sp. strain E. col BC
Protein 447 (49,076) 449 57 (74) DNAd 58 BCCP
Protein 182 (19,126) 156 39 (65) DNAd 41 S a The genes for the two subunits of ACC are unlinked in Anabaena sp. strain PCC 7120; in E. coli they are in one operon.
b Molecular weight was calculated from amino acid composition.
c From Li and Cronan, 1992.
d On the basis of amino acid ~lignm~nt.
BCCP from Anabaena sp. strain PCC 7120 was purified starting with cells from a 3-liter culture grown on BGll medium (Rippka etal., 1979). Cells were broken by sonication at 0~C in 30 ml of 0.5 m NaCl-0.1 M Tris-HCl (pH 7.5)-14 mM~-mercaptoethanol-0.2 mM phenylmethylsulfonyl fluoride. Insoluble m~tt ri:~l was15 removed by centrifugation at 31,000 x g for 30 min, and the soluble protein fraction cont~ining BCCP was precipitated by adding solid ammonium sulfate (50%
saturation). The pellet was resuspended in 15 ml of 0.2 M NaCl-50 mM Tris-HCl (pH
7.5)-10% glycerol-0.5% SDS and then mixed at room temperature for about 18 h with 0.5 ml of streptavidin-agarose suspension (GIBCO BRL). The mixture was loaded 20 onto a column, was washed with about 30 ml of 0.25 M NaCl-50 mM Tris-HCl (pH
7.5)-0.5 mM EDTA-0.2% SDS, and then was washed with 5 ml of water.
Biotinylated peptides were eluted with 3 ml of 70% formic acid, dried under vacuum, WO 96/32484 PC~JlJSgl~)l)J;~9 and separated by SDS-PAGE. The N-terrnin~l sequence of the biotin-cont~ining ~25-kDa polypeptide was dçterminPd by Edrnan degradation after transfer to - Immobilon-P~;) as described above. The sequence was PLDFN~IRQL (SE~Q ID
NO:21).

5.3 EXAMPLE 3 -- Characterization of the Synechococcus acc Genes and Purification of the Synechococcus BCCP
5.3.1 Biotin Carboxylase (accC) All carboxylases have a conserved arnino acid motif that constitutes the 10 ATP-binding site. A 1.2-kb Sst~-PstI fragment (cont~ining the ATP-binding motif) within the E. coliaccC gene was used as a probe to examine the Synechococcus PCC7942 genornic DNA by Southern hybridization at 58~C. A strongly hybri~li7ing 0.8-kb Bam HI-PstI fragment was detected and subsequently cloned by a two-stage size fractionation method.
Synechococcus PCC 7942 genomic DNA was first digested with Bam HI and electrophoresed on an agarose gel. The gel region cont~ining DNA of sizes between 1.6-kb and 3-kb was cut out and purified (using Geneclean II Kit from BiolOl). The purified DNA was then digested with PstI and electrophoresed on an agarose gel. The gel region cont~ining DNA of sizes between 0.5-kb and 2-kb was cut out and purified.
20 DNA samples (from each step of purification) were electrophoresed, transferred onto a Genescreen Plus membrane, hybridized with the E. coli accC probe to confirm that the homologous DNA fragrnent was not lost during each purification step. A library of fragments between 0.5-kb and 2-kb was created by cloning the purified fraction of Synechococcus PCC 7942 DNA into vector pBluescript(~) KS. Ampicillin-resistant 25 and white (i.e., with insert) colonies were selected by plating on LB plates cont~ining ampicillin, X-Gal and IPTG.
A total of 287 ampicillin-resi~t~nt white clones were screened; the plasmid DNA rnixture (from pools of 5 white clones per pool) were prepared, doubly-digested with PstI and BamHI, electrophoresed, transferred onto a Genescreen Plus membrane, 30 then hybridized with the E. coliaccC probe at 58~C. Positive signals appeared on 8 WO 96/32484 PCT/US96/0' ~!3S

pools. Twelve positive individual clones were identified at the second round of screening. Two (of the 12) positive clones, each with a single fragment inserted, had the inserts sequenced. Both clones had iclenti~l inserts. Sequence comparison in~ t~rl only about 60% identity at the nucleotide level between the E. coli accC
gene and the cloned Synechococcus PstI-BamHI fragment. This cloned fragrnent wasthen used as a probe to screen a Synechococcus cosmid library. Hybridization of the cosmid library was performed at 65~C. One hybridizing clone was identified and a2.4-kb BamH~-NheI fragment from this cosmid clone was isolated and sequenced.
The 1362-nucleotide DNA segment comprising the Synechococcus accC gene is given in SEQ ID NO:7. Only one signific~nt open reading frame (OR~) was found.
This ORF potentially encodes a protein of 453 amino acids. The complete trzln~J~tç~
amino acid sequence of the Synechococcus accC gene encoding BC is given in SEQ
ID NO:8.

5.3.2 Biotin Carboxyl Carrier Protein (accB) In Synechococcus PCC 7942, the accB gene is not imm~ tely upstream of accC, as it is in E. coli. Gene-specific DNA probes from both E. coli and Anabaena PCC7120 accB failed to hybridize with the Synechococcus genomic DNA by Southern analysis. A different approach was necessary.
Since biotin carboxyl carrier protein is biotinylated and streptavidin has a strong specific affinity for biotin, streptavidin was used to identify the number of biotin-cont~inin~ proteins in Synechococcus PCC 7942. The proteins (from a crudewhole protein extract) of Synechococcus PCC 7942 were first separated by standard SDS-PAGE method, then transferred onto an Immobilon-P~ transfer membrane, which was subsequently incubated with 35S-streptavidin. Only one radioactive band (corresponding to a protein of about 25 kDa) appeared on the autoradiogram. Thisresult suggests that there is only one biotin-cont~ining protein in Sy)lechococcus and its mass is similar to the reported mass of E. coli biotin carboxyl carrier protein, 22,500 Da.

WO 96132484 PCT)US9~ 3G~5 This biotin-cont~inin~ protein was purified Synechococcus cells were first broken by sonication in a buffer cont~ining NaCl, Tris, glycerol and SDS. The - supern~t~nt was separated from cell debris by centrifugation, then followed by a 50%
(NH4)2 S04 precipitation. The precipitate was dissolved in the same buffer, and was allowed to bind to ~ avidin agarose beads. The bound agarose beads were washed and the bound proteins were eluded with 70% formic acid. The formic acid-eluted portion was dried and washed with water before loading onto an acrylamide gel. After electrophoresis, the proteins were transferred from the gel to an Imrnobilon-P(~) transfer membrane. The membrane was stained briefly with Coomassie Brilliant blue dye, destained in a mixture of methanol and acetic acid, and soaked in water for na hour or so before air drying. The band corresponding to the streptavidin-b~und protein was cut out and its N-ferrnin~l amino acid sequence was determined.
Based on the amino acid sequence from the N-terminlls of the Synechococcus biotin-cont:~ining protein and the amino acid sequence around the biotinylation site in all other known BCCPs, degenerate oligonucleotide primers were designed for PCRTM
amplification studies with Synechococcus genomic DNA. The pair of primers were:
primer LE8 5'-GCTCTAGACNCARYTNAAYTT-3' (SEQ ID NO:26) primer LE7 3 '-CRNTACTTYGACNWCTTAAGCT-5' (SEQ ID NO:27) PCRTM was performed for 40 cycles (each with 1 minute at 95~C, 1 minute at 48~C, 2 minutes at 72~C), with Cetus Taq polymerase, 0.5 mglml of template DNA, 5 mg/ml of primer LE8, 40 mg/ml of primer LE7 and with 1 mM Mg2+ final concentration. Under these conditions, a specific PCRTM produce was identified.
Sequence analysis of this cloned PCRTM product indicated that it encoded a region of conserved amino acids within accB of Synechococcus PCC 7942 (compared to the amino acid sequences of the biotin carboxyl carrier protein from Anabaena PCC 7120 and E. coli). Using this PCRTM fragment as a probe in Southern hybridization, a positive clone was identified from the Synechococcus cosmid library. A 1.6-kb PstI
fragment from this positive cosmid clone was isolated and sequenced.
A 477-nucleotide DNA segment comprising the Synechococcus accB gene is given in SEQ ID NO:3. Only one .cignific~nt ORF was found. The deduced amino WO 96/3~484 PCT/U59~ 0~5 acid sequence at the N-terminus of this ORF m~tchPs the earlier ~ptermin~d N-terminal amino acid sequence of the purified Synechococcus biotin-cont~ining protein. The 158-amino acid sequence of the Synechococcus BCCP is given in SEQ
ID NO:4. Sequence ~lignment in(liczlt.o~ that the translational product of accB from Synechococcus PCC 7942 is closer to that from Anabaena PCC 7120 than that from E.coli (53% versus 31% amino acid identity).

