WO2000000585A9 - MAIZE CYTOCHROME P450 MONOOXYGENASE cDNA (CYP71C3v2) - Google Patents

Abstract

The present invention relates to an isolated and purified polynucleotide that encode maize P450 cytochrome CYP71C3v2, expression vectors containing those polynucleotides, host cells transformed with those expression vectors, and a process of conferring herbicide resistance to organisms that are transformed with these polynucleotides.

Description

MAIZE CYTOCHROME P450 MONOOXYGENASE cDNA (CYP71C3v2)

Technical Field of the Invention

The present invention relates to maize P450 cytochromes. More specifically, this invention relates to polynucleotide sequences that encode a maize P450 cytochrome and a method for using these polynucleotide sequences to confer herbicide resistance .

Background of the Invention

In conjunction with NADPH-dependent P450 reαuctases, cytochrome P450 monooxygenases (P450s) reductively cleave molecular dioxygen to produce functionalized organic substrates. These b-type cytochromes range in size from 45 to 65 kD (average 55kD) and contain a protoporphyrin IX heme prosthetic group covalently attached to the cysteine of a highly conserved F--G-R- C-G motif found near the C-terminus. Except for these few conserved amino acids, the structural variations between P450 proteins are extensive. In the case of the highly divergent bacterial P450_BM3 (CYP102) and P450_TERP (CYP108) proteins whose crystal structures have been defined, only 7% of the ammo acids are identical when the two three-dimensional structures are superimposed. Schuler, (1996) Cri t . Rev. Plan t Sci . 15:235-284. In the case of many other P450s, primary sequence alignments of P450s are so low (<20% identity) that they are often considered to be equivalent to random alignments of similar length proteins. Id. In only a few cases are primary sequence alignments of P450s so high (>97% identity) that the corresponding P450s are assumed to represent allelic variants of a single locus. Id. Structural studies have indicated that the functional significance of these ammo acid variations differs: very similar P450 sequences can be associated with radically different substrate specificities and highly divergent P450s may have similar substrate reactivities. Id. For example, mutation of three ammo acids in the mouse CYP2A5 protein abolishes coumann- 7-hydroxylase activity and enhances testosterone-15 -hydroxylase activity; mutation of one of these three ammo acids is enough to alter catalytic specificity. Id. In a converse example, the highly divergent mouse CYP2A5 and black swallowtail CYP6B1 proteins (26% ammo acid identity) metabolize the same toxic plant furanocoumarm, xanthotox . Id.

In plants, P450s are involved in the biosyntnesis of lignms, flavonoids/anthocyanms, phytoalexms, alkaloids and a variety of other plant secondary compounds as well as the detoxification of herbicides (See: Bolwell et al . , (1994) Phytochem . 37:1491-1506; Durst and O'Keefe, (1995) Drug Metab . Drug In teractions 12:171-187; Schuler, (1996) Cπ t . Rev. Plant Sci . 15:235-284). For a variety of herbicides including the sulfonylurea herbicide triasulfuron, resistance is mediated by the conversion of a herbicide to a hydroxylated, inactive product (Frear et al., (1991) Pest . Biochem . Physiol . 41:274-287; Moreland et al . , (1993) Pest. Biochem . Physiol . 47:206-214; Thalacker et al., (1994) Pest . Biochem . Physiol . 49:209-223; Persans and Schuler, (1995) Plant Physiol . 109:1483-1490) which is subsequently conjugated to carbohydrate moieties m the plant cell wall (Kreuz et al., (1996) Plant Physiol . 111:349-353).

The range of herbicides which are metabolized suggests that several different microsomal P450s are responsible for the various herbicide detoxification reactions occurring within a plant species. At present, it is unclear whether the P450s responsible for herbicide modifications represent biosynthetic P450s that are capable of hydroxylating/demethylating particular herbicides in addition to their endogenous substrates ("moonlighting enzymes") or detoxicative P450s that metabolize only herbicides ("dedicated enzymes").

Many metabolic studies have now indicated that select subsets of P450 isozymes capable of metabolizing particular herbicides are induced in response to compounds as divergent as naphthalic anhydride (a plant safener) , ethanol, manganese ions and the herbicides themselves. In wheat microsomes, triasulfuron hydroxylase activity is induced 6- to 19-fold by naphthalic anhydride, ethanol or phenobarbital and 26-fold by naphthalic anhydride in combination with ethanol (Frear et al., (1991) Pest . Biochem . Physiol . 41:274-287; Thalacker et al . , (1994) Pest . Biochem . Physiol . 49:209-223). In maize microsomes, this activity is induced 10- to 33-fold by naphthalic anhydride and synergistically up to 88-fold by naphthalic anhydride in combination with triasulfuron (Moreland et al . , (1993) Pest . Biochem . Physiol . 47:206-214.; Persans and Schuler, (1995) Plant Physiol . 109:1483-1490).

A number of full-length cDNAs for plant P450s have been cloned. For example, the CYP71A1 sequence encoding p-chloro-N- methylaniline demethylase from avocado (Bozak et al., (1990) Proc . Na tl . Acad. Sci . USA 87:3904-3918; Bozak et al . , (1992) Plant Physiol . 100:1976-1981), multiple CYP73A sequences encoding trans-cinnamic acid hydroxylases ( t-CAH) (Fahrendorf and Dixon, (1993) Arch . Biochem . Biophys . 305:509-515; Mizutani et al., (1993) Biochem . Biophys . Res . Comm . 190:875-880; Teutsch et al., (1993) Proc. Na tl . Acad. Sci . USA 90:4102-4106; Frank et al . , (1996) Plant Physiol. 110:1035-1046), a CYP75A1 sequence encoding flavonoid 3', 5 ' -hydroxylase from petunia (Holton et al., (1993) Nature 366:276-279), a CYP79 sequence encoding tyrosine Λ⁷- hydroxylase from sorghum (Koch et al., (1995) Arch. Biochem. Biophys. 323:177-186), a CYP80 sequence encoding berbamunine synthase from barberry (Kraus and Kutchan, (1995) Proc. Natl. Acad. Sci. USA 92:2071-2075) , a CYP90 sequence encoding a C23- cathasterone hydroxulase from Arabidopsis (Szekeres et al.,

(1996) Cell 85:171-182), a CYP83 sequence encoding ferulate-5- hydroxylase from Arabidopsis (Meyer et al., (1996) Proc. Natl. Acad. Sci. USA, 93:6869-6874), several CYP74A sequences encoding allene oxide synthases (Song et al., (1993) Proc. Natl. Acad. Sci. USA 90:8519-8523; Pan et al., (1995) J. Biol. Chem. 270:8487-8494; Laudert et al., (1996) Plant. Mol. Bio. 31:323- 335) and a series of CYP71C1-C4 sequences encoding DIMB0A biosynthetic enzymes from maize (Frey et al., (1995) Mol. Gen. Genet. 246:100-109; Frey et al . , (1997) Science 277:696-699). Some of the substrate reactivities for these isozymes have been defined by heterologous expression in yeast (avocado CYP71A1, Bozak et al., (1992) Plant Physiol. 100:1976-1981; alfalfa CYP73A3, Fahrendorf and Dixon, (1993) Arch. Biochem. Biophys. 305:509-515; Jerusalem artichoke CYP73A1, Urban et al., (1994) Eur. J. Biochem. 222:843-850; petunia CYP75A1 and CYP75A3, Holton et al., (1993) Nature 366:276-279; maize CYP71C1-C4, Frey et al . ,

