EP0820514A2 - COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION - Google Patents

COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION

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Publication number
EP0820514A2
EP0820514A2 EP96912726A EP96912726A EP0820514A2 EP 0820514 A2 EP0820514 A2 EP 0820514A2 EP 96912726 A EP96912726 A EP 96912726A EP 96912726 A EP96912726 A EP 96912726A EP 0820514 A2 EP0820514 A2 EP 0820514A2
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EP
European Patent Office
Prior art keywords
seq
acetyl
plant
coa carboxylase
segment
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EP96912726A
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German (de)
English (en)
Inventor
Robert Haselkorn
Piotr Gornicki
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Arch Development Corp
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Arch Development Corp
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Priority claimed from US08/422,560 external-priority patent/US5910626A/en
Priority claimed from US08/611,107 external-priority patent/US5801233A/en
Application filed by Arch Development Corp filed Critical Arch Development Corp
Priority claimed from PCT/US1996/005095 external-priority patent/WO1996032484A2/fr
Publication of EP0820514A2 publication Critical patent/EP0820514A2/fr
Ceased legal-status Critical Current

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Definitions

  • the present invention relates to the field of molecular biology. More 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.
  • ACC cyanobacterial and plant acetyl-CoA carboxylases
  • Acetyl-CoA carboxylase [ACCase; acetyl-CoA arbon dioxide ligase (ADP- forming), EC 6.4.1.2] catalyzes the first committed step in de novo fatty acid biosynthesis, the addition of CO 2 to acetyl-CoA to yield malonyl-CoA. It belongs to a group of carboxylases that use biotin as cofactor and bicarbonate as a source of the carboxyl group. ACC catalyzes the addition of CO 2 to acetyl-CoA to yield malonyl- CoA in two steps as shown below.
  • biotin becomes carboxylated at the expense of ATP.
  • the carboxyl group is then transferred to Ac-CoA (Knowles, 1989).
  • This irreversible reaction is the committed step in fatty acid synthesis and is a target for multiple regulatory mechanisms.
  • 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 eukaryotic ACC in which all the domains are located on one large polypeptide (e.g., rat, chicken, yeast, diatom and wheat).
  • BC biotin carboxylase
  • BCCP biotin carboxyl carrier protein
  • CT carboxyltransferase
  • E. coli ACC consists of a dimer of 49-kDa BC monomers, a dimer of 17-kDa BCCP monomers and a CT tetramer containing 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 et al., 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.
  • accA CT ⁇ subunit
  • accB BCCP
  • accC BC
  • accD CT ⁇ subunit
  • accC and accB form one operon, while accA and accD are not linked to each other or to accCB (Li and Cronan, 1992).
  • accC and accB are unlinked as well (Gornicki et ⁇ .., 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 consisting of 230-kDa subunits.
  • Their primary structure has been deduced from cDNA sequences (Al-feel et al., 1992; Lopez-Casillas et al., 1988; Takai et l., 1988; Roessler and Ohlrogge, 1993; Ha et ai, 1994).
  • homologs of the four bacterial genes are fused in the following order: accC, accB, accD and accA.
  • ACC activity varies with the rate of fatty acid synthesis or energy requirements in different nutritional, hormonal and developmental states.
  • ACC mRNA is transcribed using different promoters in different tissues and can be regulated by alternative splicing.
  • the rat enzyme 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 with phospholipid metabolism (Chirala, 1992; Haslacher et al., 1993).
  • At least one form of plant ACC is located in plastids, the primary site of fatty acid synthesis.
  • the gene encoding it 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 some chloroplast 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 (Sasaki et al, 1993).
  • ACCase 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 multi-subunit enzyme (e.g., in pea, soybean, tobacco and Arabidopsis).
  • the other plant ACCase, located in the cytoplasm, is of the eukaryotic type.
  • genes for both cytosolic and plastid eukaryotic-type ACCase are nuclear. No ACCase coding sequence can be found in the complete sequence of rice chloroplast DNA.
  • plastid ACCases are synthesized in the 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 been deduced from genomic or cDNA sequences ( ⁇ gli et al, 1995; Li and Cronan, 1992; Gomicki et al, 1994; Schulte et al, 1994; Shorrosh et al, 1994; Shorrosh et al, 1995; Roesler et al, 1994; Anderson et al, 1995).
  • ACC isoforms one present in plastids and another in the cytoplasm.
  • 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.
  • two isoforms were found in maize, both consisting of >200-kDa subunits but differing in size, herbicide sensitivity and immunological properties.
  • the major form was found to be located in mesophyll chloroplasts. It is also the major ACC in the endosperm and in embryos ( ⁇ gli et al, 1993).
  • Cyanobacteria are prokaryotes that carry out green plant photosynthesis, evolving O 2 in the light. They are believed to be the evolutionary ancestors of chloroplasts. Virtually nothing is known about fatty acid biosynthesis in cyanobacteria.
  • Synechococcus is a unicellular obligate phototroph with an efficient DNA transformation system.
  • Replicating vectors based on endogenous plasmids are available, and selectable markers include resistance to kanamycin, chloramphenicol, streptomycin and the PSII inhibitors diuron and atrazine.
  • Inactivation and/or deletion of Synechococcus genes by transformation with suitable cloned material interrupted by resistance cassettes is well known in the art. Genes may also be replaced by specifically mutated versions using selection for closely linked resistance cassettes.
  • Anabaena differentiates specialized cells for nitrogen fixation when the culture is deprived of a source of combined nitrogen.
  • the differentiated cells have a unique glycolipid envelope containing C26 and C28 fatty acids (Murata and Nishida, 1987), whose synthesis must start with the reaction catalyzed by ACC. Therefore ACC must be developmentally regulated in Anabaena. Powerful systems of genetic analysis exist for Anabaena as well (Golden et al, 1987).
  • cyanobacteria 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.
  • the cloned gene for the enzyme phytoene desaturase, which 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, 1992).
  • the aryloxyphenoxypropionate class comprises derivatives of aryloxyphenoxy-propionic acid such as diclofop, fenoxaprop, fluazifop, haloxyfop, propaquizafop and quizalofop.
  • Several derivatives of cyclohexane-l,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.
  • ACC is the target enzyme for both of these classes of herbicide at least in monocots.
  • Dicotyledonous plants 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.
  • herbicides of the aryloxyphenoxypropionate and cyclohexane- 1 ,3-dione groups are not useful in the agriculture of these important grain crops owing to the inactivation of monocot ACC by such chemicals.
  • 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 cyanobacterial species.
  • the invention also provides novel DNA segments encoding eukaryotic and prokaryotic ACCs, and methods and processes for their use in regulating the oil content of plant tissues, for conferring and modulating resistance 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 determining herbicide resistance and kits for identifying the presence of plant ACC polypeptides and DNA segments.
  • 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.
  • ACC acetyl-CoA carboxylase
  • polynucleotide means a sequence of nucleotides connected by phosphodiester linkages.
  • a polynucleotide of the present invention can comprise from about 2 to about several hundred thousand base pairs.
  • 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 DNA
  • RNA ribonucleic acid
  • Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U).
  • the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding polypeptides which have the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium.
  • the cyanobacterium is Anabaena or Synechococcus.
  • a preferred Anabaena is Anabaena 7120.
  • a preferred Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).
  • a polypeptide is a biotin carboxylase enzyme of a cyanobacterium. This enzyme is a subunit of cyanobacterial acetyl-CoA carboxylase and participates in the carboxylation of acetyl-CoA.
  • a BC polypeptide is encoded by a polynucleotide comprising an accC gene which has the nucleic acid sequence of SEQ ID NO:5 (Anabaena accQ or SEQ ID NO:7 (Synechococcus acc , or functional equivalents thereof.
  • the BC polypeptide preferably comprises the amino acid sequence of SEQ ID NO:6 (Anabaena BC) or SEQ ID NO:8 (Synechococcus BC), or functional equivalents thereof.
  • the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding a biotin carboxyl carrier protein of a cyanobacterium.
  • the cyanobacterium is Anabaena or Synechococcus.
  • a preferred Anabaena is Anabaena 7120.
  • a preferred Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).
  • a polypeptide is a biotin carboxyl carrier protein of a cyanobacterium.
  • This polypeptide is a subunit of cyanobacterial acetyl-CoA carboxylase and participates in the carboxylation of acetyl-CoA.
  • 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 equivalents thereof.
  • the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding a carboxyltransferase protein of a cyanobacterium.
  • the cyanobacterium is Anabaena or
  • Synechococcus A preferred Anabaena is Anabaena 7120.
  • a preferred Synechococcus is Anacystis nidulans R2 (Synechococcus sp. strain PCC 7942).