5.3.3 Carboxyltransferase a Subunit (CToc, accA) A 0.9-kb ClaI-MluI fragment of the E. coli accA gene was used as a probe to ex~min~ the Synechococcus PCC 7942 genomic DNA by Southern hybridization at 60~C. A strongly hybridizing 1.6-kb PstI fragment was detected and subsequently cloned.
Synechococcus PCC 7942 genomic DNA was digested with PstI and electrophoresed on an agarose gel. The gel region cont~ining DNA of sizes between 1.6 and 2.5-kb was cut out and purified. A size library between 1.6-kb and 2.5-kb was created by cloning the purified fraction of Synechococcus PCC 7942 DNA into vector pBR322. Tetracycline-resistant, but ampicillin-sensitive, colonies (i.e., with insert) were selected by first plating on LB plates cont~ining tetracycline, then scored on plates cont~ining ampicillin.
A total of 800 tetracycline-resi~t,.nt, but ampicillin-sensitive, clones were screened: the plasmid DNA was prepared, digested (in pools of 5 clones per pool)with PstI, electrophoresed, transferred onto a Genescreen Plus membrane, then hybridized with the E. coli accA probe at 60~C. Positive signals appeared on 3 pools.
One positive individual clone, with 2 fragments inserted, was identified at the second round of screening. The positive fragment was isolated and re-cloned. This cloned 1.6-kb PstI fragment was then used as a probe to screen the Synechococcus cosmidlibrary where 9 positive clones were icle~tific-l A 5-kb BamHI fragment from one of these 9 clones was isolated and sequenced. DNA sequence analysis of the region indicated a cluster of three ORFs in the same orientation.

wo 96~32484 PCT~U5~ 'GS~3S

The 98~nucleotide DNA segrn~nt comprising the Synechococcus accA gene is given in SEQ ID NO: 11. The first open reading frame encodes the oc subunit of the carboxyltransferase. The 327-amino acid sequence of the Synechococcus ORF is 54%iclenti~l to that of the E.coli accA gene. The arnino acid sequence of the S Synechococcus accA gene encoding CTa is given in SEQ r~ NO: 12.

.3.4 Carboxyltransferase ~ Subunit (CT,B, accD) Oligonucleotide primers, for polymerase chain reaction (PCRTM) amplification experiments with Synechococcus genomic DNA, were based on the sequence of ORF326 (which is a homolog of the E. coli accD) from a different cyanobacterium,Synechocystis PCC 6803. he pair of primers were:
LE39 5'-GAAGATCTTTATGGGCGGTAGTATG-3' (SEQ ID NO:28) LE40 3'-GGTCGAAACGGTACAACCTAGGC-5' (SEQ ID NO:29) PCRTMwas run for 40 cycles (each with 1 minute at 95~C, 1 minute at 50~C, 2 minutes at 72~C), with Boehringer-Mannheim Taq polymerase, 0.5 mg/ml of templateDNA, 5 mg/ml of each primer and with 1 mM Mg2+ final concentration. Under these conditions, a specific PCRTM product of 256 bp was identified. Sequence analysis of this cloned PCRTM fragment showed a si~nific~nt similarity between the Synechococcus and Synechocystis genomic DNAs in the region between the primers.
Using this cloned PCRTM product as a probe, 5 positive cosmid clones were identified from the Synechococcus cosmid library by Southern hybridization.

5.4 EXAMPLE 4 -- Isolation and Chara~ ,ation of the Wheat ACC
Enzyme Biotin-cont~inin~; (streptavidin-binding) proteins in extracts prepared from leaves of two-week old seel11ing~ of wheat and pea, both total protein and protein from intact chloroplasts (prepared by centrifugation on Percoll gradients as described previously in Fernandez and Lamppa, 1991), and from wheat germ (Sephadex G-100 fraction prepared as described below) were analyzed by western blotting with 35s-s~ avidin. Proteins were separated by SDS-PAGE using a 7.5% sep~atillg gel (Maniatis et al., 1982), and then were transferred onto a PVDF membrane (Immobilon-P~, Millipore) in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer (pH 11), 10% methanol, at 4~C, 40 V, overnight. The blots were blocked with 3% BSA solution in 10 mM Tris-HCl pH 7.5 and 0.9% NaCl and then incubated for 3- 16 h with 35S-Streptavidin (Amersham). The blots were washed at room telllpe,~Lu,c~ with 0.5% Nonidet-P40TM in 10 mM Tris-HCl pH 7.5 and 0.9% NaCl.
In wheat, the 220-kDa protein was present in both total and chloroplast protein. It was the major biotinylated polypeptide in the chloroplast protein (traces of smaller biotinylated polypeptides, most likely degradation products of the large one, could also be de~ccted). ACC consisting of 220-kDa subunits is the most abundantbiotin-dependent carboxylase present in wheat chloroplasts. In pea chloroplasts the biotinylated peptides are much smaller, probably due to greater degradation of the 220-kDa peptide, which could be detect.-~l only in kace amounts in some chloroplast preparations. The amount of all biotinylated peptides, estim~t~cl from band intensities on western blots (amount of protein loaded was norm~li7e-1 for chlorophyll content).
is much higher in pea than in wheat chloroplasts.
Purification of wheat germ ACC was carried out at 4~C or on ice. 200 g of wheat germ (Sigma) were homogenized (10 pulses, 10 s each) in a Waring blender with 300 ml of 100 mM Tris-HCl pH 7.5, 7 mM 2-mercaptoethanol. Two 0.3 ml aliquots of fresh 0.2 M solution of phenylmethyl-sulfonyl fluoride (Sigma) in 100%
ethanol were added immediately before and after homogenization. Soluble protein was recovered by centrifugation for 30 min at 12000 rpm. 1/33 volume of 10%
poly(ethyleneimine) solution (pH 7.5) was added slowly and the mixture was stirred for 30 min (Egin-Buhler et al., 1980), followed by centrifugation for 30 min at 12000 RPM to remove the precipitate. ACC in the supernatant was precipitated by addingsolid ammonium sulfate to 50% saturation.
The precipitate was collected by centrifugation for 30 min at 12000 rpm, dissolved in 200 ml of 100 mM KCl, 20 mM Tris-HCl pH 7.5, 20% glycerol, 7 mM
2-mercaptoethanol, mixed with 0.2 ml of phenylmethylsulfonyl fluoride solution (as Wo 96132484 PCTJU~ 5.'1~t~5 above) and loaded on a 5 cm x 50 cm Sephadex G-100 column equilibrated and eluted with the same buffer. Fractions cont~inin~ ACC activity (assayed as described below - using up to 20 ,ul aliquots of column fractions) were pooled and loaded imm.o.rli~tely on a 2.5 cm x 40 cm DEAE-cellulose column also equilibrated with the same buffer.
The column was washed with 500, 250 and 250 ml of the same buffer cont~ining 150, 200 and 250 mM KCl, respectively. Most of the ACC activity was eluted in the last wash. Protein present in this fraction was precipitated with ammonium sulfate (50%
saturation), dissolved in a small volume of 100 mM KCl, 20 mM Tris-HCl pH 7.5, 5% glycerol, 7 mM 2-melca~loethanol, and separated in several portions on two Superose columns connected in-line (Superose 6 and 12, Pharmacia). 1 ml fractlons were collected at 0.4 ml/min flow rate. Molecular mass standards were thyroglobulin, 669-kDa; ferritin, 440-kDa; aldolase, 158-kDa; albumin, 67-kDa (Pharmacia).
ACC-cont~ining fractions were concentrated using Centricon-100 concentrators (Amicon) and the proteins were separated by SDS-PAGE as described above.
By gel filtration, active ACC had an apparent molecular mass of ~ 500-kDa and the individual polypeptides have a molecular mass of 220-kDa. The 220-kDa polypeptide was the major component of this plcp~dlion as revealed by Coomassie staining of proteins separated by SDS-PAGE. This preparation also contained several smaller biotin-cont~ining peptides as revealed by western blotting with 35S-Streptavidin, most likely degradation products of the ca. 220-kDa peptide, which retained their ability to form the ~S00-kDa complex and therefore co-purified with intact ACC. The ACC preparations were active only when they contained intact 220-kDa biotinylated polypeptide. It is not possible to estimate the recovery of the active ACC, due to continuous degradation of the 220-kDa peptide during purification and to increased recovery of ACC activity in more purified preparations, probably due to separation of the enzyme from inhibitors in the cruder extracts.
The 220-kDa wheat peptide isolated as a dimer according to the above protocol was finally purified by SDS-PAGE and transferred to Irnmobilon-Pg) for sequencing. The N-terminus of the peptide appeared to be blocked. A mixture of amino acids was clçtçcte~l only after the protein was cleaved chemically with CNBr.

WO 96/32484 PCT/US~")5~!J5 The 220-kDa protein was therefore purified on an SDS gel, cleaved with CNBr, andthe resulting peptides were fractionated by gel electrophoresis basically as described (Jahnen-Dechent and Simpson, 1990), with the following modifications. A slice ofgel cont~ining about 20 ~Lg of the 220-kDa polypeptide was dried under vacuum toabout half of its original volume and then incubated overnight in 0.5 ml of 70%
forrnic acid cont~ining 25 mg of CNBr. The gel slice was dried again under vacuum to about half of its original volume and was equilibrated in 1 ml of 1 M Tris-HCl (pH
8.0). The CNBr peptides were separated by inserting the gel piece directly into a well of a tricine gel (as described in Jahnen-Dechent and Simpson, 1990; but without a spacer gel). Gels used to separate peptides for sequencing were pre-run for 30 min with 0.1 rnM thioglycolic acid in the cathode buffer. Peptides were transferred to Immobilon-P for sequencing by the Edman degradation method as described above.
Several bands of peptides, ranging in size from 4 to 16-kDa, with a well-resolved single band at about 14-kDa, were obtained. Attempts to sequence the smaller peptides failed, but the 14-kDa peptide yielded a clean results for residues 3-13.