(1997) Science 277:696-699), in baculovirus (barberry CYP80, Kraus and Kutchan, (1995) Proc. Natl. Acad. Sci. USA 92:2071- 2075) and in planta (Arabidopsis CYP83, Meyer et al . , (1996) Proc. Natl. Acad. Sci. USA 93:6869-6874; Arabidopsis CYP90, Szekeres et al., (1996) Cell 85:171-182). Additional full length cDNA sequences for two P450s from maize (CYP78, Larkin, (1994) Plant Mol. Biol. 25:343-353; CYP88, inkler and Helentjaris, (1995) Plant Cell 7:1307-1317) are now available (http: //drnelson. utmem.edu. nelsonhomepage.html; Schuler, (1996) Cri t . Rev. Plan t Sci . 15:235-284). It is suggested that maize CYP88 mediates the conversion of the gibberellic acid precursor GA₁₂ to GA₅₃ ( inkler and Helentjaris, (1995) Plant Cell 7:1307- 1317) but the limited identity of CYP78 with other plant P450s provides few clues as to its function. Information on the induction patterns for these and other plant P450 transcripts is primarily limited to t-CAH transcripts which have been shown to be induced several-fold by a variety of chemicals (e.g., naphthalic anhydride, aminopyrine, MnCl₂) and wounding in Jerusalem artichoke tuber tissue (Batard et al . , (1997) Plan t Physiol . 113:951-959), by wounding in pea stem sections (Frank et al., (1996) Plant . Physiol . 110:1035-1046), by light and wounding in Arabidopsis leaves (Bell-Lelong et al., (1997) Plant Physiol . 113:729-738; Mizutani et al., (1997) Plant Physiol . 113:755-763), and by fungal elicitors in alfalfa suspension cell cultures (Fahrendorf and Dixon, (1993) Arch . Biochem . Biophys . 305:509- 515) .

Much remains to be learned about the specificity of P450 reactions in plants and their mechanisms of regulation. Schuler, (1996) Crit. Pev. Plan t Sci . 15:235-284. Although it was originally thought that plant P450s were significantly more regiospecific than their mammalian and bacterial counterparts, it has become increasingly apparent that some endogenous plant P450s are capable of metabolizing a broader range of substrates. Id. As mentioned previously, it is unclear whether some of the P450s responsible for herbicide detoxification represent biosynthetic P450s that are capable of hydroxylating particular herbicides in addition to endogenous substrates ("moonlighting enzymes") or detoxificative P450s that only metabolize herbicides ("dedicated enzymes") . Id. Recent studies have suggested that the same P450 mediates hydroxylation of endogenous lauric acid and the exogenous herbicide diclofop and that trans-cmna ic acid hydroxylase can mediate hydroxylation of chlortoluron at very low efficiency. Id.

In the growing number of plant P450 sequences that have been cloned and characterized, none have been identified as efficient herbicide-detoxifying P450s. Some plant P450s, such as the

Jerusalem artichoke CYP73A1 (t-CAH), are capable of metabolizing the phenylurea herbicide chlortoluron at extremely low efficiency when expressed in yeast (Pierrel et al., (1994) Eur. J. Biochem . 224:835-844) .

A fusion construct bearing a chloroplast transit sequence and a bacterial P450 (CYP105A1) isolated from Streptomyces gπseolus has been engineered into the tobacco (Ni cotiana tabacum) nuclear genome from which it was expressed and targeted to chloroplasts. See U.S. Patent 5,212,296 As a result of effective coupling with the endogenous chloroplast ferrodoxm, this heterologous catabolic P450 mediates the N-dealkylation of the sulfonylurea R7402 proherbicide, thereby producing the active form of this herbicide (within the chloroplast) . Id. This patent does not disclose or suggest targeting of plant P450 cytochromes to the endogenous reticulum (e.r.) for coupling with e.r. -localized NADPH-dependent P450 reductase to confer herbicide resistance to plants.

In another study, a fusion construct containing rat CYP1A1 protein fused with the yeast NADPH-dependent P450 reductase was engineered into the tobacco nuclear genome (Shiota et al., (1994) Plan t Physiol . 106:17-23). Targeting of this heterologous P450 to microsomal membranes confers resistance to 50 μM chlortoluron (a phenyl urea herbicide) on transgenic plants. Id. To date, other prospective herbicide-detoxifying P450s have not been characterized beyond the level of metabolic activities and spectral binding analyses.

Therefore, a need exists in the art for the identification and sequencing of an efficient herbicide-detoxifying P450 and a chemically-activated (safener-inducible) promoter.

Summary of the Invention

The present invention relates to an isolated and purified polynucleotide that consists essentially of a nucleotide sequence that is a) the sequence of SEQ ID NO: 1; b) sequences that are complementary to the sequence of (a) ; c) sequences that, on expression, encode a polypeptide encoded by the sequence of (a) . The polynucleotide is a DNA molecule and has the nucleotide sequence set forth in SEQ ID NO:l. Additionally, the polynucleotide may be an RNA molecule.

The present invention also relates to an expression vector that contains the polynucleotide described above that has the nucleotide sequence set forth in SEQ ID NO:l. The expression vector contains a promoter that is operatively linked to the polynucleotide .

The present invention also relates to an oligonucleotide of from about 15 to about 50 nucleotides that contain a nucleotide sequence of at least 15 nucleotides that are identical or complementary to the contiguous sequence of the polynucleotide described above. The present invention also relates to a host cell transformed with the expression vector described above. The transformed host cell may be a yeast, plant or bacterial cell.

The present invention also relates to an isolated and purified polypeptide of about 534 ammo acids that has the ammo acid sequence of SEQ ID NO: 2.

The present invention also relates to transgenic plants that contain the polynucleotide having SEQ ID N0:1 and which are resistant to one or more herbicides.

The present invention also relates to a method for controlling undesired vegetation m a location containing agronomically useful plants that have been transformed with an isolated polynucleotide have SEQ ID NO: 1 and are resistant to one or more herbicides. The method involves applying to the location an effective amount of one or more herbicides.

The present invention also relates to a method for identifying compounds having herbicidal activity. The method involves transforming an organism with an isolated polynucleotide having SEQ ID NO:l, treating the transformed organism with one or more compounds, and finally, identifying and selecting those compounds which exhibit herbicidal activity.