  • a polypeptide is a carboxyltransferase ⁇ or ⁇ subunit protein of a cyanobacterium. These polypeptides are subunits of cyanobacterial acetyl-CoA carboxylase and participate in the carboxylation of acetyl-CoA.
  • a CT ⁇ polypeptide is encoded by a polynucleotide comprising an accA gene which has the nucleic acid sequence of SEQ ID NO: 11 (Synechococcus accA), or a functional equivalent thereof.
  • the CT ⁇ polypeptide preferably comprises the amino acid sequence of SEQ ID NO: 12 (Synechococcus CT ⁇ ), or a functional equivalent thereof.
  • the present invention contemplates isolated and purified polynucleotides comprising DNA segments encoding an acetyl-CoA carboxylase protein of a plant.
  • the plant is a monocotyledonous or a dicotyledonous plant.
  • An exemplary and preferred monocotyledonous plant is wheat, rice, maize, barley, rye, oats or timothy grass.
  • An exemplary and preferred dicotyledonous plant is soybean, rape, sunflower, tobacco, Arabidopsis, petunia, pea, canola, bean, tomato, potato, lettuce, spinach, alfalfa, cotton or carrot.
  • a preferred monocotyledonous plant is wheat, and a preferred dicotyledonous plant is canola.
  • a polypeptide is an acetyl-CoA carboxylase (ACC) protein of a plant. This polypeptide participates in the carboxylation of acetyl-CoA.
  • an ACC polypeptide is encoded by a polynucleotide comprising an ACC cDNA which has the nucleic acid sequence of SEQ ID NO:9 (wheat ACC) or SEQ ID NO: 19 (canola ACC), or functional equivalents thereof.
  • the ACC polypeptide preferably comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:31 (wheat ACC) or SEQ ID NO:20 (canola ACC), or functional equivalents thereof.
  • the present invention provides an isolated and purified DNA molecule comprising a promoter operatively linked to a coding region that 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 transcription-terminating region, whereby said promoter drives the transcription of said coding region.
  • 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.
  • a biotin carboxyl carrier protein 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.
  • the present invention also provides (1) an isolated and purified biotin carboxyl carrier protein of a cyanobacterium such as Anabaena or Synechococcus, which protein includes the amino acid residue sequence of SEQ ID NO: 2 or SEQ ID NO: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 ⁇ subunit protein of a cyanobacterium such as Synechococcus, which protein includes 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
  • 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.
  • the present invention also provides methods for identifying ACC and ACC-related polypeptides, which methods comprise contacting a sample suspected of containing such polypeptides with an immunologically effective amount of a composition comprising one or more specific anti-ACC antibodies disclosed herein.
  • Peptides that include the amino acid sequence of any of SEQ ID NO:4 through SEQ ED NO:8 and their derivatives will be preferred 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 supernatants, and the like. Such cells may be either eukaryotic (such as plant cells) or prokaryotic (such as cyanobacterial and bacterial cells).
  • 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.
  • the present invention provides a process of modulating 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 carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in a monocotyledonous plant.
  • a polypeptide is an acetyl-CoA carboxylase enzyme and, more preferably, a plant acetyl-CoA carboxylase.
  • a coding region includes the DNA sequence of SEQ ID NO:9 or SEQ ID NO: 19 and a promoter is CaMV35.
  • a cell is a cyanobacterium or a plant cell and a plant polypeptide is a monocotyledonous plant acetyl-CoA carboxylase enzyme such as wheat acetyl-CoA carboxylase enzyme.
  • the present invention also provides a transformed cyanobacterium produced in accordance with such a process.
  • the present invention still further provides a process for determining the inheritance of plant resistance to herbicides of the aryloxyphenoxypropionate or cyclohexane-l,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, detecting the presence of complexes between DNA restriction fragments and the ACC gene, and then correlating the herbicide resistance of the parental and progeny plants with the presence of particular sizes of ACC gene-containing DNA fragments as an indication of the inheritance of resistance to herbicides of these classes.
  • the acetyl-CoA carboxylase is a dicotyledonous plant acetyl-CoA carboxylase enzyme or a mutated 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.
  • a cyanobacterium is transformed with a plant ACC DNA molecule
  • that cyanobacterium can be used to identify herbicide resistant mutations in the gene encoding ACC.
  • 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 cyanobacteria;
  • step (e) characterizing DNA that encodes acetyl-CoA carboxylase from the cyanobacteria of step (d).
  • Means for transforming cyanobacteria as well as expression vectors used for such transformation are preferably the same as set forth above.
  • cyanobacteria are transformed or transfected with an expression vector comprising a coding region that encodes wheat ACC.
  • Cyanobacteria resistant to the herbicide are identified. Identifying comprises growing or culturing 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 known to be inhibited by that herbicide.
  • the present invention provides a process for identifying herbicide resistant variants of a plant acetyl-CoA carboxylase.
  • Such methods generally involve transforming a cyanobacterium or a bacterium or a yeast cell with a DNA molecule that encodes a plant acetyl-CoA carboxylase enzyme, 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.
  • the present invention provides 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 plant polypeptide having the ability to catalyze the carboxylation of acetyl-CoA, which coding region is operatively linked to a transcription-terminating 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.
  • Another aspect of the invention relates generally to transgenic plants which express genes or gene segments encoding the novel polypeptide compositions disclosed herein.
  • transgenic plants is intended to refer to plants that have incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression. It is contemplated that in some instances the genome of transgenic plants of the present invention will have been augmented through the stable introduction of the trarisgene. However, in other instances, the introduced gene will replace an endogenous sequence.
  • a preferred gene which may be introduced includes, for example, the ACC DNA sequences from cyanobacterial or plant origin, particularly those described herein which are obtained from the cyanobacterial species Synechococcus or Anabaena, or from plant species such as wheat or canola, of any of those sequences which have been genetically engineered to decrease or increase the activity of the ACC in such transgenic species.
  • Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the cDNA, gene or gene sequences of the present invention, and particularly those encoding ACC. These DNA constructs can 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 expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant.
  • 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 herbicide inactivation, e.g., a dicotyledonous ACC gene.
  • a transgenic DNA segment encoding a plant acetyl-CoA carboxylase enzyme which is resistant to herbicide inactivation, e.g., a dicotyledonous ACC gene.
  • a cyanobacterial ACC polypeptide-encoding DNA segment could also be used to prepare a transgenic plant with increased resistance to herbicide inactivation.
  • transgenic plants may be desirable having an decreased herbicide resistance. This would be particularly desirable in creating transgenic plants which are more sensitive to such herbicides.
  • a herbicide-sensitive plant could be prepared by inco ⁇ orating into such a plant, a transgenic DNA segment encoding a plant acetyl-CoA carboxylase enzyme which is sensitive to herbicide inactivation, e.g., a monocotyledonous ACC gene, or a mutated dicotyledonous or cyanobacterial ACC-encoding gene.
  • the invention concerns processes of modifying the oil content of a plant cell.
  • transgenic DNA segments encoding a plant or cyanobacterial acetyl-CoA carboxylase composition of the present invention.
  • Such processes would generally result in increased expression of ACC and hence, increased oil production in such cells.
  • ACC-encoding transgenic DNA segments or antisense (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 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 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.
  • kits for screening samples suspected of containing ACC polypeptides or ACC-related polypeptides, or cells producing such polypeptides can contain a nucleic acid segment or an antibody of the present invention.
  • the kit can contain reagents for detecting an interaction between a sample and a nucleic acid or antibody of the present invention.
  • the provided reagent can be radio-, fluorescently- or enzymatically-labeled.
  • the kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of die present invention.
  • the reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder.
  • the liquid solution is an aqueous solution.
  • the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide.
  • the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
  • the present invention concerns immunodetection methods and associated kits. It is proposed that the ACC peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect ACC or ACC-related epitope-containing 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 detecting the presence of the immunocomplex.
  • immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches.
  • the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like.
  • immunocomplex formation will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like).
  • a label such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like).
  • a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
  • any sample suspected of comprising either an ACC peptide or an ACC-related peptide or antibody sought to be detected 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.
  • the present invention contemplates the preparation of kits that may be employed to deteci the presence of ACC or ACC-related proteins or peptides and/or antibodies in a sample. Samples may include cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing ACC peptides.
  • kits in accordance with the present invention will include a suitable ACC peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody 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.
  • 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 containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. 2.6 ELISAs and Inununoprecipitation
  • ELISAs may be used in conjunction with the invention.
  • 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 a polystyrene microtiter plate.
  • 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.