5.5 EXAMPLE 5 -- Effects of the Herbicide Haloxyfop on Wheat ACC
The effect of haloxyfop, one of the aryloxyphenoxypropionate herbicides has been tested, on the activity of ACC from wheat germ and from wheat see-11ing leaves.
For the in vitro assay of ACC activity, 1-8 ,ul aliquots of ACC preparations were incubated for 45 min at 37~C with 20 ,ul of 100-200 mM KCl, 200 mM Tris-HCl pH
8.0, 10 mM MgCl2, 2 mM ATP, 2 mM DTT, 2 mM l4C-NaHCO3, and where in~ ted 1 mM Ac-CoA, in a final volume of 40 ,ul. The reaction was stopped by adding 4 ,ul of concenllaled HCl 30-40 ,ul aliquots of the reaction mixture werespotted on filter paper and dried, and acid-stable radioactivity was measured using scintillation cocktail. Haloxyfop was added as the Tris salt of the acid, generously supplied by J. Secor of Dow-Elanco.
For the in vivo assay of ACC activity, 2-week old seedlings of wheat (Triticum aestivum cv. Era) were cut about 1 cm below the first leaf and transferred to a 1.5 ml WO 96132484 PCT/USSf ~OJ~~!~S

micro tube containing l4C-sodium acetate and haloxyfop (Tris salt) for ~6 h. Theleaves were then cut into small pieces and treated with 0.5 rrll of 40% KOH for 1 h at 70~C, and then with 0.3 ml of H2SO4 and 20 ~11 of 30% TCA on ice. Fatty acids were extracted with three 0.5 ml aliquots of petroleum ether. The organic phase was washed with 1 ml of water. Incorporation of 14C-acetate into fatty acids is expressed as the percentage of the total radioactivity taken up by the see.-llin~.c, present in the organic phase.
As expected, the enzyme from wheat germ or from wheat chloroplasts was sensitive to the herbicide at very low levels. 50% inhibition occurs at about 5 and 2 ,uM haloxyfop, respectively. For comparison, the enzyme from pea chloroplasts isrelatively resistant (50% inhibition occurs at >50 :M haloxyfop). Finally, the in vivo incorporation of ~4C-acetate into fatty acids in freshly cut wheat seedling leaves is even more sensitive to the herbicide (50% inhibition occurs at <1 :M haloxyfop),which provides a convenient assay for both ACC and haloxyfop.
5.6 EXAMPLE 6 -- Cloning and Sequencing of Tr,i~icum aeshvum ACC cDNA
5.6.1 Materials and Methods 5.6.1.1 PCRTM Amplification Degenerate PCRTM primers were based on the ~ nm~nt of amino acid sequences of the following proteins (accession numbers in brackets): rat (J03808) and chicken (J03541) ACCs; E. coli (M80458, M79446, X14825, M32214), Anabaena 7120 (L14862, L14863) and Synechococcus 7942 BCs and BCCPs; rat (M22631) and human (X14608) propionyl-coenzyme A carboxylase (" subunit); yeast (J03889) pyruvate carboxylase; Propionibacterium shermanii (M11738) transcarboxylase (1.3S subunit) and Klebsiella pneumonia (J03885) oxaloacetate decarboxylase (a subunit). Each primer consisted of a 14-nucleotide specific sequence based on the amino acid sequence and a 6- or 8-nucleotide extension at the S'-end.
Poly(A)+ RNA from 8-day old plants (Triticum aestivum var. Era) was used for the synthesis of the first strand of cDNA with random hexamers as primers for 30 AMV reverse transcriptase (Hayrnerle et al., 1986). Reverse transcriptase was WO 96/32484 PCT/USgr'~ 5 inactivated by in~ub~fion at 90~C and low molecular weight m~teri~l was removed by filtration. All co~ onents of the PCRTM (Cetus/Perkin-Elmer), except the Taq DNApolymerase, were incubated for 3-5 min at 95~C. The PCRTM was initi~teA by the addition of polymerase. Conditions were optimized by amplification of the BC gene from Anabaena 7120. Amplification was for 45 cycles, each 1 min at 95~C, 1 min at 42-46~C and 2 min at 72~C. MgCl2 concentration was 1.5 mM. Both the reactions using Anabaena DNA and the single-stranded wheat cDNA as template yielded the expected 440-bp products. The wheat product was separated by electrophoresis on LMP-agarose and reamplified using the same primers and a piece of the LMP-agarose slice as a source of the template. That product, also 440-bp, was cloned into the Invitrogen vector pCR1000 using their A/T tail method, and sequenced.
In eukaryotic ACCs, the BCCP domain is located about 300 amino acids downstream from the end of the BC domain. Therefore, it was possible to amplify the cDNA encoding that interval between the two domains using primers, one from the C-termin~l end of the BC domain and the other from the conserved biotinylation site.
The expected 1. l-kb product of the first low yield PCRTM with primers m and IV was separated by electrophoresis on LMP-agarose and reamplified by another round of PCRTM, then cloned into the Invitrogen vector pCRII(~ and sequenced. The PCRTM
conditions were the same as those described above.
5.6.1.2 Isolation and Analysis of ACC cDNA
A wheat cDNA library (Triticum aestivum, var. Tam 107, Hard Red Winter, 13-day light grown see-1lingc) was purchased from Clontech. This 8gtl 1 library was prepared using both oligo(dT) and random primers. Colony ScreenPlus(~ (DuPont) membrane was used according to the m~n~lf~turers~ protocol (hybridization at 65~C
in 1 M NaCl and 10% dextran sulfate). The library was first screened with the l.l-kb PCRTM-amplified fragment of ACC-specific cDNA. Fragments of clones 39-1, 45-1 and 24-3 were used in subsequent rounds of screening. In each case, ~2.5 x 106 plaques were tested. More than fifty clones cont~ining ACC-specific cDNA

WO 96/32484 l~C'rll~S9~n!;C~95 fr~gmPnt~ were purified, and EcoRI fr~m~nt~ of the longest cDNA inserts were subcloned into pBluescriptSK~) for further analysis and sequencing. A subset of the - clones was sequenced on both strands by the dideoxy chain termin~tion method with Sequenase(~) (United States Biocht-mic~l~) or using the Perkin Elmer/Applied 5 Biosystems Taq DyeDeoxy Termin~tor cycle sequencing kit and an Applied Biosystems 373A DNA Sequencer.

5.6.1.3 RNA and DNA
Total RNA from 10-day old wheat plants was prepared as described in 10 (Haymerle et al., 1986). RNA was separated on a glyoxal c~cn~t--ring gel (Sambr~ok etal., 1989). GeneScreen Plus~) (DuPont) blots were hybridized in lM NaCI and 10% dextran sulfate at 65~C (wheat RNA and DNA) or 58-60~C (soybean and canola DNA). All cloning, DNA manipulation and gel electrophoresis were as described (Sambrook et al., 1989).
5.6.2 Results 5.6.2.1 PCRTM Cloning of the Wheat (Trilicum a~liv~l,.) ACC cDNA
A 440-bp cDNA fragment encoding a part of the biotin carboxylase domain of wheat ACC and a l.l-kb cDNA fragment encoding the interval between the biotin 20 carboxylase domain and the conserved biotinylation site were amplified. Thesefr~gm~nt~ were cloned and sequenced. In fact, three different 1. l-kb products, corresponding to closely related sequences that differ from each other by 1.5%, were identified. The three products most likely represent transcription products of three different genes, the minimllm number expected for hexaploid wheat. These two 25 overlapping DNA fr~gm~nt~ (total length of 1473 nucleotides) were used to screen a wheat cDNA library.

5.6.2.2 Isolation and Sequence Analysis of Wheat ACC cDNAs A set of overlapping cDNA clones covering the entire ACC coding sequence 30 was isolated and a subset of these clones has been sequenced. The nucleotide WO 96/32484 PCT/US9~ 9 sequence within overlapped regions of clones 39-1, 20-1 and 45-1 differ at l.l~o of the nucleotides within the total of 2.3 kb of the overlaps. The sequence within the overlap of clones 45-1 and 24-3 is identical. The sequence contains a 2257-aminoacid reading frame encoding a protein with a calculated m~lec~ r mass of 251 kDa.
5 In wheat germ the active ACC has an apparent molecular mass of ~500 kDa and the individual polypeptides have an apparent molecular mass (measured by SDS-PAGE) of about 220 kDa (Gornicki and T~çlkorn, 1993). The 220-kDa protein was also present in both total leaf protein and protein from intact chloroplasts. In fact, it was the major biotinylated polypeptide in the chloroplast protein. The cDNAs (total length 7.4 kb) include 158 bp of the 5'-untr~n~l~te-l and 427 bp of the 3'-untr~n~l~ted sequence.
The 7360-nucleotide DNA segment comprising the wheat ACC cDNA is given in SEQ ID NO:9. The 2257-amino acid tr~n.cl~tt~l wheat ACC sequence is given in SEQ ID NO: 10.
5.6.2.3 Northern Analysis of ACC mRNA
Northern blots with total RNA from 10-14 day old wheat leaves were probed using different cDNA fragments (the l.l-kb PCRTM-amplified fragment and parts ofclones 20-1, 24-3 and 01-4). In each case the only hybridizing mRNA species was 7.9 kb in size. This result shows clearly that all the cDNA clones correspond to mRNA of large, eukaryotic ACC and that there are no other closely related biotin-dependent carboxylases, con~i~ting of small subunits that are encoded by smaller mRNAs, inwheat.
Northern analysis of total RNA prepared from different sectors of 10-day old wheat see-lling.c indicates very high steady-state levels of ACC-specific mRNA in cells of leaf sectors I and II near the basal meristem. The ACC mRNA level is .~ignific~ntly higher in sectors I and II than in sectors m-VI. This cannot be explained r by dilution of specific mRNA by increased levels of total RNA in older cells. Based on published results (Dean and Leech, 1982), the increase in total RNA between sectors I and VI is expected to be only about two-fold.