Brief Description of the Figures

Figure 1 shows the nucleotide and derived ammo acid sequence for CYP71C3v2 cDNA. The underlined bold letters (F—G- R-C-G) designate highly conserved ammo acid sequences found in most P450 heme binding domains. The start and stop codons as well as the putative polyadenylation signals are simply underlined. The lower case sequence representing six base pairs of 5' nontranslated sequence and the first coding nucleotide was derived from a RACE-amplified DNA clone primed with the 3' Sal2 primer. The locations of the two CYP71C3v2 introns are designated by triangles located above the CYP71C3v2 nucleotide sequence. Multiple polyadenylation sites detected in the CYP71C3v2 cDNA clones are designated with arrows. The sequences of CYP71C3v2 introns 1 and 2 are shown at the bottom of the figure. The slashes at the beginning and end of these sequences designate the 5' and 3' splice sites for each intron. Intron sequences containing adenosine and thymidine (AT) -rich tracts (AU in mRNA) are underlined.

Figure 2 shows the derived amino acid sequences for the NA PCR 1-5 clones generated by reverse transcription-PCR amplification of naphthalic anhydride-treated 6.5-day-old seedling mRNAs . The asterisks designate highly conserved amino acids in the P450 heme binding domain. Amino acids represented in the 5' PN-3 PCR primer are above the sequence.

Figure 3 shows the structure of the CYP71C3v2 cDNA. The open box designates the coding sequence and the thick lines designate the 5' and 3' nontranslated regions. The positions and lengths of the two introns present in the CYP71C3v2 gene are depicted above the cDNA. The second of these introns is retained in the CYP71C3v2a cDNA. Restriction sites and nucleotide positions are designated above the cDNA diagram. Amino acid positions and conserved P450 motifs are designated below the cDNA diagram.

Figure 4 shows the alignment of the CYP71C3v2 amino acid sequence with the maize CYP71C3vl, CYP71C1, CYP71C2, CYP71C4, CYP78, and CYP88 sequences described by Frey et al . , (1995) Mol . Gen . Genet . 246:100-109, Larkin, (1994) Plan t Mol . Biol . 25:343- 353, inkler and Helentjaris (1995) Plan t Cell 7:1307-1317. The asterisks designate highly conserved amino acids in the P450 heme binding domain. The alignment of the amino acid sequences in this Figure was generated using the Clustal sequence alignment program.

Figure 5 shows the DNA sequences for the following oligonucleotide primers that are used in the present invention: 5' PN-3, 3' PC-1, Notl oligo (dT) , 3' pYES SEQ, 5' ENG, INT PR1, INT PR2, INT PR3, INT PR , INT PR5, INT PR6, 3' ENG, 3' Sal2, tCAH 5 ' and tCAH 3 ' .

Figure 6 shows 1 ug poly (A) + mRNA isolated from control (C) , naphthalic anhydride-treated (NA) or naphthalic anhydride plus triasulfuron-treated (NA/T) 2.5-day or 6.5-day seedlings electrophoresed on 1.2% agarose-formaldehyde gels, transferred to Hybond-N nylon membrane and probed with the first 580 bp of CYP71C3v2 cDNA and subsequently with the constitutive maize 1055 cDNA (Sachs, M. , (1991) Molecular Response to Anoxic Stress in Maize. In Plant Life Under Oxygen Deprivation. M.B. Jackson, D.D. Davies, and H. Lambers, eds. (Netherlands, Academic Publishing), pp. 129-139). CYP71C3v2 mRNA levels were quantified by phosphorimager analysis and normalized relative to the 1055 mRNA level. The level of induction for each treatment is reported below each lane relative to the level of CYP71C3v2 mRNA in control seedlings of the same age.

Figure 7 shows 1 ug poly (A) + mRNA isolated from control (C) , naphthalic anhydride-treated (NA) or naphthalic anhydride plus triasulfuron-treated (NA/T) 2.5-day or 6.5-day seedlings electrophoresed on 1.2% agarose-formaldehyde gels, Northern blotted and probed with the CYP73A7 RT-PCR product and subsequently with the constitutive maize 1055 cDNA (Sachs, M., (1991) Molecular Response to Anoxic Stress in Maize. In Plant Life Under Oxygen Deprivation. M.B. Jackson, D.D. Davies, and H. Lambers, eds. (Netherlands, Academic Publishing), pp. 129-139). CYP73A (t-CAH) mRNA levels were quantified by phosphorimager analysis and normalized relative to the 1055 mRNA level. The level of induction for each treatment is reported below each lane relative to the level of CYP73A mRNA in control seedlings of the same age.

Figure 8 shows maize B73 genomic DNA restriction digested with BamHI (lane 3), EcoRI (lane 4), Hindlll (lane 5), Xhol (lane 6) and Xbal (lane 7) was electrophoresed on a 0.8% agarose gel, blotted to Hybond-N nylon membrane and hybridized with a ³²P- labeled CYP71C3v2 cDNA 3' end probe (bp 1350-1800) at high stringency. The sizes of the molecular weight standards in lane 1 are shown at the left.

Figure 9 shows CYP7lC3v2 genomic DNA and cDNA PCR amplified with the INT PR primer sets depicted in the diagram at the bottom of the figure. The bands PCR amplified from genomic DNA with the INT PR 1+6 and 2+5 primer sets were cloned and sequenced. These PCR products were found to contain CYP71C3v2 introns 1 and 2, respectively.

Figure 10 shows the expression of CYP71C3v2 mRNA in yeast. The CYP71C3v2 cDNA was cloned into the pYES (Invitrogen) yeast expression vector. mRNA was isolated from W(R) yeast containing the pYES plasmid only and from W(R), AT11, and AT21 yeast containing the CYP71C3v2 cDNA in the pYES vector. Approximately 1 ug of each mRNA was Northern blotted and probed with the CYP71C3v2 CDNA.

Figure 11 shows the carbon monoxide (CO) difference spectra of yeast microsomes. Microsomes were isolated from 8.5 hour galactose-induced W(R), AT11, and WAT21 yeast containing the pYES plasmid with and without the CYP71C3v2 CDNA.

Figure 12 shows the complementation of yeast with the CYP71C3v2 cDNA. Figure 12(A) shows W(R) yeast (containing pYES ± CYP71C3v2) grown to saturation (O.D. 2.0-2.2) at 30°C, diluted to 1 x 10⁴ cells/ml, plated (100 μl ) on YGIM plates ± 60 μM nerbicide and regrown on selection media for 6 days at 30°C. Figure 12(B) shows DBY2616 yeast (containing pYES ± CYP71C3v2) grown to saturation (O.D. 2.0-2.2) at 30°C, diluted to 1 x 10⁴ cells/ml, plated (100 μl) on YGIM plates ± 40 μM herbicide and regrown on selection media for 6 days at 30°C. Figure 12(c) shows DBY2616 yeast (cured of the pYES and CYP71C3v2/pYES plasmids by 5-FOA treatment) grown to saturation (O.D. 2.0-2.2) at 30°C, diluted to 1 x 10" cells/ml, plated (100 μl) on YGIM plates ± 40 μM herbicide and regrown on selection media for 6 days at 30°C. Figure 12(D) shows W(R) yeast (cured of the pYES and CYP71C3v2/pYES plasmids by 5-FOA treatment) grown to saturation (O.D. 2.0-2.2) at 30°C, diluted to 1 x 10⁴ cells/ml, plated (100 μl) on YGIM plates ± 60 μM herbicide and regrown on selection media for 6 days at 30°C.