  • BSA bovine serum albumin
  • casein casein
  • 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.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBSVTween®. These added agents also tend to assist in the reduction of nonspecific background.
  • the layered 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 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 ween®, or borate buffer.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS Tween®).
  • 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-benzthiazoline)-6-sulfonic acid (ABTS) and H O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • 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 discriminate or isolate minute amounts of protein.
  • For the isolation of membrane proteins cells must be solubilized into detergent micelles.
  • Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
  • 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.
  • 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 identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • 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.
  • the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross- reacting signal.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
  • the present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which inco ⁇ orates an epitope that is immunologically cross-reactive with one or more anti- ACC antibodies.
  • the term "inco ⁇ orating an epitope(s) that is immunologically cross-reactive with one or more anti-ACC antibodies” is intended to refer to a peptide or protein antigen which includes 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 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.
  • ACC immunodominant epitopes and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter.
  • 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 "epitopic core sequences" may then be readily inco ⁇ orated into peptides, either through the application of peptide synthesis or recombinant technology.
  • Preferred peptides for use in accordance with the present invention will generally be on the order of 8 to 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic ACC-derived peptides will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution. It is proposed tiiat 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 sequences are identified herein in particular aspects as hydrophihc 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 stimulation, and, hence, elicit specific antibody production.
  • An epitopic core sequence 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.
  • the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
  • the smallest useful core sequence anticipated by me present disclosure 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.
  • this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
  • peptides 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.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5.
  • agents which will inhibit microbial growth such as sodium azide or Merthiolate.
  • the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.
  • 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 prove to encode proteins, polypeptides, subunits, functional domains, and the like of ACC- related or other non-related gene products.
  • these DNA segments may be synthesized entirely in vitro using methods that are well-known to those of skill in the art.
  • 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 fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
  • 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 elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences.
  • 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 term 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 region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to die segment by the hand of man.
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode an ACC peptide species that includes within its amino acid sequence an amino acid sequence essentially as set forth in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ED NO: 12, SEQ ID NO:20, and SEQ ID NO:31.
  • sequence essentially as set forth in any of SEQ ED 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 means that the sequence substantially corresponds to a portion of the sequence of either SEQ ED 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 or SEQ ID NO:31, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences.
  • 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 ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ED NO: 12, SEQ ID NO:20, and SEQ ID NO:31 will be sequences that are "essentially as set forth in any 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.”
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino 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 maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • nucleic acid segments of the present invention 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, witii the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments may be prepared that include a short contiguous stretch encoding either of the peptide sequences disclosed in any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ED NO:8, SEQ ID NO: 10, SEQ ED NO: 12, SEQ ID NO:20 and SEQ ID NO:31, or that are identical 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: 11, SEQ ID NO: 19, or SEQ ID NO:30.
  • 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 contemplated to be useful.
  • intermediate lengths 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.
  • this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which 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: 19, 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 redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • 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 properties of the amino acids being exchanged. Changes designed 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 mutants in order to examine activity at the molecular level.
  • 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 purification or immunodetection pu ⁇ oses (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • 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 segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter.
  • the promoter may be in the 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.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding an ACC peptide in its natural environment.
  • Such promoters may include 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 molecular biology, for example, see Sambrook et al, 1989.
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate 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 include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
  • 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 useful.
  • Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequences from any of SEQ ED NO:2, SEQ DD NO:4, SEQ DD NO:6, SEQ DD NO:8, SEQ DD NO: 10, SEQ DD NO: 12, SEQ DD NO:20, or SEQ DD NO:31.
  • 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 embodiments.
  • 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 nucleotide long contiguous DNA segment any of SEQ DD NO: 1, SEQ DD NO:3, SEQ DD NO:5, SEQ DD NO:7, SEQ DD NO:9, SEQ DD NO: 11, SEQ DD NO: 19, and SEQ ID NO.30 will find particular utility.
  • Longer contiguous identical or complementary sequences 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 intermediate lengths and up to and including full-length sequences will also be of use in certain embodiments.
  • 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.
  • sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • Nucleic acid molecules having sequence regions consisting 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 DD NO:3, SEQ ED NO:5, SEQ DD NO:7, SEQ DD NO:9, SEQ ED NO: 11, SEQ DD NO: 19, and SEQ DD NO:30 are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting.
  • hybridization 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 preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molev;ules obtained.
  • fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion.
  • Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • fragments 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 inco ⁇ orated 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.
  • the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.
  • one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
  • relatively stringent conditions e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C.
  • Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating ACC- encoding DNA segments.
  • nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
  • the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA 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 example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
  • specific hybridization is detected, or even quantitated, by means of the label.
  • 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 changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons listed in Table 1.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with 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 properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, inco ⁇ orate 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, and the like.
  • Each amino 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); phenylalanine (+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); histidine (-3.2); gluta ⁇ uite (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5).
  • 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.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+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).
  • 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.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, 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, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants 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.
  • 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.
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • 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 such as the Ml 3 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 eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • 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 mutated sequence is prepared, generally synthetically. This primer is then annealed with the single- stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation- bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • 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.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.2.12 Monoclonal Antibody Generation
  • mAbs monoclonal antibodies
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
  • an immunogenic composition in accordance with the present invention
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.
  • a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary 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 include glutaraldehyde, -maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • complete Freund's adjuvant a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis
  • incomplete Freund's adjuvants a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis
  • aluminum hydroxide adjuvant aluminum hydroxide adjuvant.
  • polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. 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 immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified ACC protein, polypeptide or peptide.
  • a selected immunogen composition e.g., a purified or partially purified ACC protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, 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 preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol.
  • B cells B lymphocytes
  • 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 preferred, 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.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • 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).
  • the immunized animal is a mouse
  • 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 preferred murine myeloma cell is the NS-1 myeloma cell line (also termed
  • P3-NS-l-Ag4-l 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-azaguanine-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 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 medium.
  • the selective medium 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 azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, 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.
  • HPRT hypoxanthine phosphoribosyl transferase
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to tiiree weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, 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 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 medium from which they can 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. 3.
  • FIG. 1 Structure of the cytosolic ACCase gene from wheat. Arrows indicate fragments of the genomic clones analyzed in more detail. Sequenced fragments are marked in black. The localization of the ACCase functional domains was established by amino acid sequence comparison with other biotin-dependent carboxylases (Gomicki et al, 1994). BC, biotin carboxylase; BCC, biotin carboxyl carrier; CT, carboxyltransferase .
  • FIG. 2 Alignment 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 underlined. Asterisks 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' flanking sequence of the ACCase 1 gene (about 3 kb upstream of the translation initiation codon, of clone 71L. The sequence is shown in SEQ ID NO:32.
  • FIG. 6 Shown is the 5' flanking sequence of the ACCase 2 gene designated 153. The sequence is shown in SEQ ID NO: 33.
  • 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 that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates 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. Transformation: 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 inco ⁇ orated into a chromosome or is capable of autonomous replication.
  • exogenous DNA sequence e.g., a vector, a recombinant DNA molecule
  • 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.
  • a plasmid is an exemplary vector.
  • Primer 2 5'-GCTCTAGAGKRTGYTCNACYTG-3' (SEQ ID NO: 14); where N is A, C, G or T; H is A, C or T; R is A or G; Y is T or C and K is G 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 the primer to provide anchors for rounds of amplification after the first round and to provide convenient restriction sites for analysis and cloning.
  • 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-terminal 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 D_) 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
  • the MKM primer (primer 4) was first checked by determining whether it would amplify the fabE 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 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 me correct fragment of the Anabaena fabE gene, which was used to identify cosmids that contained the entire fabE gene and flanking DNA.
  • An about 4-kb Xb ⁇ l fragment containing the gene was cloned into the vector pBluescriptKS® for sequencing.
  • Primers 3 and 4 were then used to amplify the intervening 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 pCRU®.
  • the amino acid sequence of the polypeptide predicted from the cDNA sequence for this entire fragment of wheat cDNA 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.
  • E. coli ACC 33 rat propionyl CoA carboxylase 32 31 yeast pyruvate carboxylase 31
  • DNA sequence information provided by the invention 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.
  • nucleic acid probes of an appropriate length are prepared based on a consideration of a selected ACC gene sequence, e.g., a sequence such as that shown in SEQ DD NO: 9 or SEQ DD NO: 19, or a selected gene sequence encoding a subunit of a cyanobacterial ACC, e.g., a sequence as that shown in SEQ DD NO:l, SEQ DD NO:3, SEQ DD NO:5, SEQ DD NO:7, or SEQ ID NO: 11.
  • nucleic acid probes to specifically hybridize to an ACC gene sequence lend them particular utility in a variety of embodiments.