WO 96132484 PCI~/US96~0S~g~i All cell division occurs in the basal meristem and cells in other sectors are indirrt;~ l stages of development. Differences between these young cells and the - mature cells at the tip of the leaf include cell size, number of chloroplasts and amount of total RNA and protein per cell (Dean and Leech, 1982). Expression of some genes 5 is correlated with the cell age (e.g., Lampa et al., 1985). It is not surprising that the level of ACC-specific mRNA is highest in dividing cells and in cells with increasing number of chloroplasts. The burst of ACC mRNA synthesis is necessary to supply enough ACC to meet the demand for malonyl-coenzyme A. The levels of ACC
mRNA decrease signific~ntly in older cells where the demand is much lower. The 10 same differences in the level of ACC specific mRNA between cells in Llirre~ tsectors were found in plants grown in the dark and in plants illllmin~t~d for one day at the end of the dark period.

5.6.2.4 Southern Analysis of Plant DNA
Hybridization, under stringent conditions, of wheat total DNA digests with wheat ACC cDNA probes revealed multiple bands. This was expected due to the hexaploid nature of wheat (Triticum aestivum). Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants. The specificity of this hybridization was demonstrated by sequencing several fragments of canola genornic 20 DNA isolated from a library using wheat cDNA probe 20-1 and by Northern blot of total canola RNA using one of the canola genomic clones as a probe. The Northernanalysis revealed a large ACC-specific message in canola RNA similar in size to that found in wheat.

25 5.6.2.5 ACC mRNA
The putative translation start codon was assigned to the first methionine of theopen reading frame. An in-frame stop codon is present 21 nucleotides up-stream from this AUG. The nucleotide sequence around this AUG fits quite well with the consensus for a monocot translation initiation site derived from the sequence of 93 30 genes, except for U at position +4 of the consensus which was found in only 3 of the CA 022l8l39 l997-lO-l4 W O 96/32484 PCTrUS9~'0~5 93 sequences. The ACC mRNA stop codon UGA is also the most frequently used stop codon found in monocot genes, and the surrounding sequence fits the consensus well.

5 5.6.2.6 Homologies with Other Carboxylases A cu,l,~alison of the wheat ACC amino acid sequence with other ACCs shows sequence conservation among these carboxylases. The sequence of the polypeptide predicted from the cDNA described above was compared with the amino acid sequences of other ACCs, and about 40% identity are with the ACC of rat, diatom and 10 yeast (about 40%). Less extensive similarities are evident with subunits of bacterial ACCs. The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is about 50% identical to the ACC of yeast, chicken, rat and diatom, but only about 27% identical to the biotin carboxylases of E. coli and Anabaena 7120. The biotin attachment site has the typical 15 sequence of eukaryotic ACCs. Several conserved amino acids found in the carboxyltransferase domains previously icl~ntified (Li and Cronan, 1992) are also present in the wheat sequence. Surprisingly, none of the four conserved motifs cont~ining serine residues, which correspond to phosphorylation sites in rat, chicken and human ACCs (Ha etal., 1994),is present at a similar position in the wheat 20 polypeptide.

5.6.2.7 Lack of Targeting Sequence in Wheat ACC cDNA
The wheat cDNA does not encode an obvious chloroplast targeting sequence unless this is an extremely short peptide. There are only 12 amino acids preceding the 25 first conserved amino acid found in all eukaryotic ACCs (a serine residue). The conserved core of the BC domain begins about 20 amino acids further down-stream.The appalt;nt lack of a transit peptide poses the question of whether and how the ACC
described in this paper is transported into chloroplasts. It was shown recently that the large ACC polypeptide purifies with chloroplasts of wheat and maize (Gornicki and Haselkorn, 1993; Egli etal., 1993). No obvious chloroplast transit peptide between -wo 96132484 PC~JUS91)~J~

the ER signal peptide and the mature protein was found in diatom ACC either (Roessler and Ohlrogge, 1993).
- The number of ACC genes in wheat have been ~sess~l by Southern analysis and by sequence analysis of the 5'- and 3'-untr;~n~l~tecl portions of ACC cDNA
S representing transcripts of dirre~ t genes. These cDNA fragments may be obtained by PCRTM amplification using the 5'- and 3'-RACE methodology. The genome structure of wheat (Triticum aestivum) suggests the presence of at least three copies of the ACC gene, i.e. one in each ancestral genome. Sequence analysis of the 5'-untranscribed parts of the gene may determine whether any f~mili~r promoter and 10 regulatory elements are present. The structure of introns within the control region and in the 5'-fragment of the coding sequence is also of interest.
The plant ACC genes are full of introns and their transcripts undergo ~ltern~tive splicing. In some plant genes, introns have been found both within the sequence encoding the transit peptide, and at the junction between the transit peptide 15 and the mature protein.
In plants, variant cytoplasmic and plastid isoenzymes could arise, for example, by zllt~rn~tive splicing or by transcription of two independent genes. This problem is especially intriguing as it was not possible to identify a transit peptide in the sequences of wheat ACC obtained so far. The two possibilities can be distinguished 20 by sequence analysis of the ap~lup~iate fragment of the ACC genes (clones from genomic library) and mRNAs (as cDNA). The sequence of these 5 '- and 3'-untranscribed and untr~ncl~tecl fr~qgmt-ntc of the gene are usually .cignific~ntly different for different alleles so they may also be used as specific probes to follow expression of individual genes.
.7 EXAMPLE 7 -- DNA Compositions Comprising a Wheat Cytosolic ACC
This example describes the cloning and DNA sequence of the entire gene encoding wheat (var. Hard Red Winter Tam 107) acetyl-CoA carboxylase (ACCase).
Comparison of the 12-kb genomic sequence (SEQ n~ NO:30) with the 7.4-kb cDNA
30 sequence reported in Exarnple 6 revealed 29 introns. Within the coding region (SEQ

WO 9f '3?~1~q PCTIUS9~/0"J('7 '7 ID NO:31), the exon sequence is 98% iclenti~l to the wheat cDNA sequence (SEQ IDNO:XX). A second ACCase gene was j~lentifled by sequencing fr~gm~,nts of genomicclones that include the first two exons and the first intron. Additional transcripts were detected by 5'- and 3'-RACE analysis. One set of transcripts had 5'-end sequence5 identical to the cDNA found previously and another set was identical to the gene reported here. The 3'-RACE clones fall into four distinguishable sequence sets, bringing the number of ACCase sequences to six. None of these cDNA or genomic clones encode a chloroplast targeting signal. Identification of six different sequences suggests that either the cytosolic ACCase genes are duplicated in the three 10 chromosome sets in hexaploid wheat or that each of the six alleles of the cytosolic ACCase gene has a readily distinguishable DNA sequence.

5.7.1 Materials and Methods 5.7.1.1 Isolation and Analysis of ACCase Genomic Clones A wheat genomic library (T. aestivum, var. Hard Red Winter Tam 107, 13-day light grown see~llin,~.c) was purchased from Clontech. This 8 EMB03 library was prepared from genomic DNA partially digested with Sau3A. Colony ScreenPlus (DuPont) membrane was used according to the manufacturers' protocol (hybridization at 65~C in lM NaCl and 10% dextran sulfate). The library was screened with a 20 440-bp PCRTM-amplified fragment of ACCase-specific cDNA and with cDNA clone 24-3 (Gornicki et al., 1994). In each case, ~1.2 x 106 plaques were tested. 24 clones containing ACCase-specific DNA fragments were purified and mapped. Selected restriction fr~m~,nt~ of these genomic clones were subcloned into pBluescriptSK(~) for further analysis and sequencing. The 3'-termin~l fragment of the gene (clone 145) 25 was arnplified by PCRTM using wheat genomic DNA as a template. Primers were based on the sequence of genomic clone 233, 5'-CGCTATAGGGAAACGTTAGAAGGATGGG-3' (SEQ ID NO:34) and 3'-RACE
clone 4, 5'-ATCGATCGGCCTCGGCTCCAATTTCATT-3' (SEQ ID NO:35).
All PCRTM components except Taq polymerase were incubated for 5 min. at 30 95~C. The reactions were initi~t~cl by the addition of the polymerase followed by 35 WO. 961324~4 PCT/US~ J~YS