Figure 13 shows the results when 1 μg poly (A) + mRNA isolated from nutrient broth control (Nut Br C) , Erwinia stuartii-treated (E. stuartii ) or Acidovorax avenae-treated (A. avenae) 6.5-day- old seedling shoots were electrophoresed on 1.2% agarose- formaldehyde gels, transferred to Hybond-N nylon membrane and probed at high stringency with the 5' terminal sequence (base pairs 1-580 relative to the first coding nucleotide) of the CYP71C3v2 cDNA and subsequently with the constitutive maize 1055 cDNA (Sachs, 1991 "Molecular Response to Anoxic Stress In Maize. In Plant Life Under Oxygen Deprivation", MB Jackson, D.D. Davies, and H. Lambers, eds. (Netherlands Academic Publishing) pp. 129- 139) . CYP71C3v2 mRNA levels were quantified by phosphorimager analysis and normalized relative to the 1055 mRNA levels. The relative induction levels for CYP71C3v2 mRNA after each treatment compared to the CYP71C3v2 mRNA in control seedlings of the same age are shown in each lane.

Figure 14 shows type I binding spectra demonstrating that CYP71C3v2 protein expressed in yeast microsomes of the (R) strain binds 50 μM triasulfuron whereas the endogenous protein in yeast microsomes of the (R) strain do not bind to this herbicide.

Figure 15 shows that transgenic Arabidopsis plants carrying 35S:CYP71C3v2 cDNA construct delivered with the pKYLX vector (as described in Maiti et al., PNAS (1993) 90:6110-6114) are capable of growing and flowering in the presence of 0.167 μM triasulfuron when the CYP71C3v2 transcript is expressed (see the arrows) .

Sequence Listing

The present application also contains a sequence listing that contains 19 sequences. The sequence listing contains nucleotide sequences and amino acid sequences. For the nucleotide sequences, the base pairs are represented by the following base codes: Svmbol Meaning

A A; adenine

C C; cytosine

G G; guanine

T T; thymine

U U; uracil

M A or C

R A or G

W A or T/U

S C or G

Symbol Meaning

Y C or T/U K G or T/U

V A or C or G; not T/U H A or C or T/U; not G D A or G or T/U; not C B C or G or T/U; not A N (A or C or G or T/U)

The amino acids shown in the application are in the L-form and are represented by the following amino acid-three letter abbreviations :

Abbreviation Amino acid name

Ala L-Alanine

Arg L-Arginine

Asn L-Asparagine

Asp L-Aspartic Acid

Asx L-Aspartic Acid or Asparagine

Cys L-Cysteine

Glu L-Glutamic Acid

Gin L-Glutamine

Glx L-Glutamine or Glutamic Acid

Gly L-Glycine

His L-Histidine

He L-Isoleucine

Leu L-Leucine

Lys L-Lysine

Met L-Methionine

Phe L-Phenylalanine

Pro L-Proline

Ser L-Serine

Thr L-Threonine

Trp L-Tryptophan

Tyr L-Tyrosine

Val L-Valine

Xaa L-Unknown or other Detailed Description of the Invention

The present invention provides isolated and purified polynucleotides that encode the maize P450 cytochrome CYP71C3v2, expression vectors containing those polynucleotides, host cells transformed with those expression vectors, and a process of conferring herbicide resistance to organisms such as yeast, plants and bacteria, that are transformed with these polynucleotides .

In one aspect, the present invention provides an isolated and purified polynucleotide that encodes the maize cytochrome P450 designated CYP71C3v2. A polynucleotide of the present invention that encodes CYP71C3v2 is an isolated and purified polynucleotide that comprises (a) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:l, (b) sequences that are complementary to the sequence of (a) , and sequences that, when expressed, encode a polypeptide of (a) .

In a second aspect, the present invention also contemplates naturally occurring allelic variations and mutations of the DNA sequences set forth above so long as those variations and mutations code, on expression, for an CYP71C3v2 of this invention as set forth hereinafter.

In a third aspect, the present invention also includes DNA sequences which hybridize under stringent hybridization conditions to the DNA sequences set forth above. Stringent hybridization conditions are well known in the art and define a degree of sequence identity greater than 70% to 80%.

As set forth above, SEQ ID NO:l, is a full length cDNA clone of CYP71C3v2. As is well known in the art, because of the degeneracy of the genetic code, there are numerous other DNA and RNA molecules that can code for the same polypeptide as those encoded by SEQ ID N0:1. The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode for the polypeptide encoded by SEQ ID NO:l and its allelic variants. Having identified the amino acid sequence of CYP71C3v2, and with knowledge of all triplet codons for each particular amino acid, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid are within the scope of this invention. Also within the scope of this invention are those nucleic acid sequences which code for natural and synthetic allelic variants of the CYP71C3v2 protein sequence.

A simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure of the encoded polypeptide. By way of example, it can be seen from SEQ ID NO:l (See Figure 1) that a CCC codon for proline exists at nucleotide positions (See e.g., nucleotide positions 103-105). It can also been seen from that same sequence, however that proline can be encoded by a CCG codon (See e.g., nucleotide positions 166-168) and the CCT codon (See e.g., nucleotide positions 175-177) . Substitution of the latter CCG or CCT codons for proline, or vice versa, does not substantially alter the DNA sequence of SEQ ID NO:l and results in expression of the same polypeptide. In a similar manner, substitutions of codons for other amino acid residues can be made in a like manner without departing from the true scope of the present invention.

The present invention also contemplates oligonucleotides from about 15 to about 50 nucleotides in length, which serve as primers and hybridization probes for the screening of DNA libraries and the identification of DNA or RNA molecules that encode CYP71C3v2 and related sequences. Such primers and probes are characterized in that they will hybridize to polynucleotide sequences encoding CYP71C3v2 or related cytochrome P450 proteins. An oligonucleotide probe or primer contains a nucleotide sequence of at least 15 nucleotides that is identical to, nearly identical to or complementary to a contiguous sequence of a CYP71C3v2 polynucleotide of the present invention. Thus, where an oligonucleotide probe is 25 nucleotides in length, at least 15 of those nucleotides are identical or complementary to a sequence of the CYP71C3v2 polynucleotide of the present invention.

A detailed description of the preparation, isolation and purification of polynucleotides encoding CYP71C3v2 is set forth below.

As set forth in detail hereinafter in the Examples, the strategy employed to isolate the CYP71C3v2 involved using reverse transcription coupled with polymerase chain reaction amplification (RT-PCR). More specifically, poly (A) ^τ mRNA prepared from 6.5-day-old naphthalic anhydride-treated maize seedlings was used for reverse transcription coupled with polymerase chain reaction amplification (RT-PCR) cloning. mRNA was reverse transcribed and PCR amplified using a 3' oligo (dT) primer (3' PC-1, shown in Figure 5 and SEQ ID NO: 6) complementary to the poly (A) tract of mRNAs and a 1024-fold degenerate primer (5' PN- 3, shown in Figure 5 and SEQ ID NO: 5) encoding part of a conserved amino acid sequence (EEF-PERF) located approximately 30 amino acids upstream from the heme-binding cysteine. The resulting RT-PCR products were cloned using BamHI and EcoRI sites included in the 5' and 3' RT-PCR primers and 90 transformants with inserts in the 300-500 bp range were sequenced from their 5' end to identify clones containing the conserved F—G-R-C-G P450 heme-binding motif and those containing this set of amino acids were fully sequenced. Five distinct P450 clones (NA PCR 1-5) were identified within this group of 90 clones (see Figure 2) . Comparison of the degenerate 5' PCR primer regions in these clones and the poly (A) addition sites indicated that NA PCR 1 had been independently isolated seven times and the remaining clones (NA PCR 2-5) had been isolated once in this screening.