  • the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample.
  • oligonucleotide primers 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 mutating a defined segment of an ACC gene from a cyanobacterium or a plant using PCRTM technology. Segments of ACC genes from other organisms may also be amplified by PCRTM using such primers.
  • 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 in SEQ ID NO:l, SEQ ID NO:3, SEQ DD NO:5, SEQ DD NO:7, SEQ DD NO:9, SEQ DD NO: 11, or SEQ DD NO: 19.
  • 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 complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and ti ereby improve the quality and degree of specific hybrid molecules obtained.
  • Such fragments may be readily prepared by, for example, directly synthesizing 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 inco ⁇ orated by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction sites.
  • a nucleotide sequence of the invention can be used for its ability to selectively form duplex molecules with complementary stretches of the gene.
  • relatively stringent conditions for applications requiring a high degree of selectivity, one will typically desire to employ relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. These conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.
  • a polynucleotide of the present invention in combination with an appropriate label for detecting hybrid formation.
  • appropriate labels include radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
  • a hybridization probe described herein is useful both as a reagent in solution hybridization as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is 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).
  • specific hybridization is detected, or even quantitated, by means of the label.
  • an expression vector comprising a polynucleotide of the present invention.
  • an expression vector is an isolated and purified DNA molecule comprising a promoter operatively linked to an coding region that encodes a polypeptide having the ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.
  • operatively linked means that a promoter is connected 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.
  • 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 preferred promoter for the cyanobacterium Synechococcus is the psbAI gene promoter.
  • the cyanobacterial ace gene promoters themselves can be used.
  • 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.
  • the promoter is a near-constitutive promoter such as CaMV 35S
  • increases in polypeptide expression are found in a variety of transformed plant tissues (e.g., callus, leaf, seed and root).
  • the effects of transformation can be directed to specific plant tissues by using plant integrating vectors containing a tissue- specific promoter.
  • 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 (Lei) 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 characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al, 1983; Lindstrom et al, 1990.)
  • An expression vector containing 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 the effects of transformation in more than one specific tissue.
  • tissue-specific promoters are com sucrose synthetase 1 (Yang et al, 1990), com alcohol dehydrogenase 1 (Vogel et al, 1989), com light harvesting complex (Simpson, 1986), com heat shock protein (Odell et al, 1985), pea small subunit RuBP Carboxylase (Poulsen et al, 1986; Cashmore 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
  • a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively 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).
  • Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S promoter.
  • the vector used to express the polypeptide includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker.
  • a drug resistance selection marker is the gene whose expression results in kanamycin resistance; i.e., the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase D and nopaline synthase 3' nontranslated 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 terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
  • mRNA messenger RNA
  • 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 d e ability to catalyze the carboxylation of a biotin carboxyl carrier protein of a cyanobacterium 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.
  • a polypeptide has the amino acid residue sequence of SEQ ID NO:6 or SEQ DD NO:8, or a functional equivalent of those sequences.
  • a coding region comprises the entire DNA sequence of SEQ ED NO:5 or the DNA sequence of SEQ DD NO:5 comprising the Anabaena accC gene.
  • a coding region comprises the entire DNA sequence of SEQ DD NO:7 or the DNA sequence of SEQ DD NO:7 comprising the Synechococcus accC gene.
  • 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 DD NO:2 or SEQ DD NO:4, or functional equivalents thereof.
  • a coding region comprises the entire DNA sequence of SEQ DD NO: 1 or the DNA sequence of SEQ DD NO:l comprising the Anabaena accB gene.
  • a coding region comprises the entire DNA sequence of SEQ DD NO: 3 or the DNA sequence of SEQ DD NO: 3 comprising the Synechococcus accB gene.
  • an expression vector comprises a DNA segment that encodes a carboxyltransferase protein of a cyanobacterium. That carboxyltransferase protein preferably includes a CT ⁇ or CT ⁇ subunit, and preferably includes the amino acid residue sequence of SEQ ID NO: 12, or a functional equivalent thereof.
  • a coding region comprises the entire DNA sequence of SEQ DD NO: l 1 or the DNA sequence of SEQ DD NO: l 1 comprising the Synechococcus ace A gene.
  • 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 plant acetyl-CoA carboxylase enzyme.
  • a preferred monocotyledonous plant polypeptide encoded by such a coding region is preferably wheat ACC, which ACC includes the amino acid residue sequence of SEQ DD NO: 10 or SEQ DD NO: 31 or functional equivalents thereof.
  • a preferred coding region includes the DNA sequence of SEQ DD NO:9 or SEQ DD NO:30.
  • a preferred dicotyledonous plant ACC such as canola ACC, is also preferred.
  • Such an ACC enzyme is encoded by the DNA segment of SEQ DD NO: 19 and has the amino acid sequence of SEQ ID NO: 20.
  • the present invention provides novel polypeptides that define a whole or a portion of an ACC of a cyanobacterium or a plant.
  • 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 Anabaena or Synechococcus.
  • a biotin carboxyl carrier protein from Anabaena includes the amino acid sequence of SEQ ED NO:2, with such amino acid sequence listing encoded by the DNA segment of SEQ DD NO: 1.
  • 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 DD NO:2.
  • the present invention provides an isolated polypeptide comprising a biotin carboxylase protein of a cyanobacterium such as Anabaena or Synechococcus.
  • a biotin carboxylase protein from Anabaena includes the amino acid sequence of SEQ DD NO:6, with such amino acid sequence listing encoded by the DNA segment of SEQ DD NO:5.
  • a biotin carboxylase protein from Synechococcus includes the amino acid sequence of SEQ ID NO:8, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:7.
  • the present invention provides an isolated polypeptide comprising a carboxyltransferase protein of a cyanobacterium such as Synechococcus.
  • a carboxyltransferase protein comprises a CT ⁇ or CT ⁇ subunit and includes the amino acid sequence of SEQ DD NO: 12, with such amino acid sequence listing encoded by the DNA segment of SEQ DD NO: 11.
  • the present invention contemplates an isolated and purified plant polypeptide having a molecular weight of about 220 kDa, dimers of which have the ability to catalyze the carboxylation of acetyl-CoA.
  • a polypeptide preferably includes the amino acid residue sequence of SEQ DD NO: 10 or SEQ DD NO:31, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO:9 or SEQ DD NO:30.
  • 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 amino acid residue sequence of SEQ ID NO:20, with such amino acid sequence listing encoded by the DNA segment of SEQ ID NO: 19.
  • a cyanobacterium, a yeast cell, or a plant cell or a plant transformed with an expression vector of the present invention is also contemplated.
  • a transgenic 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 similar 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 of log-phase cells with DNA. (Golden et al, 1987)
  • Methods for DNA transformation of plant cells include Agrobacterium- mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs, injection into immature embryos and particle bombardment.
  • Agrobacterium-mediated plant transformation protoplast transformation
  • gene transfer into pollen injection into reproductive organs
  • injection into immature embryos and particle bombardment Each of these methods has distinct advantages and disadvantages.
  • one particular method of introducing genes into a particular plant strain may not necessarily be the most effective for another plant strain, but it is well known which methods are useful for a particular plant strain.
  • Suitable methods are 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-mediated transformation of protoplasts (Omirulleh et al, 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc.
  • acceleration methods are preferred and include, for example, microprojectile bombardment and the like.
  • 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-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
  • the introduction of DNA by means of electroporation, is well-known to those of skill in the art.
  • certain cell wall-degrading enzymes such as pectin- degrading enzymes
  • recipient cells are made more susceptible to transformation, by mechanical wounding.
  • friable tissues such as a suspension culture of cells, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly.
  • pectolyases pectolyases
  • Such cells would then be recipient to DNA transfer by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly inco ⁇ orated DNA.
  • a further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment.
  • particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
  • An illustrative embodiment of a method for delivering DNA into 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 com cells cultured in suspension.
  • 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 aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cefls by projectiles that are too large.
  • cells in suspension are preferably concentrated on filters or solid culture medium.
  • immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • one or more screens are also positioned between the acceleration device and the cells to be bombarded.
  • bombardment transformation one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum 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 manipulation of cells before and immediately 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 linearized DNA or intact supercoiled plasmids. It is believed mat pre-bombardment manipulations are especially important for successful transformation of immature embryos.
  • TRFs trauma reduction factors
  • Agrobacterium-mediaied transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast.
  • the use of Agr ⁇ b ⁇ cter. wm-mediated 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 integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Shmann et al, 1986; Jorgensen et al, 1987).
  • Modem 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 Ajjrob ⁇ cteriu -mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate 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 suitable for present pu ⁇ oses.
  • Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Aj rob ⁇ cter.Mm-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
  • a transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene.
  • heterozygous usually implies the presence of a complementary gene at the same locus of the second chromosome of a pair of chromosomes, and there is no such gene in a plant containing 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 segregates independently during mitosis and meiosis.
  • transgenic plant that is homozygous for the added stmctural 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 transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
  • transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate 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 calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, for example, Potrykus et al, 1985; Lorz et al,
  • 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.
  • the amount of a gene coding for a polypeptide of interest can be increased in monocotyledonous plants such as com by transforming those plants using particle bombardment methods (Maddock et al, 1991).
  • an expression vector containing an coding region for a dicotyledonous ACC and an appropriate selectable marker is transformed into a suspension of embryonic maize (com) 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 used to successfully 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 into the cells of immature embryos and the rehydration of desiccated embryos as described (Neuhaus et al, 1987; Benbrook et al, 1986).
  • 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 used to pollinate regenerated plants.
  • a transgenic plant of the present invention containing 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 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 preferred transgenic plant is an independent segregant and can transmit that gene and its activity to its progeny.
  • a more preferred transgenic plant is homozygous for that gene, and transmits that gene to all of its offspring on sexual mating.
  • 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 resistance, preferably in the field, under a range of environmental conditions.
  • the commercial value of a transgenic plant with increased herbicide resistance or with altered fatty acid production is enhanced 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 traits.
  • 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 resistance. Because of this, herbicide resistance is preferably bred into a large number of parental lines so that many hybrid combinations can be produced.
  • Herbicides such as aryloxyphenoxypropionates and cyclohexane-l,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 organisms and prokaryotic organisms are resistant to those compounds.
  • 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 the herbicide resistance of a monocotyledonous plant comprising transforming the plant 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-terminating region, whereby the promoter is capable of driving the transcription of the coding region in a monocotyledonous plant.
  • 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 transcription-terminating region are preferably the same as set forth above.
  • Transformed monocotyledonous plants can be identified using herbicide resistance.
  • a process for identifying a transformed monocotyledonous plant cell involves transforming the monocotyledonous plant cell with a DNA molecule that encodes a dicotyledonous acetyl-CoA carboxylase enzyme, and determining the resistance of the plant cell to a herbicide and thereby the identification oi the transformed monocotyledonous plant cell.
  • Means for transforming a monocotyledonous plant cell are the same as set forth above.
  • the resistance of a transformed plant cell to a herbicide is preferably determined by exposing such a cell to an effective herbicidal dose of a preselected herbicide and maintaining 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 herbicide that retards growth or kills plant cells not containing 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.
  • a herbicide used in such a process is an aryloxyphenoxypropionate or cyclohexanedione herbicide.
  • ACC catalyzes the carboxylation of acetyl-CoA.
  • the carboxylation of acetyl-CoA in a cyanobacterium or a plant can be altered by, for example, increasing an ACC gene copy number or changing the composition (e.g., nucleotide sequence) of an ACC gene.
  • Changes in ACC gene composition may alter gene expression at either the transcriptional or translational level.
  • changes in gene composition can alter ACC function (e.g., activity, binding) by changing primary, secondary or tertiary structure of the enzyme.
  • certain changes in ACC structure are associated with changes in the resistance 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 appropriate expression vector comprising a DNA molecule that encodes ACC.
  • 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 operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cyanobacterium.
  • 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.
  • the present invention provides a process for determining the inheritance of plant resistance to herbicides of the aryloxyphenoxypropionate or cyclohexanedione class. That process involves measuring resistance to herbicides of the aryloxyphenocypropionate or cyclohexanedione class in a parental plant line and in progeny of the parental plant line and detecting the presence of a DNA segment encoding ACC in such plants.
  • phenotypic traits such as herbicide resistance
  • RFLPs Restriction fragment length polymo ⁇ hisms
  • RFLPs Restriction fragment length polymo ⁇ hisms
  • the herbicide resistant variant of acetyl-CoA carboxylase is a dicotyledonous plant acetyl-CoA carboxylase enzyme or a portion thereof.
  • the herbicide resistant variant of acetyl- CoA carboxylase is a mutated 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.
  • Restriction fragment length polymo ⁇ hism analyses are conducted, for example, by Native Plants Inco ⁇ orated (NPI). This service is available to the public on a contractual basis.
  • NPI Native Plants Inco ⁇ orated
  • the genetic marker profile of the parental inbred lines is determined. If parental lines are essentially 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 those parents, 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 appropriate 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. 4.10 Oil Content of Seeds
  • Manipulation of the oil content and quality of seeds may benefit from knowledge of this gene's structure and regulation. Understanding the basis of resistance 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 resistance.
  • 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.
  • genes encoding ACC may alter the oil content of the grain, and thus may be of significant value. Increases in oil content may result in increases in metabolizable-energy-content and -density of the seeds for uses in feed and food.
  • 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.
  • 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.
  • oil properties may also be altered to improve its performance 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 properties 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 possessing them or by increasing levels of native fatty acids while possibly reducing levels of precursors.
  • introduction of DNA segments which are complementary to the DNA segments disclosed herein into plant cells may bring about a decrease in ACC activity in vivo and lower the level of fatty acid biosynthesis in such transformed cells. Therefore, transgenic plants containing such novel constructs may be important due to their decreased oil content in such cells. Introduction of specific mutations in either the DNA segments disclosed, or in their complements, may result in transformed plants having intermediate ACC activity.
  • the gene for the BC subunit was cloned with a fragment of the E. colifabG gene as a heterologous hybridization probe.
  • the 3.1-kb H dEQ fragment identified by this probe in the Anabaena sp. strain PCC 7120 DNA digest was purified by gel electrophoresis and then was digested with Nhel, yielding a 1.6-kb Nhel-HindlU fragment that hybridized with the same fabG probe.
  • the 1.6-kb fragment was purified by gel electrophoresis and cloned into Xb ⁇ l-HindDI-digested pUC18. The ends of the insert were sequenced.
  • the N-terminal amino acid sequence of BCCP was used to design an upstream
  • PCRTM primer The downstream primer was targeted to the conserved sequence encoding d e biotinylation site.
  • PCRTM was carried out as described in the GeneAmp® kit manual (Perkin-Elmer Cetus). All components of the PCRTM except the Taq DNA polymerase were incubated for 3 to 5 min at 95°C. The PCRTM was then initiated by the addition of polymerase. Amplification was for 45 cycles, each 1 min at 95°C, 1 min at 42 to 45°C, and 2 min at 72°C, with 0.5 to 1.0 ⁇ g of template DNA per ml and 50 ⁇ g of each primer per ml. The PCRTM amplification yielded a product -450 bp in size (i.e., the correct size for the anticipated fragment of the Anabaena sp. strain PCC 7120 BCCP gene deduced from the E.
  • the PCRTM product was cloned into the Invitrogen vector pCRlOOO with the A/T tail method and was sequenced to confirm its identity.
  • the fragment of the Anabaena sp. strain PCC 7120 BCCP gene was then used as a probe to identify cosmids that contain the entire gene and flanking DNA. Three such cosmids were detected in a 1,920-member library (same as described above).
  • a 4.2-kb Xbal fragment containing the BCCP gene was subcloned into pBluescriptD®, and its HindHl-Nhel fragment was sequenced with specific primers as described above.
  • the 1458-nucleotide DNA segment comprising the Anabaena accB gene is given in SEQ DD NO:l.
  • the 182-amino acid translation of the accB gene encoding the Anabaena BCCP is given in SEQ DD NO:2.
  • the amino acid sequence deduced from the DNA sequence of the BCCP gene exactly matches the N-terminal sequence obtained for purified protein. Likely translation initiation codons were identified by comparison with E. coli.
  • the AUG start codon is not preceded by an obvious ribosome-binding site. There is a stop codon in ti e same open reading frame one codon upstream from the AUG codon, excluding the possibility of additional amino acids at the N terminus.
  • the GUG start codon for BCCP immediately precedes codons for the amino acids identified by protein sequencing of the N terminus of purified BCCP.
  • a putative 5-nucleotide ribosome-binding site, GAGGU is located 11 nucleotides upstream of the 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 sitess from translation. This excludes the possibility that the purified BCCP polypeptide lacks more than one amino acid (Met) because of rapid proteolytic degradation.
  • strain PCC 7120 BC including the ATP binding site motif and the conserved sequence including Cys-230 as a part of the bicarbonate binding site.