cycles of incubation at 94~C for lmin, 55~C for 2 min and 72~C for 2 min. A 1.8-kb PCRTM product was gel-purified, reamplified using the same primers, cloned into the Invitrogen vector pCRIITM and sequenced.
s-5 5.7.1.2 Analysis of mRNA by rapid ~mrlifi~t;on of cDNA ends (RACE) Two sets of l5 and 20 cDNA fr~gm~nts corresponding to mRNA 5'- and 3'-ends, respectively, were prepared by T/A cloning of RACE products into the vector pCRlI. Total RNA from l5-day old wheat (Triticum aestivum var. Tam 107, Hard Red Winter) plants was prepared as described in Chirgwin etal. (1979). A Gibco 10 BRL 5'-RACE kit was used according to the m~n~lfA~tllrers' protocol. For the 5'-end amplification, the first strand of cDNA was prepared using a gene-specific primer:
5'-GTTCCCAAAGGTCTCCAAGG-3' (SEQ ID NO:36); followed by the addition of a homopolymeric dA-tail.
dT-Anchor primer: 5'-GCGGACTCGAGTCGACAAG(~'1"1"1"1 1"1"1"1''1"1"1"1"1"1"1"1"1'-3' l5 (SEQ ID NO:37); and a gene-specific primer, 5'-ACGCGTCGACTAGTA
GGTGCGGATGCTGCGCATG-3' (SEQ ID NO:38) were used in the first round of PCR
Universal primer, 5'-GCGGACTCGAGTCGACAAGC-3' (SEQ ID NO:39) and another gene-specific primer, 5'-ACGCGTCGACCATCCCA
20 TTGTTGGCAACC-3' (SEQ ID NO:40) were used for reamplification. The gene-specific primers were targeted to a stretch of 5 '-end coding sequence identical in clones 39 and 7 l that were available.
Clone 7 l was isolated from a 8gtl l cDNA library as described before using a fragment of cDNA 39 as probe (Example 4). The same dT-anchor primer and 25 universal primer together with a gene specific primer 5'-GACTCATTGAGATCAAGTTC-3' (SEQ ID NO:41) were used for the first strand cDNA synthesis and 3 '-end amplification. The latter primer was targeted to the 3'-end of the ACCase open reading frame.
All cloning, DNA manipulations and gel electrophoresis were as described 30 (Sambrook et al., 1989). DNA was sequenced on both strands by the dideoxy chain WO 96/32484 PCT/U~.3. ~a~s termin~tion method using 35S-[dATP] with Sequenase (United States Bio(~h~mic~
or using the Perkin Elmer/Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kit and an Applied Biosystems 373A DNA Sequencer.

5 5.7.2 Re~ults 5.7.2.1 Analysis of wheat cytosolic ACCase gene~
Two cDNA fr~gm~nt~, one encoding a part of the biotin carboxylase domain of wheat ACCase and the other a part of the carboxyltransferase, were used to isolate a set of overlapping DNA fr:~gmt-nt~ covering the entire ACCase gene. Some of these 10 genornic fragments were sequenced as in~1ic~ted in FIG. 1. Where they overlap, the nucleotide sequences of clones 31, 191 and 233 are identical. These obviously derive from the same gene. cDNA clone 71 (see below) represents the transcription product of this gene (430-nucleotide identical sequence). The sequence of clone 145 obtained by PCRTM to cover the rern~inin~ 3'-end part of the gene differs from clone 233 by 5 of 400 nucleotides of the overlap located within the long exon 28 (FIG. 1). It must therefore derive from a different copy of the ACCase gene. 3'-RACE clone 4 (3'-4, see below) differs at 6 of 490 nucleotides in the overlap.
The sequence was deposited in GenBank (as accession number U39321), and is a composite of these three very closely related sequences. Its 5'-end corresponds to 20 the 5'-end of clone 71 and the 3 '-end corresponds to the poly(A) attachment site of the 3'-RACE clone 4. It was ~sllm~cl that no additional introns are present at the very end of the gene.
C(lrnr~ri.~on of the genomic sequence with the cDNA sequence in Example 4 revealed 29 introns. Intron location is conserved among all three known plant 25 ACCase genes except for two introns not present in wheat but found in rape (Schulte etal., 1994), A. thaliana (Roesler etal., 1994) and soybean (Anderson etal., 1995) (FIG. 1). The nucleotide sequence at splice sites fits well with the consensus for monocot plants. The A+T content of the gene exons and introns is 52% and 63%, respectively, compared to 42% and 61 % found for other monocot plant genes (VVhite et al., 1992). The exon coding sequence is 98% iclenti~ ~l to that of the cDNA

WO 96132484 PCTJUS~ b~95 sequence reported earlier. This is the same degree of identity as found previously for dirr~ t transcripts of the cytosolic ACCase genes in hexaploid wheat (~xample 4).
The 11-amino acid sequence obtained previously for a CNBr-generated internal fragment of purified 220-kDa wheat germ ACCase (Gornicki and Haselkorn, 1993) 5 differs from the sequence encoded by these cDNA and genomic clones at one position, but it is identic~l with the corresponding cDNA sequence of the plastid ACCase from maize (Egli et al., 1995), excluding one amino acid which could not be zl~.cigne-l unambiguously in the sequence.
Two additional genomic clones, 153 and 231, were also partially sequenced 10 (I;IG. 1). The sequenced fr~gm~nt~ include parts of the first two exons and the first intron. Although cDNA corresponding exactly to genomic clone 153 is not avai,able, the bolln-l~fies of the first intron could easily be identified by sequence comparison with cDNA clone 71 (corresponding to genomic clone 31). Clone 153 encodes a polypeptide that differs by only one out of the first 110 amino acids of the ACCase 15 open reading frame. The sequence of the S'-leader was also well conserved but the 5'-part of the first intron of clone 153 is significantly different from that of genomic clone 31.
On the other hand, only the 3'-splice site of an intron could be identified by sequence comparison in this part of clone 231. The sequence immediately upstream20 of the 3'-splice site and that of the following exon is identical to that of clone 31. No sequence related to that found upstream of the first intron of clone 191 could be identified in clone 231 by hybridization (including a ~6 kb fragment upstream of the ACCase open reading frame) or by sequen~ing (~ 2 kb of the upstream fragment). It is possible that the first intron in this gene is much larger (additional upstream introns 25 can not be excluded) or that the upstream exon(s) and untranscribed part of the gene has a completely different sequence. A cloning artifact can not be ruled out. Indeed clone 31 contained such an unrelated sequence at its 5'-end (probably a ligationartifact).
Identific~tion of three additional genomic clones with sequence closely related 30 to the other ACCase genes but cont~ining no introns at several tested locations WO 96/32484 PCT/U:~5C/0~0~5 suggests the exi~t~nce of a pseudogene in wheat. A fragment of clone 232 that was sequenced is represented in the diagram shown in F~G. 1. It is 93% and 96% identical with clone 233 at the nucleotide and arnino acid level, respectively.
Shown in FIG. 5 is ~e 5' fl~nking sequence of the ACCase 1 gene (about 3 5 kb u~ edlll of the translation ir~itiation codon, of clone 71L (SEQ ID NO:32). The 5' fl~nkinp sequence of the ACCase 2 gene desi~n~ted 153 (SEQ ID NO:33! is shown in FIG. 6.

5.7.2.2 Analysis of mRNA ends In the original library screen (Gornicki etal., 1994) it was not possible to isolate any cDNA clones corresponding to the very ends of the ACCase mRNA. With the new sequence available it becarne possible to generate the mi~cing pieces byRACE. Two sets of 5'-end RACE clones, 71L and 39L, were identified. Their sequence is identical to the sequence of cDNA clones 71 (this work) and 39 (Gornicki et al., 1994), respectively. The two sequences extend 239 and 312 nucleotides upstream of the ACCase initiation codon and define an approximate position of the transcription start site. None of the genomic clones corresponds to 39L. The presence of the first intron in the corresponding gene could not therefore be confirmed. All three coding sequences are very sirnilar (they differ by only one three-amino acid deletion or one E to D substitution found within the first 110 amino acids) and none of them encodes additional amino acids at the N-terminl~, i.e., none of them encodes a potential chloroplast transit peptide.
The sequences of the 5'-leaders differ signific~ntly although they share some distinctive structural features. They are relatively long (at least 239-312 nucleotides as indicated by the lengths of 39L and 71L, respectively), G+C rich (67%) and contain upstream AUG codons. The open reading frames found in the leaders are 70-90 arnino acids long and they end within a few nucleotides of the ACCase initiationcodon. A similar arrangement was found in the sequence of genomic clone 153. Thethree upstream AUG codons are conserved and the presence of deletions, most of which are a multiple of three nucleotides, suggests at least some conservation of the WO 96132484 PCTMS~CJ~51 open reading frames at the amino acid level. This arrangement, found in the cytosolic ACCase genes, contrasts with the majority of 5'-untr~nsl~te~l leaders found in plants.
Although much longer leader sequences cont~ining upstream AUG codons have been reported in plants (e.g., Shorrosh et al., 1995), they are rare. In most cases, the ~Irst 5 AUG codon is the site of initiation of translation of the major gene product. The upstream AUGs are believed to affect the efficiency of mRNA translation and as such may be important in the regulation of expression of some genes (Roesler et al., 1994;
Anderson etal., 1995). They are often found in mRNAs encoding transcription factors, growth factors and receptors, all important regulatory proteins (Kozak, 1991).
10 They are also found in some plant mRNAs encoding heat shock proteins (Joshi and Nguyen, 1995). The ~800 nucleotide long leader intron found in both genes (clones 153 and 191) may also be important for the level and pattern of gene expression (e.g., Fu et al., 1995).
Four different sequences and two different polyadenylation sites ~300 and 15 ~500 nucleotides downstream of the translation stop codon, respectively, weredetected among the 3'-end RACE clones (FIG. 2). The sequence of the cDNA
reported previously (Gornicki et al., 1994) and the sequence of genornic clone 145 are also different in this region, bringing the total number of different sequences to six.
3-14 nucleotide differences were found in pairwise comparisons among these six 20 sequences within two stretches that include 282 nucleotides at the 5'-end of the 3 '-RACE clones and 204 nucleotides at the 3 '-end (FIG. 2).