Upon further analysis, it was discovered that NA PCR 1 shared 98%, 65%, 60% and 46% amino acid identity with maize CYP71C3vl, CYP71C2, CYP71C1 and CYP71C4 sequences (hydroxylases in the DIMBOA biosynthetic pathway) (Frey et al., (1995) Mol . Gen . Genet . 246:100-109; Frey et al . , (1997) Science 277 : 696-699) , 35% amino acid identity with the maize CYP78 sequence Larkin, (1994) Plant Mol . Biol . 25:343-353), 21% amino acid identity with the maize CYP88 sequence (potential hydroxylase for GA₁₂) ( inkler and Helentjaris, (1995) Plant Cell 7 :1307-1317) , and 53% amino acid identity with the avocado CYP71A1 sequence (p-chloro-N- methylaniline demethylase) (Bozak et al . , (1990) Proc . Na tl . Acad. Sci . USA 87:3904-3908). NA PCR 2 has nucleotide and amino acid sequences identical to those defined for NA PCR 1 except for the presence of 12 alternate nucleotides and an EcoRI site at the 3' end of the clone; as a result of these differences, NA PCR 2 lacks seventeen C-terminal amino acids encoded in NA PCR 1. NA PCR 3 shares 56% amino acid identity with the tobacco CYP92A2 and CYP92A3 sequences (Czernic et al., (1996) Plant Mol . Bio . 31:255- 265), 45% amino acid identity with the maize CYP78 sequence (Larkin, (1994) Plant Mol . Biol . 25:343-353), 43% amino acid identity with the Thalaspi arvense CYP71B1 sequence (Udvardi et al., (1994) Plant Physiol . 105:755-756) and 42% amino acid identity with the maize CYP71C3vl sequence (Frey et al., (1995) Mol . Gen . Genet . 246:100-109). NA PCR 4 shares 43% amino acid identity with the Ca tharanthus roseus CYP72 sequence (Vetter et al., (1992) Plan t Physiol . 100:998-1007) and only 13-22% amino acid identity with other sequenced maize P450s. The short NA PCR 5 sequence, extending from the 5' PN-3 primer to an internal BamHI site, shares limited amino acid identity (32%) with the C. roseus CYP72 sequence.

Intercomparisons of these maize RT-PCR clones indicated that they were significantly different from one another: NA PCR 1 was 32% identical at the amino acid level to NA PCR 3, 14% identical to NA PCR 4 and only 6% identical to NA PCR 5; NA PCR 3 was 19% identical to NA PCR 4 and 5% identical to NA PCR 5; NA PCR 4 is 10% identical to NA PCR 5. Northern analysis of NA- and NA/T- treated poly (A) ⁺ mRNA with the ³²P-labeled NA PCR 1 probe suggested NA PCR 1 (and related) transcripts were induced 2.8- fold in 2.5-day-old NA-treated seedlings (relative to control seedlings) and 5.0-fold in 2.5-day-old NA/T-treated seedlings. This analysis further suggested that NA PCR 1 transcripts were induced to the same extent (2.0-fold) in 6.5-day-old NA-and NA/T- treated seedlings.

In a second cloning, control mRNA from 6.5-day old etiolated seedlings was RT-PCR amplified using the t-CAH 5' primer (See Figure 5 and SEQ ID NO: 18) encoding amino acids 320-326 that occur upstream of the conserved heme-binding region and the t-CAH 3' primer (See Figure 5 and SEQ ID NO: 19) complementary to nucleotides encoding amino acids 463-469 that occur downstream of the conserved heme-binding region. Cloning of the individual RT- PCR products and subsequent DNA sequence analysis identified several clones that were identical to the published maize CYP13A1 sequence encoding one t-CAH isoform and that one clone was identical to the maize CYP73A6 sequence encoding another t-CAH isoform (Potter et al., (1995) Drug Metabol . Drug Interact . , 12:317-327) .

Full length cDNAs corresponding to NA PCR 1 were isolated from a cDNA library constructed with 2.5-day-old NA/T-treated seedling mRNA in the pYES yeast expression vector (Invitrogen) . Of 34 positives detected with the ³²P-labeled NA PCR 1 probe at high stringency, six clones potentially representing NA PCR 1 or NA PCR 2 were 1.6 kb or larger. Sequencing of these indicated that five were identical derivatives of the same coding and 3' nontranslated sequence, designated CYP71C3v2. The sole difference between these five sequences occurred in the 3' nontranslated region where three alternate polyadenylation sites were used to generate individual transcripts. The remaining clone, designated CYP71C3v2a , retained a 124 nucleotide intron between amino acids 325 and 326 in the CYP71C3v2 sequence. The six nucleotide 5' nontranslated region and first coding adenosine designated by lower case letters in Figure 1 were derived by RACE amplification of the cDNA library with a CΥP72C3v.2-specific primer, 3' Sal2 (shown in Figure 5 and SEQ ID NO:17), and pYES vector primers (T7 or 3' pYES SEQ; Figure 5).

Nucleotide sequencing of the CYP71C3v2 cDNA clone revealed a 1847 bp cDNA. As discussed previously, the body of the corresponding CYP71C3v2 gene sequence is interrupted by two introns which subdivide the coding region into three sections. Figure 1 shows the nucleotide sequence of the cDNA clone of CYP71C3v2. The sequences of the two introns present in clones PCR-amplified from inbred 73 genomic DNA are shown at the bottom of the figure. Without the two introns, the full length of the cDNA sequence (5 'NT, coding, 3'NT, short poly (A) tract) is 1846 bp. SEQ ID NO: 1 contains the sequence of CYP71C3v2 without the introns .

The first intron and second introns are shown at the bottom of Figure 1 and in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The first introns occurs between amino acids 178 and 179 and the second intron occurs between amino acids 325 and 326.

In another aspect, the present invention provides a CYP71C3v2 polypeptide. The CYP71C3v2 polypeptide of the present invention is a polypeptide of about 534 amino acids. The amino acid sequence of CYP71C3v2 is shown in SEQ ID NO: 2.

The present invention also contemplates amino acid residue sequences that are substantially duplicative of the sequences set forth herein such that those sequences demonstrate similar biological activity to disclosed sequences. Such contemplated sequences include those sequences characterized by a minimal change in amino acid residue sequence or type (e.g., conservatively substituted sequences) .

It is well known in the art that modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide. For example, certain amino acids can be substituted for other amino acids in a given polypeptide without any appreciable loss of function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like. As detailed in United States Patent No. 4,554,101, incorporated herein by reference, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); He (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4). It is understood that an amino acid residue can be substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0) and still obtain a biologically equivalent polypeptide.