  • Mitochondrial enzymes rat propionyl-CoA carboxylase (Browner et al, 1989) and yeast pyruvate carboxylase (Lim et al, 1988), are only 45 to 47% identical. Similarities with carbamoyl-phosphate synthetases 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 for Anabaena 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 D. The structure of this part of the BCCP gene was confirmed by sequencing the corresponding PCRTM-cloned fragment of Anabaena sp. strain PCC 7120 DNA.
  • 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.
  • RNA Northern
  • the major hybridizing 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 rnRNAs 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 containing C 26 and C 28 fatty acids (Murata and Nishida, 1987).
  • 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 screened with probes specific for BCCP and BC).
  • 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 tr 30 amino acids.
  • Anabaena sp. strain PCC 7120 BCCP is longer by 20 tr 30 amino acids.
  • the unusual electrophoretic properties of the E. coli protein shows a slightly lower mobility than E. coli BCCP (-22.5 kDa), suggesting that Anabaena sp. strain PCC 7120 BCCP is longer by 20 tr 30 amino acids.
  • the unusual electrophoretic properties of the E. coli protein shows a slightly lower mobility than E. coli BCCP (-22.5 kDa), suggesting that Anabaena sp. strain PCC 7120 BCCP is longer by 20 tr 30 amino acids.
  • the unusual electrophoretic properties of the E. coli protein shows a slightly lower mobility than E. coli BCCP (-22.5 kDa), suggesting that Anabaena sp
  • DNA'' 41 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 alignment.
  • BCCP from Anabaena sp. strain PCC 7120 was purified starting with cells from a 3-liter culture grown on BG11 medium (Rippka et al, 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 material was removed by centrifugation at 31,000 x g for 30 min, and the soluble protein fraction containing 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 (G ⁇ BCO BRL).
  • the mixture was loaded onto a column, was washed with about 30 ml of 0.25 M NaCl-50 mM Tris-HCl (pH 7.5)-0.5 mM ⁇ DTA-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, and separated by SDS-PAGE.
  • the N-terminal sequence of the biotin-containing ⁇ 25-kDa polypeptide was determined by Edman degradation after transfer to Immobilon-P® as described above.
  • the sequence was PLDFNEIRQL (SEQ
  • the gel region containing DNA of sizes between 1.6-kb and 3-kb was cut out and purified (using Geneclean D Kit from BiolOl). The purified DNA was then digested with PstI and electrophoresed on an agarose gel. The gel region containing DNA of sizes between 0.5-kb and 2-kb was cut out and purified. 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 fragment 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 and white (i.e., with insert) colonies were selected by plating on LB plates containing ampicillin, X-Gal and IPTG.
  • the 1362-nucleotide DNA segment comprising the Synechococcus accC gene is given in S ⁇ Q DD NO:7. Only one significant open reading frame (ORF) was found.
  • the proteins (from a crude whole protein extract) of Synechococcus PCC 7942 were first separated by standard SDS-PAG ⁇ method, then transferred onto an Immobilon-P® transfer membrane, which was subsequently incubated with 35 S-streptavidin. Only one radioactive band (corresponding to a protein of about 25 kDa) appeared on the autoradiogram. This result suggests that there is only one biotin-containing protein in Synechococcus and its mass is similar to the reported mass of E. coli biotin carboxyl carrier protein, 22,500 Da. This biotin-containing protein was purified Synechococcus cells were first broken by sonication in a buffer containing NaCl, Tris, glycerol and SDS.
  • the supernatant was separated from cell debris by centrifugation, then followed by a 50% (NH_ t ) 2 S0 4 precipitation.
  • the precipitate was dissolved in the same buffer, and was allowed to bind to streptavidin 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 Immobilon-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-bound protein was cut out and its N-terminal amino acid sequence was determined.
  • degenerate oligonucleotide primers were designed for PCRTM amplification studies with Synechococcus genomic DNA.
  • primer LE8 5 '-GCTCTAGACNCARYTNAAYTT-3 ' SEQ ID NO:26
  • primer LE7 3 '-CRNTACTTYGACNWCTTAAGCT- ' 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 mg/ml of template DNA, 5 mg/ml of primer LE8, 40 mg/ml of primer LE7 and with 1 mM Mg 2+ final concentration. Under these conditions, a specific PCRTM produce was identified.
  • a 477 -nucleotide DNA segment comprising the Synechococcus accB gene is given in SEQ DD NO: 3. Only one significant ORF was found. The deduced amino acid sequence at the N-terminus of this ORF matches the earlier determined N-terminal amino acid sequence of the purified Synechococcus biotin-containing protein. The 158-amino acid sequence of the Synechococcus BCCP is given in SEQ DD NO:4. Sequence alignment indicated 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).
  • a 0.9-kb Clal-Mlul fragment of the E. coli accA gene was used as a probe to examine the Synechococcus PCC 7942 genomic DNA by Southern hybridization at 60°C. A strongly hybridizing 1.6-kb Pstl fragment was detected and subsequently cloned.
  • Synechococcus PCC 7942 genomic DNA was digested with Pstl and electrophoresed on an agarose gel. The gel region containing 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 containing tetracycline, then scored on plates containing ampicillin.
  • a total of 800 tetracycline-resistant, but ampicillin-sensitive, clones were screened: the plasmid DNA was prepared, digested (in pools of 5 clones per pool) with Pstl, electrophoresed, transferred onto a Genescreen Plus membrane, then hybridized with the E. coli accA probe at 60°C. Positive signals appeared on 3 pools.
  • the 327-amino acid sequence of the Synechococcus ORF is 54% identical to that of the E. coli accA gene.
  • the amino acid sequence of the Synechococcus accA gene encoding CT ⁇ is given in SEQ ID NO: 12.
  • 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:
  • PCRTM was mn 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 template DNA, 5 mg/ml of each primer and with 1 mM Mg 2+ final concentration. Under these conditions, a specific PCRTM product of 256 bp was identified.
  • Proteins were separated by SDS-PAGE using a 7.5% separating gel (Maniatis et al, 1982), and then were transferred onto a PVDF membrane (Immobilon-P®, Millipore) in 10 mM 3-(cyclohexylamino)-l-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 35 S-Streptavidin (Amersham). The blots were washed at room temperature with 0.5% Nonidet-P40TM in 10 mM Tris-HCl pH 7.5 and 0.9% NaCl.
  • 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 detected). ACC consisting of 220-kDa subunits is the most abundant biotin-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 detected only in trace amounts in some chloroplast preparations. The amount of all biotinylated peptides, estimated from band intensities on western blots (amount of protein loaded was normalized for chlorophyll content), is much higher in pea than in wheat chloroplasts.
  • the precipitate was collected by centrifugation for 30 min at 12000 ⁇ m, 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 above) and loaded on a 5 cm x 50 cm Sephadex G-100 column equilibrated and eluted with the same buffer. Fractions containing ACC activity (assayed as described below using up to 20 ⁇ l aliquots of column fractions) were pooled and loaded immediately 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 containing 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-mercaptoethanol, and separated in several portions on two Superose columns connected in-line (Superose 6 and 12, Pharmacia). 1 ml fractions were collected at 0.4 ml/min flow rate.
  • ACC-containing 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 preparation as revealed by Coomassie staining of proteins separated by SDS-PAGE.
  • This preparation also contained several smaller biotin-containing peptides as revealed by western blotting with 35 S-Streptavidin, most likely degradation products of the ca. 220-kDa peptide, which retained their ability to form the -500-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 Immobilon-P® for sequencing.
  • the N-terminus of the peptide appeared to be blocked.
  • a mixture of amino acids was detected only after the protein was cleaved chemically with CNBr.
  • the 220-kDa protein was therefore purified on an SDS gel, cleaved with CNBr, and the resulting peptides were fractionated by gel electrophoresis basically as described (Jahnen-Dechent and Simpson, 1990), with the following modifications.
  • a slice of gel containing about 20 ⁇ g of the 220-kDa polypeptide was dried under vacuum to about half of its original volume and then incubated overnight in 0.5 ml of 70% formic acid containing 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 mM thioglycolic acid in the cathode buffer. Peptides were transferred to Immobilon-P for sequencing by the Edman degradation method as described above.
  • haloxyfop one of the aryloxyphenoxypropionate herbicides has been tested, on the activity of ACC from wheat germ and from wheat seedling leaves.
  • 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 ⁇ M haloxyfop, respectively.
  • the enzyme from pea chloroplasts is relatively resistant (50% inhibition occurs at >50 :M haloxyfop).
  • the in vivo inco ⁇ oration of 14 C-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.