5.7.2.3 Cytosolic ACC
A gene encoding eukaryotic-type cytosolic ACCase from wheat, very similar 25 in sequence to the cDNA in Example 4, was cloned and sequenced. Nucleotide identity between the cDNA and the gene within the coding sequence is 98%. The putative translation start codon was assigned in the original cDNA sequence to the first methionine of the open reading frame. An in-frame stop codon is present 21nucleotides upstream from this AUG and the conserved core of the biotin carboxylase 30 domain begins about 20 amino acids further down-stream. The gene, shown in FIG. 3 (SEQ ID NO:30), encodes a 2260-~mino acid protein with a calculated molecular mass of 252 kDa (FIG. 4 and SEQ ID NO:31). The wheat cDNA did not encode an obvious chloroplast targeting sequence. The same is true for all the cDNA and genomic sequences described in this paper. The cDNA for maize plastid ACCase, 5 reported recently (Egli et al., 1995), does encode a chloroplast transit peptide.
Comparison of the ACCase sequence encoded by the gene reported in this paper with the sequence of the wheat ACCase of Example 4 and with other representative biotin-dependent carboxylases is shown in Table 4. Wheat ACCase is most similar to other eukaryotic-type plant ACCases. Identity with other eukaryotic 10 carboxylases is also ~ignific~nt The core sequence of the most conserved ACCase domain, biotin WO 96/32484 PCT~JS9'J55095 oo C
.

C p~
P ~,~
~ C _ ~~~

C U U ~ -- --~0 ~ 'a x~ O ~0 WO 96/32484 PCT/US9G~C'~95 V~
~w ~ ~ ~ c X
C 5 ~ ~ o W
o ~ ~
E o ~-1 o ~ ~ ~ E
W C ~ ~ ~ ,~ ~ '~ 'w ~ ~ c E ~ ~ ~ o ~ ~ ~ C C c D O O O
3 C ~ 3 '~ _0 ~, ~ 3 30 ~30 1 ~3 WO 96132484 PCT)US9C,'u:5~iS

carboxylase, is well conserved in both eukaryotic and prokaryotic biotin-dependent - carboxylases. The other functional domains are less conserved (Example 4). Among plant eukaryotic-type ACCases, the wheat cytosolic ACCase is no more similar to the S maize plastid ACCase (both monocots) than it is to cytosolic ACCases from dicot plants. Clearly, cytosolic and plastid eukaryotic-type ACCases are quite distinct proteins. Another wheat ACCase for which partial sequence is available (Elborough et al., 1994) is most likely a plastid isozyme. It is more similar to the maize plastid ACCase than to the wheat cytosolic enzyme. The plant prokaryotic-type plastid 10 enzyme is more similar to bacterial, most notably cyanobacterial ACCases and to biotin-dependent carboxylases found in mitochondria, than to any of the plant cytosolic ACCases.
Sequence comparison of fr~gment~ of cDNA and genomic clones from the 3Nend of the gene brings the total number of different genes encoding cytosolic 15 ACCase in wheat to six, int1ic~ting that in hexaploid wheat there are at least two distinguishable coding sequences for the cytosolic ACCase in each of the three ancestral chromosome sets. Those two sequences might correspond to the alleles of the ACCase gene present in each ancestral chromosome set. On the other hand, it is possible that each pair of alleles has identical sequences, since the bread wheat studied 20 is extensively inbred. If that is the case, then one or more ancestral genes has been duplicated.

5.8 EXAMPLE 8 -- Developmental Analysis of ACC Genes Methods have been developed for analyzing the regulation of ACC gene 25 expression on several levels. With the cDNA clones in hand, the first may be obtained by preparing total RNA from various tissues at different developmental stages e.g., from different segments of young wheat plants, then probing Northern blots to deterrnine the steady-state level of ACC mRNA in each case. cDNA probesencoding conserved fr7lgment~ of ACC may be used to measure total ACC mRNA
30 level and gene specific probes to determine which gene is functioning in which tissue.

CA 022l8l39 l997- lO- l4 WO 96/324~4 PCT/USg~ 0~, In parallel, the steady-state level of ACC protein (by western analysis using ACC-specific antibodies and/or using labeled streptavidin to detect biotinylatedpeptides) and its enzymatic activity may be measured to identify the most important stages of synthesis and reveal m~.ch~ni~m.~ involved in its regulation. One such study 5 evaluates ACC expression in fast growing leaves (from see~llings at different age to mature plants), in the presence and in the absence of light.

5.9 EXAMPLE 9 -- Isolation of Herbicide-R~ci~t~nt Mutants Development of herbicide-resistant plants is an important aspect of the present 10 invention. The availability of the wheat cDNA sequence facilitates such a process.
By insertion of the complete ACC cDNA sequence into a suitable yeast vector in place of the yeast ACC coding region, it is possible to complement a FAS3 mutation in yeast using procedures well-known to those of skill in the art (see e.g., Haslacher etal., 1993). Analysis of the function of the wheat gene in yeast depends first on 15 tetrad analysis, since the FAS3 mutation is lethal in homozygotes.
Observation of four viable spores from FAS3 tetrads containing the wheat ACC gene may confirm that the wheat gene functions in yeast, and extracts of thecomplemented FAS3 mutant may be prepared and assayed for ACC activity. These assays may indicate the range of herbicide sensitivity, and in these studies, haloxyfop 20 acid and clethodim may be used as well as other related herbicide compounds.
Given that the enzyme expressed in yeast is herbicide-sensitive, the present invention may be used in the isolation of herbicide-resistant mnt~nt~. If spontaneous mutation to resistance is too infrequent, chemical mutagenesis with DES or EMS may be used to increase such frequency. Protocols involving chemical mutagenesis are25 well-known to those of skill in the art. Resistant mllt:~nt~, i.e., strains capable of growth in the presence of herbicide, may be assayed for enzyme activity in vitro to verify that the mutation to resistance is within the ACC coding region.
Starting with one or more such verified mnt~nt~, several routes may lead to the identification of the mllt~terl site that confers re.si~t~n~e. Using the available 30 restriction map for the wild-type cDNA, chimeric molecules may be constructed WC~ 96132484 ~CT~US9~ 50~5 _99_ cont~inin~ half, quarter and eighth fragments, etc. from each mutant, then checked by transformation and tetrad analysis whether a particular chimera confers resi.~t~nc.e or not.
~lt~ tively a series of fr~ nt~ of the mutant DNA may be prepared, end-5 labeled, and ~nn~le~l with the corresponding wild-type fragments in excess, so that all mutant fr~ment.~ are in heterozygous molecules. Brief Sl or mung bean nuclease digestion cuts the heterozygous molecules at the position of the mi~m~tch~cl base pair.
Electrophoresis and autoradiography is used to locate the position of the mi.cm~tch within a few tens of base pairs. Then oligo-primed sequencing of the mutant DNA is 10 used to identify the mutation. Finally, the mutation may be inserted into the wild- type sequence by oligo-directed mutagenesis to confirm that it is sufficient to confer the resistant phenotype.
Having identified one or more mutations in this manner, the corresponding parts of several dicot ACC genes may be sequenced (using the physical maps and 15 partial sequences as guides) to tletermine their structures in the corresponding region, in the expectation that they are now herbicide resi~t~nt 5.10 EXAMPLE 10 -- Isolation and Sequence Analysis of Canola ACC cDNA
Wheat ACC cDNA probes were used to detect DNA encoding canola ACC.
20 Southern analysis indicated that a wheat probe hybridizes quite strongly and cleanly with only a few restriction fragments that were later used to screen canola cDNA and genomic libraries (both libraries provided by Pioneer HiBred Co [Johnson City, IA]).
About a dozen positive clones were isolated from each library.
Sequence analysis was performed for several of these genomic clones.
25 Fragments containing both introns and exons were identified. One exon sequence encodes a polypeptide which is 75% identical to a fragment of wheat ACC. This isvery high conservation especially for this fragment of the ACC sequence which is not very conserved in other eukalyotes. The 398-nucleotide DNA segment comprising a portion of the canola ACC gene is given in SEQ ID NO:l9. The 132-amino acid WO 96132484 PCTtUS96/05095 tr~n~l~tecl sequence comprising a portion of the canola ACC polypeptide is given in SEQ ID NO:20.
One of the other genomic clones (6.5 kb in size) contains the 5' half of the canola gene, and ~ lition~l screening of the genomic library may produce other clones 5 which contain the promoter and other potential regulatory elements.