In a similar manner, substitutions can be made on the basis of similarity in hydropathic index. Each amino acid residue has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those hydropathic index values are: He (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (- 3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (- 4.5). In making a substitution based on the hydropathic index, a value of within plus or minus 2.0 is preferred.

Alignments of the CYP71C3v2 amino acid sequence with the closely related maize CYP71C3vl sequence and the more divergent maize CYP71C1, CYP71C2 and CYP71C4 sequences as well as the CYP78 and CYP88 sequences (Larkin, 1994; Frey et al., 1995; Winkler and Helentjaris, (1995) Plant Cell 7:1307-1312) are shown in Figure 4. The CYP71C3v2 and CYP71C3vl sequences that are derived from the divergent maize inbred lines B73 and CI31A, differ in 9/534 amino acid positions, 14/1604 coding nucleotides and 10/198 3' nontranslated nucleotides. These numbers refer to differences detected in a comparison with the CYP71C3vl Genebank's accession deposit. The inventors know that at least some of the Frey et al. 1995 sequence deposition is incorrect since there are unpredicted RFLP differences between CYP71C3v2 and CYP71C3vl.

To determine the expression patterns of CYP71C3v2 transcripts in maize seedlings, poly (A) ⁺ mRNAs from 2.5-day-old and 6.5-day-old control, NA-treated and NA/T-treated maize seedlings (inbred line B73 which was released to the public about twenty years ago by Dr. Arnold Hallauer at Iowa State University) seedlings were hybridized with the CYP71C3v2 cDNA probe using high stringency conditions that prevent cross-hybridization of NA PCR 1, 3, 4 and 5 cDNA sequences with one another. Subsequent hybridization with a maize 1055 cDNA probe for a constitutive transcript (Sachs, 1991 "Molecular Response to Anoxic Stress In Maize. In Plant Life Under Oxygen Deprivation", MB Jackson, D.D. Davies, and H. Lambers, eds. (Netherlands Academic Publishing) pp. 129-139) , indicated that CYP71C3v2 transcripts are induced 2.8-fold in 2.5-day-old NA-treated seedlings and 5.0-fold in 2.5- day-old NA/T-treated seedlings relative to control seedlings of the same ages (see Figure 6, lanes 1-3) . CYP71C3v2 transcripts are induced to the same extent (2.0-fold) in NA- and NA/T-treated 6.5-day-old seedlings (see Figure 6, lanes 4-6). In contrast, CYP71C3v2 transcripts whose endogenous levels are not developmentally regulated, maize CYP73A7 (t-CAH) transcripts are 4.2-fold more abundant in 6.5-day-old seedlings than in 2.5-day- old seedlings (see Figure 7, lanes 1 versus 4). Also in contrast to CYP71C3v2 transcripts, CYP73A7 transcripts are not significantly induced in 2.5-day and 6.5-day-old seedlings by either NA- or NA/T-treatment .

To define the genomic copy number of the CYP71C3v2 gene, genomic DNA from maize (inbred B73) was restriction digested and hybridized at high stringency with a probe representing the 3' terminus of the CYP71C3v2 cDNA (base pairs 1320-1800 relative to the first coding nucleotide) (See Figure 8) or a probe representing the 5' terminus of CYP71C3v2 cDNA (base pairs 1- 580) . Digestion of the genomic DNA with a series of restriction enzymes that do not cleave within the CYP71C3v2 cDNA sequence and hybridization with either of these probes identified a single genomic DNA fragment hybridizing with CYP71C3v2 cDNA. On the basis of these results, it was concluded that CYP71C3v2 is encoded by a single copy P450 gene or a small number of closely linked P450 genes.

To define the internal organization of the CYP71C3v2 gene in the B73 inbred, maize genomic DNA (inbred B73) was PCR amplified with primers spanning different regions of the gene. PCR amplification of genomic DNA with the 5' ENG (shown in Figure 5 and SEQ ID NO: 9) and 3' ENG (shown in Figure 5 and SEQ ID NO: 16) primers positioned at either end of the CYP71C3v2 coding sequence generate products that are approximately 220 nucleotides larger than those generated from the CYP71C3v2 cDNA and 100 nucleotides larger than that generated from the CYP71C3v2 cDNA (See Figure 9 right, lanes 1-3) . PCR amplification of genomic DNA and CYP71C3v2a cDNA with the INT PRl/INT PR6 primer set generates products that are approximately 100 nucleotides larger than the product generated with the CYP71C3v2 cDNA (See Figure 9 left, lanes 2 and 3) . PCR amplification of genomic DNA with the INT PR2/INT PR5 primer set generates a product that is approximately 125 nucleotides larger than the product generated with either of the cDNAs (See Figure 9 left, lanes 3 and 4). The subsequent cloning and sequencing of the larger amplified products indicated that two introns of 97 and 124 nucleotides occur within the CYP71C3v2 coding sequence. The first of these introns occurs between amino acids 178 and 179 and the second intron occurs between amino acids 325 and 326. Chromosomal mapping of the CYP71C3v2 gene in recombinant inbred populations has mapped this sequence to a single locus on the short arm of maize chromosome 4.

In yet another aspect, the present invention also provides DNA constructs comprising all or part of the polynucleotide sequence encoding CYP71C3v2. A "construct" as used herein, is a polynucleotide comprising nucleic acid sequences not normally associated in nature, such as a prokaryotic sequence and a eukaryotic sequence. Typically, a "construct" comprises a vector, such as a plasmid, viral, and/or episomal origin, and a sequence to be transcribed.

Generally, the DNA construct will contain at least one promoter. The promoters may be heterologous, meaning that they are not naturally operably linked to the CYP71C3v2 gene. The promoter selected for use in the DNA construct will depend upon the type of organism in which the gene is to be expressed. For example, promoters useful for expression in plants are known in the art and can be inducible, constitutive, tissue specific, derived from eukaryotes, procaryotes or viruses, or have various combinations of these characteristics. Examples of promoters that are suitable for use in plants include the cauliflower mosaic virus 35S promoter, the phytohemagglutinin (PHA) promoter, ribulose-1, 5-bisphosphate carboxylase (rbcs) promoters and chlorophyll a/b binding protein (Cab) promoters.

Selection of an appropriate vector is relatively simple, as the constraints are minimal. The minimal traits of the vector are that the desired nucleic acid sequence be introduced in a relatively intact state. For example, if plant cells are to be transformed with the DNA construct, any vector that will produce a plant carrying the introduced DNA sequence should be sufficient .

Thus, suitable vectors include the Ti plasmid vectors, shuttle vectors designed merely to maximally yield high numbers of copies, episomal vectors containing minimal sequences necessary for ultimate replication once transformation has occurred, transposon vectors, homologous recombination vectors, mini-chromosome vectors, and viral vectors. The se.ection of vectors and methods to construct them are commonly nown to persons of ordinary skill in the art and are described in general technical references, such as Sambrook et al., (1989) Molecular Cloning, A Labora tory Manual , Second Edi tion, Cold Spring Harbor Laboratory Press, Vols. 1-3, which is incorporated nerem by reference .