  • PCRTM primers were based on the alignment 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 (Ml 1738) 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 5 '-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 AMV reverse transcriptase (Haymerle et al, 1986). Reverse transcriptase was inactivated by incubation at 90°C and low molecular weight material was removed by filtration. All components of the PCRTM (Cetus/Perkin-Elmer), except the Taq DNA polymerase, were incubated for 3-5 min at 95°C. The PCRTM was initiated 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. MgCl 2 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 pCRlOOO using their A T tail method, and sequenced.
  • 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- terminal end of the BC domain and the other from the conserved biotinylation site.
  • the expected 1.1 -kb product of the first low yield PCRTM with primers ID and IV was separated by electrophoresis on LMP-agarose and reamplified by another round of PCRTM, then cloned into the Invitrogen vector pCRD® and sequenced.
  • the PCRTM conditions were the same as those described above.
  • a wheat cDNA library (Triticum aestivum, var. Tarn 107, Hard Red Winter, 13-day light grown seedlings) was purchased from Clontech. This 8gtl l library was prepared using both oligo(dT) and random primers. Colony ScreenPlus® (DuPont) membrane was used according to the manufacturers' protocol (hybridization at 65°C in 1 M NaCl and 10% dextran sulfate). The library was first screened with the 1.1 -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 10 6 plaques were tested.
  • More than fifty clones containing ACC-specific cDNA fragments were purified, and EcoRI fragments 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 termination method with Sequenase® (United States Biochemicals) or using the Perkin Elmer/Applied Biosystems Taq DyeDeoxy Terminator cycle sequencing kit and an Applied Biosystems 373A DNA Sequencer.
  • RNA from 10-day old wheat plants was prepared as described in (Haymerle et al, 1986). RNA was separated on a glyoxal denaturing gel (Sambrook et al, 1989). GeneScreen Plus® (DuPont) blots were hybridized in IM NaCl 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).
  • a 440-bp cDNA fragment encoding a part of the biotin carboxylase domain of wheat ACC and a 1.1 -kb cDNA fragment encoding the interval between the biotin carboxylase domain and the conserved biotinylation site were amplified. These fragments were cloned and sequenced. In fact, three different 1.1 -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 minimum number expected for hexaploid wheat. These two overlapping DNA fragments (total length of 1473 nucleotides) were used to screen a wheat cDNA library.
  • a set of overlapping cDNA clones covering the entire ACC coding sequence was isolated and a subset of these clones has been sequenced.
  • the nucleotide sequence within overlapped regions of clones 39-1, 20-1 and 45-1 differ at 1.1% 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-amino acid reading frame encoding a protein with a calculated molecular mass of 251 kDa.
  • 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 (Gomicki and Haselkorn, 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
  • the 7360-nucleotide DNA segment comprising the wheat ACC cDNA is given in SEQ DD NO:9.
  • the 2257-amino acid translated wheat ACC sequence is given in SEQ DD NO: 10.
  • 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 is correlated with the cell age (e.g., Lampa et al, 1985). It is not su ⁇ rising 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 significantly in older cells where the demand is much lower. The same differences in the level of ACC specific mRNA between cells in different sectors were found in plants grown in the dark and in plants illuminated for one day at the end of the dark period.
  • the putative translation start codon was assigned to the first methionine of the open 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 genes, except for U at position +4 of the consensus which was found in only 3 of the 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.
  • a comparison 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 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 sequence of eukaryotic ACCs.
  • 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 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 apparent 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 (Gomicki and Haselkorn, 1993; ⁇ gli et al, 1993). No obvious chloroplast transit peptide between the ER signal peptide and the mature protein was found in diatom ACC either (Roessler and Ohlrogge, 1993).
  • ACC genes in wheat have been assessed by Southern analysis and by sequence analysis of the 5'- and 3 '-untranslated portions of ACC cDNA representing transcripts of different genes. These cDNA fragments may be obtained by PCRTM amplification using the 5'- and 3 '-RACE methodology.
  • the genome structure of wheat 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 familiar promoter and 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 alternative 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 and the mature protein.
  • variant cytoplasmic and plastid isoenzymes could arise, for example, by alternative splicing or by transcription of two independent genes. This problem is especially interesting 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 by sequence analysis of the appropriate fragment of the ACC genes (clones from genomic library) and mRNAs (as cDNA). The sequence of these 5'- and 3'-untranscribed and untranslated fragments of the gene are usually significantly different for different alleles so they may also be used as specific probes to follow expression of individual genes.
  • This example describes the cloning and DNA sequence of the entire gene encoding wheat (var. Hard Red Winter Tarn 107) acetyl-CoA carboxylase (ACCase).
  • ACCase acetyl-CoA carboxylase
  • transcripts had 5 '-end sequence 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 chromosome sets in hexaploid wheat or that each of the six alleles of the cytosolic ACCase gene has a readily distinguishable DNA sequence.
  • PCRTM components except Taq polymerase were incubated for 5 min. at 95°C. The reactions were initiated by the addition of the polymerase followed by 35 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.
  • dT-Anchor primer 5'-GCC jACTCGAGTCGACAAGCTTTTTTTTTTTrTTTTTTT-3' (SEQ DD NO:37); and a gene-specific primer, 5'-ACGCGTCGACTAGTA
  • Universal primer, 5'-GCGGACTCGAGTCGACAAGC-3' (SEQ ID NO:39) and another gene-specific primer, 5'-ACGCGTCGACCATCCCA 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 71 that were available.
  • Clone 71 was isolated from a 8gtl 1 cDNA library as described before using a fragment of cDNA 39 as probe (Example 4). The same dT-anchor primer and 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
  • cDNA clone 71 represents the transcription product of this gene (430-nucleotide identical sequence).
  • the sequence of clone 145 obtained by PCRTM to cover the remaining 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.
  • GenBank accession number U39321
  • GenBank accession number U39321
  • 3 '-end corresponds to the poly(A) attachment site of the 3 '-RACE clone 4. It was assumed that no additional introns are present at the very end of the gene.
  • Example 4 Comparison of the genomic sequence with the cDNA sequence in Example 4 revealed 29 introns. Intron location is conserved among all three known plant ACCase genes except for two introns not present in wheat but found in rape (Schulte et al, 1994), A. thaliana (Roesler et al, 1994) and soybean (Anderson et al, 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 (White et al, 1992).
  • the exon coding sequence is 98% identical to that of the cDNA sequence reported earlier. This is the same degree of identity as found previously for different transcripts of the cytosolic ACCase genes in hexaploid wheat (Example 4).
  • the 11 -amino acid sequence obtained previously for a CNBr-generated internal fragment of purified 220-kDa wheat germ ACCase (Gomicki and Haselkorn, 1993) differs from the sequence encoded by these cDNA and genomic clones at one position, but it is identical with the corresponding cDNA sequence of the plastid ACCase from maize (Egli et al, 1995), excluding one amino acid which could not be assigned unambiguously in the sequence.
  • genomic clones 153 and 23 Two additional genomic clones, 153 and 231, were also partially sequenced (FIG. 1).
  • the sequenced fragments include parts of the first two exons and the first intron.
  • cDNA corresponding exactly to genomic clone 153 is not available, the boundaries 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 open reading frame.
  • the sequence of the 5 '-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.
  • the first intron in this gene is much larger (additional upstream introns 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 ligation artifact).
  • a fragment of clone 232 that was sequenced is represented in the diagram shown in FIG. 1. It is 93% and 96% identical with clone 233 at the nucleotide and amino acid level, respectively.
  • FIG. 5 Shown in FIG. 5 is the 5' flanking sequence of the ACCase 1 gene (about 3 kb upstream of the translation initiation codon, of clone 71L (SEQ ID NO: 32).
  • the 5' flanking sequence of the ACCase 2 gene designated 153 (SEQ ID NO:33) is shown in FIG. 6.
  • sequences of the 5 '-leaders differ significantly 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 amino acids long and they end within a few nucleotides of the ACCase initiation codon.
  • a similar arrangement was found in the sequence of genomic clone 153.
  • the three 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 open reading frames at the amino acid level.
  • the gene, shown in FIG. 3 (SEQ DD NO:30), encodes a 2260-amino acid protein with a calculated molecular mass of 252 kDa (FIG.
  • 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 reported recently (Egli et al, 1995), does encode a chloroplast transit peptide.
  • Biotin carboxylase subunit of ACCase 4 Biotin carboxylase-biotin carboxyl carrier subunit of ACCase. 5 Biotin carboxylase-biotin carboxyl carrier subunit (a) of propionyl-CoA carboxylase . 6 Pyruvate carboxylase. 7 Biotin carboxylase-biotin carboxyl carrier subunit of methylcrotonyl-CoA carboxylase.
  • the plant prokaryotic-type plastid 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.
  • 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 determine the steady-state level of ACC mRNA in each case.