5.11 EXAMPLE 11-- Methods for Obtaining ACC Mutants In E. coli, only conditional mutations can be isolated in the acc genes. The reason is that although the bacteria can replace the fatty acids in triglycerides with 10 exogenously provided ones, they also have an çc~çnti~l wall component called lipid A, whose $-hydroxy myristic acid can not be supplied externally.
One aspect of the present invention is the isolation of Anacystis mllt~nt~ in which the BC gene is interrupted by an antibiotic rç~i~t~nce c~sette. Such techniques are well-known to those of skill in the art (Golden et al., 1987). Briefly, the method 15 involves replacing the cyanobacterial ACC with wheat ACC, so it is not absolutely necessary to be able to m~int~in the mllt~nt~ without ACC. The wheat ACC clone may be introduced first and then the endogenous gene can be inactivated without loss of viability.
By replacing the endogenous herbicide resistant ACC in cyanobacteria with 20 the wheat cDNA, resulting cells are sensitive to the herbicides haloxyfop andclethodim, whose target is known to be ACC. Subsequently, one may isolate mllt~nt~
resistant to those herbicides. These methods are known to those of skill in the art (Goldenetal., 1987).
The transformation system in Anacystis makes it possible to pinpoint a very 25 small DNA fragment that is capable of conferring herbicide reci~t~nce. DNA
sequencing of wild type and resistant mllt~nt~ then reveals the basis of resistance.
~ ltern~tively, gene replacement may be used to study wheat ACC activity and herbicide inhibition in yeast. Mutants may be selected which overcome the normalsensitivity to herbicides such as haloxyfop. This will yield a variant(s) of wheat ACC
30 that are tolerant/resistant to the herbicides. The mllt~te-l gene (cDNA) present on the WC~ 96/32484 PCT~JS9 ' '~5C!35 plasmid can be recovered and analyzed further to define the sites that confer herbicide resi~t~n-~e As for the herbicide selection, there is a possibility that the herbicide may - be inactivated before it can inhibit ACCase activity or that it may not be transported into yeast. There are general schemes for treatment of yeast with permeabilizing5 antibiotics at sublethal concentrations, which are known to those of skill in the art.
Such treatments allow otherwise impermeable drugs to be used effectively. For these studies haloxyfop acid and clethodim may be used.
Characterization of the site(s) conferring herbicide resistance generally involves assaying extracts of the complem~nte-1 ACCl mutant for ACCase activity.10 Both spontaneous mutation and ch~Qmic~l mutagenesis with DES or EMS, may be used to obtain resistant mllt~nt~7 i.e., strains capable of growth in the presence of herbicide. These may be assayed for enzyme activity in vitro to verify that the mutation to resistance is within the ACCase coding region. Starting with one or more such verified mut~nt~, the mllt~t~l site that confers resi~t~nce may be analyzed. Using 15 the available restriction map for the wild-type cDNA, chimeric molecules may be constructed which cont:~ining half, quarter and eighth fr~gm~ont~, etc., from each mutant, and then checked by transformation and tetrad analysis to determine whether a particular chimera confers resistance or not.
An alternative method involves preparing a series of fragments of the mutant 20 DNA, end-labeling, and ~nnto~ling with the corresponding wild-type fragments in excess, so that all mutant fragments are in heterozygous molecules. Brief S 1 or mung bean nuclease digestion cuts the heterozygous molecules at the position of the micm~t-~h within a few tens of base pairs. Then oligo-primed sequencing of the mutant DNA is used to identify the mutation. Finally, the mutation can be inserted 2~ into the wild-type sequence by oligo-directed mutagenesis to confirm that it is sufficient to confer the resistant phenotype. Having identified one or more mutations in this manner, the corresponding parts of several dicot ACCase genes to determine their structures in the corresponding region, in the expectation that they would be "resistant".

Another method for the selection of wheat ACCase ~ tolerant or resistant to dirre,~nt herbicides involves the phage display technique. Briefly, in the phage display technique, foreign peptides can be expressed as fusions to a capsid protein of fil~ tQus phage. Generally short (6 to 18 amino acids), variable amino 5 acid sequences are displayed on the surface of a bacteriophage virion (a population of phage clones makes an epitope library). However, filamentous bacteriophages havealso been used to construct libraries of larger proteins such as the human growth hormone, alkaline phosphatase (Scott, 1992) or a 50-kDa antibody Fab domain (Kang etal., 1991). In those cases, the foreign inserts were spliced into the major coat 10 protein pvm of the M13 phagemid. A complementary helper phage supplying wild-type pvm has to be cotransferred together with the phagemid. Such "fusion phages" retained full infectivity and the fused proteins were recognized by monoclonal antibodies. These results demonstrate that foreign domains displayed by phage can retain at least partial native folding and activity.
Phage libraries displaying wild-type fr~gments of the wheat ACCase of 250 to 300 arnino acids in size may be constructed without "panning" for phage pllrific~tion.
The mechanism of purifying phages by panning involves reaction with biotinylatedmonoclonal antibodies, then the complexes are diluted, immobilized on streptavidin-coated plates, washed extensively and eluted. Generally, a few rounds of 20 panning are recomm~cle~l Tn~te~-1, fragments bearing the ATP-binding site may be obtained by using Blue Sepharose CL-6B affinity chromatography, which was shown to bind plant ACCs (Betty et al., 1992; Egin-Buhler et al., 1980). Herbicides bound to Sepharose serve for ca~lu,hlg those phages which display amino acid fr~gm~nt~ involved in 25 herbicide binding. Such herbicide affinity resins may also be employed. Afteridentifying peptide fragments that bind herbicides, ATP or acetyl-CoA, the phages bearing those peptides may be subjected to random mutagenesis, again using phagedisplay and binding to the a~lopliate support to select the interesting variants.
Sequence analysis then is used to identify the critical residues of the protein required 30 for binding.

WO 96132484 PCTJU~5CJ0~ !95 5.12 EXAMPLE 12 -- Preparation of ACC-specific antibodies Another aspect of the present invention is the pl~a dtion of antibodies reactive against plant ACC for use in immuno~l~cipi~alion, affinity chromatography, and immlln~electron microscopy. The antisera may be prepared in rabbits, using methods that are well-known to those of skill in the art (see e.g., Schneider and HaseL~orn, 1988).
Briefly, the procedure encomp~cses the following aspects. Gel-purified protein is electroeluted, dialyzed, mixed with complete Freund's adjuvant and injected in the footpad at several locations. Subsequent boosters are given with incomplete adjuvant and finally with protein alone. Antibodies are partially purifiec, by precipitating lipoproteins from the serum with 0.25% sodium dextran sulfate and 80 mM CaCl2. Immunoglobulins are precipitated with 50% saturating ammonium sulfate, suspended in phosphate-buffered saline at 50 mg/ml and stored frozen. The antisera prepared as described may be used in Western blots of protein extracts from wheat, pea, soybean, canola and sunflower chloroplasts as well as total protein.
5.13 EXAMPLE 13 -- Protein Fusions, Transgenic Plants and Transport Mutants Analysis of promoter and control elements with respect to their structure as well as tissue specific expression, timing etc., is performed using promoter fusions (e.g. with the GUS gene) and applo~liate in situ assays. Constructs may be made which are useful in the preparation of transgenic plants.
For identifying transport of ACC, model substrates cont~3ining different length N-termin~l fr~gm~.nts of ACC may be prepared by their expression (and labeling) in E. coli or by ill vitro transcription with T7 RNA polymerase and translation (and labeling) in a reticulocyte lysate. Some of the model substrates will include the functional biotinylation site (located ~800 amino acids from the N-terminus of the ~- mature protein; the miniml-m biotinylation substrate will be defined in parallel) or native ACC epitope(s) for which antibodies will be generated as described above.

CA 022l8l39 l997- lO- l4 WO 96/32484 PCT/U.,~C~'~,S'~

Adding an antibody tag at the C-te.rrninlls will also be very helpful. These substrates will be purified by affinity chromatography (with antibodies or ~LI~t~vidin) and used for in vitro assays.
For modification of ACC protein transport, model substrates consisting of a 5 transit peptide (or any other chloroplast targeting signals) to f~rilit~t~ import into chloroplasts, fused to different ACC domains that are potential targets for modification, may be used. Modified polypeptides from cytoplasmic andlor chloroplast fractions will be analyzed for modification. For example, protein phosphorylation (with 32p) can be followed by immunoprecipitation or by PAGE.
10 Antibodies to individual domains of ACC may then be employed. The same experim~nt~l set-up may be employed to study the possible regulation of plant ACC
by phosphorylation (e.g., Witters and Kemp, 1992). Biotinylation may be followed by Western analysis using 35S-streptavidin for detection or by PAGE when radioactive biotin is used as a substrate.
5.14 EXA~PLE 14 -- Expression Systems for Preparation of ACC
Polypeptides The entire plant ACC cDNA and its fragments, and BC, BCCP and the CT
gene clones from cyanobacteria may be used to prepare large amounts of the 20 corresponding proteins in E. coli. This is most readily accomplished using the T7 expression system. As designed by Studier, this expression system consists of an E.
coli strain carrying the gene for T7 lysozyme and for T7 RNA polymerase, the latter controlled by a lac inducible promoter. The expression vector with which this strain can be transformed contains a promoter recognized by T7 RNA polymerase, followed25 by a multiple cloning site into which the desired gene can be inserted (Ashton et al., 1994).
Prior to induction, the strain grows well, because the few molecules of RNA
polymerase made by basal transcription from the lac promoter are complexed with T7 lysozyme. When the inducer IPTG is added, the polymerase is made in excess and the 30 plasmid-borne gene of interest is transcribed abundantly from the late T7 promoter.

W09613~ 4 PCTlUS9'~50~5 This system easily makes 20% of the cell protein the product of the desired gene. A
benefit of this system is that the desired protein is often sequestered in inclusion bodies that are impossible to dissolve after the cells are lysed. This is an advantage in the present invention, because biological activity of these polypeptides is not required S for purposes of raising antisera. Moreover, other expression systems are also available (Ausubel et al., 1989).

F REN ES

6. RE E C
The references listed below and all references cited herein are incorporated herein by reference to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or composicions employed herein.