The vector may also include any additional attached polynucleotide sequences which will confer resistance to the degradation of the polynucleotide fragment to be introduced, which assists in the process of genomic integration or which provides a means to easily select for transformed cells or plants are advantageous and greatly decrease the difficulty of selecting useable transgenotes . Commonly, expression vectors will contain selection markers, such as kanamyc resistance, hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA sequences (see U.S. Patent Number 4,704,362, which is herein incorporated by reference.)

Useful vectors will generally contain sequences that allow replication in a prokaryotic host useful for cloning the DNA sequences of the present invention. The most commonly used prokaryotic hosts are strains of Escherichia coli , although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used, and are well known m the art. Useful vectors may also contain other sequence elements useful for cloning (for example restriction sites) or expression (for example, enhancer sequences) .

In order to provide for the expression of the polynucleotide encoding CYP71C3v2, polynucleotides of the present invention will be cloned in the sense orientation into expression vectors so that they are expressed as essentially full length polypeptides. Useful expression vectors are well known m the art and are readily available. Typically, expression vectors containing polyadenylation sites and translation regulatory sequences (such as translation start sites), and may also contain introns and splice sites, enhancer sequences (which can be inducible, tissue specific or constitutive), and 5' and 3' regulatory and flanking sequences .

To achieve expression, it is necessary to introduce the appropriate construct into at least some cells of a host organism, such as by transformation. As used herein, the term "transformation" means alteration of the genotype (including episomal genes) of a target organism by the introduction of a nucleic acid sequence. The nucleic acid sequence need not necessarily originate from a different source, but it will, at some point, have been external to the cell into which it is to be introduced.

The host organism can be yeast cells, such as Sa ccharomyces cerevisiae, plant cells such as maize cells, insect cells such as Tn5 cells and bacterial cells such as E. coli and Pseudomanas . In the present invention, it is preferred that the host cells to be transformed with the expression vector hereinbefore described contain a gene that encodes NADPH-dependent P450 reductase. The NADPH-dependent P450 reductase may correspond to the host cell's endogenous copy or to an exogenous copy derived from a heterologous organism. For example, Sa ccharomyces cerevisia e cells may contain a DNA construct that allow them to overexpress Arabidopsis NADPH-dependent P450 reductase isoform 1. For optimal P450 expression, it is most preferable that the host cells overexpress either the endogenous or exogenous NADPH- dependent P450 reductase.

The transformation of plants may be carried out in essentially any of the various ways known to those skilled in the art of plant molecular biology. See, in general, Methods in Enzymo logy Vol. 153 ("Recombinant DNA Part D") 1987, Wu and Grossman Eds., Academic Press, incorporated herein by reference. As used herein, the term "plant" refers to whole plants and plant-derived tissues. As used herein, "plant-derived tissues" refers to differentiated and undifferentiated tissues of plants, including, but not limited to roots, shoots, leaves, pollen, ovules, seeds, tumor tissue, and various forms of cells in culture such as intact cells, protoplasts, embryos and callus tissue. Plant-derived tissues may be in planta or in organ, tissue or cell culture. A "monocotyledonous plant" refers to a plant whose seeds have only one cotyledon, or organ of the embryo that stores and absorbs food. A "dicotyledonous plant" refers to a plant whose seeds have two cotyledons. A "protoplast" refers to a plant cell without a cell wall or extracellular matrix. The foreign nucleic acid, which comprises the construct of the present invention, may be introduced into a host organism a number of different ways, which are well known in the art. For example, in plants, the foreign nucleic acid may be introduced into plant cells by microinjection, by using polyethylene glycol (Paszkowski et al. (1984) EMBO J. 3:2717-2722) , by electroporation (Fromm et al. (1985) Proc . Na tl . Acad. Sci . USA 82:5824-5828), by nigh ballistic penetration by small particles with the nucleic acid either withm the matrix of small beaos or particles, or on the surface (Klein et al., (1987) Na ture 327:70- 73) .

A preferred method of introducing the nucleic acid segments into plant cells is to infect a plant cell, an explant, a meristem or a seed with a genetically engineered Agroba cteri um tumefaciens or Agroba cterium rhizogenes strain carrying the segment. Within the T-DNA segment of its full-size Ti plasmid or on an abbreviated binary Ti plasmid vector containing the T-DNA boundary sequences the Agroba cterium tumafaciens Ti plasmid is, if used, the wild-type Ti plasmid and must be "disarmed", i.e., have its tumor-inducing activity removed, prior to use. To facilitate selection of transgenic plant cells, it is preferable that the gene segment be linked to a selectable marker, for example, kanamycm resistance. In some species, such as Arabidopsis thaliana , this Agrobacterium infection process is facilitated by vacuum infiltration of embryonic tissue (as in Becktold et al., (1993) C. R . Acad. Sci . Paris 316:1194-1199). Examples of Agroba cterium tumefa ciens strains that can be used include LBA4404, as described by Hoekema et al . , (1983) Na ture 303:179-180, and EHA101 as described by Hoot et al . , (1986) J. Bacteriol . 168:1297-1301. A preferred Agrobacterium rhizogenes strain is 15834, as described by Birot et al., (1987) Plan t Physiol . Biochem . 25:323-325. The Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Horsch et al., (1984) Science 233:496-498; Fraley et al., (1983) Proc . Na tl . Acad. Sci . USA 80:4803-4807). Under appropriate conditions known m the art, the transformed plant cells are placed under antibiotic selection and grown in tissue culture media to form culture shoots, roots, and eventually intact plants which can be propagated in soil.

Ti plasmids contain two regions essential for the production of transformed cells. One of these, named transfer DNA (T-DNA), induces tumor formation. The other, termed virulent region, is essential for the introduction of the T-DNA into plants. The transfer DNA region, which transfers to the plant genome, can be increased in size by the insertion of the foreign nucleic acid sequence without its transferring ability be affected. By removing the tumor-causing genes so that they no longer interfere, the modified Ti plasmid an then be used as a vector for the transfer of the gene constructs of the invention into an appropriate plant cell, such being a "disabled Ti vector".

There are presently at least three different ways to transform plant cells with Agrobacterium :

(1) co-cultivation of Agroba cteri um with cultured isolated protoplasts;

(2) transformation of cells or tissues with Agrobacterium; or

(3) transformation of seeds, apices, meπstems or whole plants with Agrobacterium . Method (1) requires an established culture system that allows plant regeneration from cultured protoplasts. Method (2) requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants. Method (3) requires regeneration or micropropagation or simply "propagation" of Arabidopsis seeds transformed with a vector.

In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid. Any one of a number of T-DNA containing plasmids can be used, the only requirement is that one be able to select independently for each of the two plasmids.

As a result of this plant cell transformation process, a Ti plasmid segment carrying the desired DNA segment is integrated in the nuclear chromosome and transformed cells can be selected by using a selectable marker linked to the desired DNA segment. These selectable markers include, but are not limited to, antibiotic resistance, herbicide resistance or visually-assayable activities. Other selectable markers known in the art may be used in this invention.