  • cDNA probes encoding conserved fragments of ACC may be used to measure total ACC mRNA level and gene specific probes to determine which gene is functioning in which tissue.
  • the steady-state level of ACC protein (by western analysis using ACC-specific antibodies and/or using labeled streptavidin to detect biotinylated peptides) and its enzymatic activity may be measured to identify the most important stages of synthesis and reveal mechanisms involved in its regulation.
  • One such study evaluates ACC expression in fast growing leaves (from seedlings at different age to mature plants), in the presence and in the absence of light.
  • Observation of four viable spores from FAS 3 tetrads containing the wheat ACC gene may confirm that the wheat gene functions in yeast, and extracts of the complemented FAS3 mutant may be prepared and assayed for ACC activity. These assays may indicate the range of herbicide sensitivity, and in these studies, haloxyfop acid and clethodim may be used as well as other related herbicide compounds.
  • the present invention may be used in the isolation of herbicide-resistant mutants. 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 are well-known to those of skill in the art. Resistant mutants, 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.
  • chimeric molecules may be constructed containing half, quarter and eighth fragments, etc. from each mutant, then checked by transformation and tetrad analysis whether a particular chimera confers resistance or not.
  • mutant DNA may be prepared, end- labeled, and annealed with the corresponding wild-type fragments in excess, so that all mutant fragments are in heterozygous molecules.
  • SI or mung bean nuclease digestion cuts the heterozygous molecules at the position of the mismatched base pair.
  • Electrophoresis and autoradiography is used to locate the position of the mismatch within a few tens of base pairs. Then oligo-primed sequencing of the mutant DNA is 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.
  • the corresponding parts of several dicot ACC genes may be sequenced (using the physical maps and partial sequences as guides) to determine their structures in the corresponding region, in the expectation that they are now herbicide resistant.
  • Wheat ACC cDNA probes were used to detect DNA encoding canola ACC. 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.
  • One of the other genomic clones (6.5 kb in size) contains the 5' half of the canola gene, and additional screening of the genomic library may produce other clones which contain the promoter and other potential regulatory elements.
  • One aspect of the present invention is the isolation of Anacystis mutants in which the BC gene is interrupted by an antibiotic resistance cassette.
  • Such techniques are well-known to those of skill in the art (Golden et al, 1987). Briefly, the method involves replacing the cyanobacterial ACC with wheat ACC, so it is not absolutely necessary to be able to maintain the mutants without ACC.
  • the wheat ACC clone may be introduced first and then the endogenous gene can be inactivated without loss of viability.
  • gene replacement may be used to study wheat ACC activity and herbicide inhibition in yeast. Mutants may be selected which overcome the normal sensitivity to herbicides such as haloxyfop. This will yield a variant(s) of wheat ACC that are tolerant/resistant to the herbicides.
  • the mutated gene (cDNA) present on the plasmid can be recovered and analyzed further to define the sites that confer herbicide resistance.
  • 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.
  • Characterization of the site(s) conferring herbicide resistance generally involves assaying extracts of the complemented A C1 mutant for ACCase activity. Both spontaneous mutation and chemical mutagenesis with DES or EMS, may be used to obtain resistant mutants, 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 mutants, the mutated site that confers resistance may be analyzed.
  • chimeric molecules may be constructed which containing half, quarter and eighth fragments, 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 DNA, end-labeling, and annealing 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 mismatch 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 into the wild-type sequence by oligo-directed mutagenesis to confirm that it is sufficient to confer the resistant phenotype.
  • phage display technique Another method for the selection of wheat ACCase mutants tolerant or resistant to different herbicides involves the phage display technique. Briefly, in the phage display technique, foreign peptides can be expressed as fusions to a capsid protein of filamentous phage. Generally short (6 to 18 amino acids), variable amino acid sequences are displayed on the surface of a bacteriophage virion (a population of phage clones makes an epitope library).
  • filamentous bacteriophages have also 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 et al, 1991).
  • the foreign inserts were spliced into the major coat protein pVID of the Ml 3 phagemid.
  • a complementary helper phage supplying wild-type pVID has to be cotransferred together with the phagemid.
  • Such "fusion phages” retained full infectivity and the fused proteins were recognized by monoclonal antibodies.
  • 300 amino acids in size may be constructed without "panning" for phage purification.
  • the mechanism of purifying phages by panning involves reaction with biotinylated monoclonal antibodies, then the complexes are diluted, immobilized on streptavidin-coated plates, washed extensively and eluted. Generally, a few rounds of panning are recommended.
  • 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 capturing those phages which display amino acid fragments involved in herbicide binding.
  • Such herbicide affinity resins may also be employed.
  • the phages bearing those peptides may be subjected to random mutagenesis, again using phage display and binding to the appropriate support to select the interesting variants. Sequence analysis then is used to identify the critical residues of the protein required for binding. 5.12 EXAMPLE 12 -- Preparation of ACC-specific antibodies
  • Another aspect of the present invention is the preparation of antibodies reactive against plant ACC for use in immunoprecipitation, affinity chromatography, and immunoelectron 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
  • the procedure encompasses 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 purified by precipitating lipoproteins from the serum with 0.25% sodium dextran sulfate and 80 mM CaCl 2 .
  • 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.
  • 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 appropriate in situ assays. Constructs may be made which are useful in the preparation of transgenic plants.
  • model substrates containing different length N-terminal fragments of ACC may be prepared by their expression (and labeling) in E. coli or by in 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 minimum biotinylation substrate will be defined in parallel) or native ACC epitope(s) for which antibodies will be generated as described above. Adding an antibody tag at the C-terminus will also be very helpful.
  • These substrates will be purified by affinity chromatography (with antibodies or streptavidin) and used for in vitro assays.
  • model substrates consisting of a transit peptide (or any other chloroplast targeting signals) to facilitate import into chloroplasts, fused to different ACC domains that are potential targets for modification, may be used.
  • Modified polypeptides from cytoplasmic and/or chloroplast fractions will be analyzed for modification. For example, protein phosphorylation (with P) can be followed by immunoprecipitation or by PAGE. Antibodies to individual domains of ACC may then be employed. The same experimental 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 S-streptavidin for detection or by PAGE when radioactive biotin is used as a substrate.
  • 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 corresponding proteins in E. coli. This is most readily accomplished using the T7 expression system.
  • 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, followed by a multiple cloning site into which the desired gene can be inserted (Ashton et al, 1994).
  • the strain 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.
  • the inducer 1PTG When the inducer 1PTG is added, the polymerase is made in excess and the plasmid-borne gene of interest is transcribed abundantly from the late T7 promoter.
  • 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 for purposes of raising antisera.
  • other expression systems are also available (Ausubel et al, 1989).
  • Haslacher et al J. Biol. Chem., 268: 10946-10952, 1993.
  • AAAGTGATGA TTTTGAACTA ACGGTGCGTA AAGCTGTTGG TGTGAATAAT AGTGTTGTGC 900 _
  • TTACCCGCTC CAACCCCTGC GGCAGCACCG CCTGCTGGAC CTCTGGGTGG CGAGAAGTTC 240
  • AAATGTCATC ACTCCCAATA CAGGGCCAAG AATCCAAACG CTCAGGTTAA CACCAGTCAT 3000

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Abstract

Cette invention concerne des polynucléotides isolés et purifiés qui codent pour des polypeptides de plante supérieure et de cyanobactérie participant à la carboxylation d'acétyl-CoA. Elle concerne également des polypeptides de plante supérieure et de cyanobactérie isolés catalysant la carboxylation d'acétyl-CoA. Des procédés visant à modifier une carboxylation d'acétyl-CoA, à accroître la résistance de plantes aux herbicides et à recenser des variants d'acétyl-CoA, résistants aux herbicides, sont également présentés.
EP96912726A 1995-04-14 1996-04-12 COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION Ceased EP0820514A2 (fr)

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US422560 1995-04-14
US08/422,560 US5910626A (en) 1992-10-02 1995-04-14 Acetyl-CoA carboxylase compositions and methods of use
US08/468,793 US6177267B1 (en) 1992-10-02 1995-06-06 Acetyl-CoA carboxylase from wheat
US468793 1995-06-06
US61154696A 1996-03-05 1996-03-05
US08/611,107 US5801233A (en) 1992-10-02 1996-03-05 Nucleic acid compositions encoding acetyl-coa carboxylase and uses therefor
US611546 1996-03-05
PCT/US1996/005095 WO1996032484A2 (fr) 1995-04-14 1996-04-12 COMPOSITIONS A BASE D'ACETYL-CoA CARBOXYLASE ET PROCEDES D'UTILISATION

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