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WO 96J32484 PCr~U~J6,', ~;~9gS

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(B) LOCATION:one-of(11, 14) (D) OTHER INFORMATION:/mod_base= OTHER
~~ /note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:20 (D) OTHER INFORMATION:/mod-base= OTHER
/note= "R = A or G"
(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION: 17 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "H = A, C, or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (~) TOPOLOGY: l~near (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(3, 9) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"
(ix) FEATURE:

CA 022l8l39 l997-l0-l4 W096132484 PCT~S96/05095 (A) NAME/KEY: modified_base (B) LOCATION:6 (D) OTHER INFORMATION: /mod_base= OTHER
/note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:13 (D) OTHER INFORMATION: /mod_base= OTHER
/note= "K = G or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:12 (D) OTHER INFORMATION: /mod_base= OTHER
/note= " R = A or G"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: li near (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

(2) INFORMATION FOR SEQ ID NO: 16:

CA 022l8l39 l997- lO- l4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(3, 9) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:6 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:12 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:13 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "K = G or T"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

(2) INFORMATION FOR SEQ ID NO: 17:

CA 022l8l39 l997- lO- l4 WO 96/32484 PCT/US~i/030~5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(9, 11, 14) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:18 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:21 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "H = A, C, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:22 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "M = A or C"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

(2) INFORMATION FOR SEQ ID NO: 18:

CA 022l8l39 l997- lO- l4 WO 96/32484 PCT/U~3~ 095 (i) SEQUENCE CHARACTERISTICS:
(A) LBNGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:2 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"

(ix) FEATURE:
(A) NAME/KBY: modified_base (B) LOCATION:one-of(3, 13) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:9 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:14 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "W = A or T"
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

(2) INFORMATION FOR SEQ ID NO: 19:

o o o o o o a ~D ~ CO~ O ~ a~

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WO9~ 1 PCT~S96/0~095 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2l:

Pro Leu Asp Phe Asn Glu Ile Arg Gln Leu l 5 l0 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Leu Asp Phe Asn Glu Ile Arg l 5 (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(9, ll, 14) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base CA 022l8l39 l997- lO- l4 WO 96/32484 PCT/U~3G/O!iO!i~

(B) LOCATION: 18 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"

(ix) FEATURE: f (A) NAME/KEY: modi~ied_base (B) LOCATION:21 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "H = A, C, or T"
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base (B) LOCATION:22 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "M = A or C"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Asn Met Lys Met Xaa (2) INFORMATION FOR SEQ ID NO: 25:

W096/32484 PCTIU'~56/ Sl/!~:;

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:2 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R - A or G"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-o~(3, 13) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modi~ied_base (B) LOCATION:9 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(ix) FEATURE:
(A) NAME/KEY: modi~ied_base (B) LOCATION:14 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "W = A or T"
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 25:

(2) INFORMATION FOR SEQ ID NO: 26:

W 096/32484 PCTrUS96/05095 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(10, 16) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:13 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(14, 19) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 022l8l39 l997-l0-l4 w~s6/3~s4 PCT~S9~'0~5 (ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:2 (D) OTHER INFORMATION:/mod_base= OTHER
s 5 /note= "R = A or G"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:one-of(3, 13) (D) OTHER INFORMATION:/mod_base= OTHER
/note= "N = A, C, G, or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:9 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "Y = C or T"

(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:14 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "W = A or T"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 022l8l39 l997-lO-l4 W096/32484 PCT~Sg~105 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11994 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: modi~ied_base (B) LOCATION: 10357 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"

(ix) FEATURE:
(A) NAME/KEY: modi~ied_base (B) LOCATION:one-o~(10198, 10472, 10501, 11698) (D) OTHER INFORMATION:/mod_base= OTHER

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CA 022l8l39 l997-l0-l4 W096l32484 PCT~S96JOS09S

(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

GCGGACTCGA GTCGACAAGC 'l"l"ll"l"l"l"l"l"l~l"l"l"l~l"l"l~ 37 _ (2) INFORMATION FOR SEQ ID NO: 37:

CA 022l8l39 l997-l0-l4 W096/3~84 PCT~S96/05095 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

Wos6/32484 PCTJU~J~ S

(2) INFORMATION FOR SEQ ID NO: 40:

-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experim.ont~tion in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be a~alent to those of skill in the art that variations may be applied S to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.
More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications 10 apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims (44)

Claims

1. An isolated canola acetyl-CoA carboxylase enzyme.

2. The enzyme according to claim 1, comprising the amino acid sequence of SEQ ID NO:20.

3. A purified DNA segment encoding canola acetyl-CoA
carboxylase.

4. The DNA segment of claim 3, further defined as encoding the amino acid sequence of SEQ ID NO:20.

5. The DNA segment of claim 4, further defined as comprising SEQ ID NO:19.

6. The DNA segment of claim 3, defined further as a recombinant vector.

7. The DNA segment of claim 3, wherein said DNA is operatively linked to a promoter, said promoter expressing the DNA segment.

8. A recombinant host cell comprising the DNA segment of claim 3.

9. The recombinant host cell of claim 8, defined further as being a prokaryotic cell.

10. The recombinant host cell of claim 9, further defined as a bacterial or cyanobacterial host cell.

11. The recombinant host cell of claim 8, defined further as being a eukaryotic cell.

12. The recombinant host cell of claim 11, further defined as a yeast cell or a plant host cell.

13. The recombinant host cell of claim 12, wherein said cell is a monocotyledonous plant cell.

14. The recombinant host cell of claim 10, wherein the bacterial host cell is E. coli.

15. The recombinant host cell of claim 10, wherein the cyanobacterial host cell is Synechococcus or Anabaena.

16. The recombinant host cell of claim 8, wherein the DNA
segment is introduced into the cell by means of a recombinant vector.

17. The recombinant host cell of claim 8, wherein the host cell expresses the DNA segment to produce the encoded acetyl-CoA carboxylase protein or peptide.

18. A recombinant host cell comprising a DNA segment encoding canola or cyanobacterial acetyl-CoA carboxylase, wherein the expressed acetyl-CoA carboxylase protein or peptide includes a contiguous amino acid sequence from SEQ ID
NO:4; SEQ ID NO:12; or SEQ ID NO:20.

19. An isolated nucleic acid segment characterized as:

(a) a nucleic acid segment comprising a sequence region that consists of at least 14 contiguous that have the same sequence as, or are complementary to, 14 contiguous nucleotides of SEQ ID NO:3; SEQ ID NO:11; or SEQ ID NO:19: or (b) a nucleic acid segment of from 14 to about 10,000 nucleotides in length that hybridizes to the nucleic acid segment SEQ ID NO:3; SEQ ID NO:11; or SEQ ID
NO:19; or the complements thereof, under standard hybridization conditions.

20. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 20 nucleotides; or wherein the segment is about 20 nucleotides in length.

21. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 30 nucleotides; or wherein the segment is about 30 nucleotides in length.

22. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 50 nucleotides; or wherein the segment is about 50 nucleotides in length.

23. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 100 nucleotides; or wherein the segment is about 100 nucleotides in length.

24. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 200 nucleotides; or wherein the segment is about 200 nucleotides in length.

25. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 500 nucleotides; or wherein the segment is about 500 nucleotides in length.

26. The nucleic acid segment of claim 19, wherein the segment comprises a sequence region of at least about 1000 nucleotides; or wherein the segment is about 1000 nucleotides in length.

27. The nucleic acid segment of claim 19, wherein the segment is up to 10,000 basepairs in length.

28. The nucleic acid segment of claim 19, wherein the segment is up to 5,000 basepairs in length.

29. The nucleic acid segment of claim 19, wherein the segment is up to 3,000 basepairs in length.

30. A nucleic acid detection kit comprising, in suitable container means, an isolated canola or cyanobacterial acetyl-CoA carboxylase-encoding nucleic acid segment according to claim 3 or 19 and a detection reagent.

31. The nucleic acid detection kit of claim 30, wherein the detection reagent is a detectable label that is linked to said acetyl-CoA carboxylase nucleic acid segment.

32. An enzyme composition, free from total cells, comprising a purified acetyl-CoA carboxylase that includes a contiguous amino acid sequence from SEQ ID NO:4; SEQ ID
NO:12; or SEQ ID NO:20.

33. The composition of claim 32, comprising a peptide that includes a 15 to about 50 amino acid long sequence from SEQ ID NO:4; SEQ ID NO:12; or SEQ ID NO:20.

34. The composition of claim 32, comprising a peptide that includes a 15 to about 150 amino acid long sequence from SEQ ID NO:4; SEQ ID NO:12; or SEQ ID NO:20.

35. The composition of claim 32, wherein the protein or peptide is a recombinant protein or peptide.

36. A process of modifying the oil content of a plant cell, comprising expressing in a plant cell a DNA segment according to claim 3 or 19 that encodes a canola or cyanobacterial acetyl-CoA carboxylase or the complement of said DNA segment.

37. The process according to claim 36, comprising incorporating into said plant cell a DNA segment that encodes a canola or cyanobacterial acetyl-CoA carboxylase polypeptide, wherein said cell expresses the acetyl-CoA

carboxylase enzyme.

38. The process according to claim 37, wherein said plant cell is a monocotyledonous plant cell.

39. A process of increasing the herbicide resistance of a monocotyledonous plant, comprising incorporating into said plant a transgene comprising a DNA segment according to claim 3 or 19 encoding a canola or cyanobacterial acetyl-CoA carboxylase polypeptide resistant to herbicide inactivation, the plant expressing the polypeptide.

40. The process according to claim 39, wherein said canola acetyl-CoA carboxylase polypeptide comprises the amino acid sequence of SEQ ID NO:20.

41. The process according to claim 39, wherein said canola acetyl-CoA carboxylase polypeptide is encoded by the DNA
sequence comprising SEQ ID NO:19.

42. The process according to claim 39, wherein said cyanobacterial acetyl-CoA carboxylase polypeptide comprises the amino acid sequence of SEQ ID NO:12.

43. The process according to claim 39, wherein said cyanobacterial acetyl-CoA carboxylase polypeptide is encoded by the DNA sequence comprising SEQ ID NO:11.

44. A transgenic plant having incorporated into its genome a transgene comprising a DNA segment according to claim 3 or 19 that encodes a canola or cyanobacterial acetyl-CoA
carboxylase.