Normally, regeneration will be involved in obtaining a complete, transgenic organism from the transformation process. For example, in plants, the term "transgenote" refers to the immediate product of the transformation process and to resultant whole transgenic plants. The term "regeneration" as used herein, means growing a whole or complete transgenic organism. For example, in plants, the term regeneration relates to growing a whole plant form a plant cell, a group of plant cells, a plant part, a plant piece (e.g., from a protoplast, callus, or tissue part) , or the propagation of seeds transformed with Agrobacteri um by vacuum infiltration.

In plants, regeneration from cultured protoplasts is described in Evans et al . , Protoplast Isola tion and Cul ture in Handbook of Plan t Cell Cul tures 1:124-176 (MacMillan Publishing Co. New York 1983); M.R. Kavey, "Recent Developments the Culture and Regeneration of Plant Protoplasts, " Protoplasts (1983) -Lecture Proceedings, pp. 12-29, (Birkhauser, Basal 1983); P.J. Dale, "Protoplasts Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops", Protoplasts ( 1983) -Lecture Proceedings, pp.31-41 (Birkhauser, Basel (1983); and H. Binding, "Regeneration of Plants," Plant protoplasts pp. 21-73 (CRC Press, Boca Raton 1985) .

Identification, selection or confirmation of transgenic organisms is typically based on an assay or assays. Transgenic organisms (such as transgenotes) can be screened by biochemical, molecular biological, and other assays. For example, various assays may be used to determine whether a particular plant, plant part, or transgenote cell shows an increase (i.e., overexpression) or reduction (i.e., suppression) of the CYP71C3v2 gene. Typically the change m expression or activity of the transgenote will be compared to levels found in wHd-type (e.g., untransformed) plants of the same type. Preferably, the effect of the introduced construct (transgene) on the level of expression or activity of the endogenous gene will be established from a comparison of sibling plants with and without the construct containing the desired DNA fragment. mRNA levels can be measured by Northern blotting, primer extension, πbonuclease protection, quantitative or semi-quantitative PCR (polymerase chain reaction) , and other methods well known the art (see, e.g., Sambrook et al., (1989)). Protein can be measured in a number of ways including immunological methods such as by ELISA or Western blotting.

The inventors of the present invention have found that organisms containing a gene encoding NADPH-dependent P450 reductase which are transformed with an expression vector containing the polynucleotide shown in SEQ ID NO:l, exhibit tolerance to herbicides such as triasulfuron. Therefore, the present invention can be used for creating transgenic organisms such as yeast, plants and bacteria that are resistant to herbicides as herbicidal compounds. As used herein, the term "resistant" refers to the capability of an organism or cell to grown in the presence of selective concentrations of an inhibitor. As used herein, the term "herbicide" refers to a compound which inhibits the metabolism, growth, or replication of the cells or whole organism. Additionally, the present invention contemplates a method for making herbicide resistant organisms. The method involves transforming organisms with a polynucleotide having SEQ ID NO: 1.

In the present invention the strategy for constructing the DNA constructs, expression vectors and method for conferring herbicide resistance to an organism is set for below.

As set forth in detail hereinafter in the Examples, cDNA for galactose-inducible expression in Saccharomyces cerevisiae was engineered using the pYES vector (Stratagene) . The engineered CYP71C3v2/pYES construct and the empty pYES vector (lacking a cDNA segment) were transformed into the DBY2616 yeast strain (ura^~, his^", lys^", sue^") and into the W(R), WAT11 and WAT21 yeast strains. The DBY2616, a publically available strain, expresses endogenous levels of yeast NADPH-dependent P450 reductase. The W(R) strain, derived from the W303-1B strain, overexpresses yeast NADPH P450 reductase from a hybrid GAL10/CYC1 promoter preceding the yeast NADPH-dependent P450 reductase coding sequence (Pompon et al., (1996) Meth . Enzymol . 272:51-64) . The WAT11 AND WAT21 strains, which are deleted for the endogenous yeast NADPH P450 reductase gene, overexpress Arabidopsis NADPH-dependent P450 reductase isoforms 1 and 2 from the same hybrid GAL10/CYC1 promoter fused to the Arabidopsis NADPH-dependent P450 reductase coding sequences.

To determine the level of CYP71C3v2 transcript expressed, mRNAs isolated from these various yeast strains were initially hybridized with the maize CYP71C3v2 cDNA probe and subsequently with a constitutive yeast DPMI cDNA probe (Orlean et al., (1988) J. Biol . Chem . 263:17499-17507). Under the hybridization conditions used for these analyses, mRNA isolated from the control W(R) strain containing the pYES vector did not hybridize with the CYP71C3v2 cDNA probe (see Figure 10, lane 1) indicating that endogenous yeast P450 transcripts do not crossreact with the maize CYP71C3v2 cDNA. mRNA isolated from galactose- duced W(R), WAT11 and WAT21 strains transformed with the engineered CYP71C3v2/pYES construct hybridized with the CYP71C3v2 cDNA probe to varying degrees (Figure 10, lanes 2-4) indicating that this maize P450 transcript is expressed at different levels each of these strains. Normalization against the constitutive yeast DPMI transcript indicates that CYP71C3v2 transcripts levels were galactose-mduced at least 228-, 12- and 15-fold m the W(R), WAT11, and WAT21 strains, respectively (Figure 10, lanes 2-4), over the control levels in the W(R) strain transformed with the pYES vector (lane 1) . To define the quantity and quality of expressed CYP71C3v2 protein, microsomal protein obtained from each of these yeast strains grown under the same conditions used for Northern analysis was monitored by carbon monoxide (CO) -difference analysis (Omura and Sato, (1964) J. Biol . Chem . 239:2370-2378). By this analysis (See Figure 11, top), the W(R) strain transformed with the engineered CYP71C3v2/pYES construct expressed a significant amount of P450 protein with a CO- difference maximum at 420 nm. The W(R) strain transformed with the pYES vector alone expressed significantly less endogenous P450 protein and no quantifiable P420 protein. CO-difference quantitation (See Figure 11, bottom) indicates that the WM strain containing the engineered CYP71C3v2/pYES construct expresses 432 pmol P450/mg microsomal protein compared to 164 pmol P450/mg microsomal protein in the W(R) strain containing the pYES vector. Comparable quantitation of the P450 protein levels in the WATll and WAT21 strains express significantly less exogenous P450 protein (about 4-5 fold) and more P420 protein than the W(R) strain (See Figure 11, bottom). Even so, the levels of P450 protein in the WATll and WAT21 strains containing the engineered CYP71C3v2/pYES construct are at least two-fold over the endogenous P450 levels in the WATll and WAT21 strains containing the pYES vector alone.

To analyze the metabolic capabilities of the CYP71C3v2 protein, the engineered CYP71C3v2/pYES construct and the pYES vector were analyzed first in the DBY2616 yeast strain that expresses endogenous levels of yeast NADPH-dependent P450 reductase. As demonstrated by their sensitivity to 40 μM triasulfuron, the DBY2616 yeast contain an acetolactate synthase (ALS) sensitive to triasulfuron. On minimal media, galactose- mduced DBY2616 yeast expressing the CYP71C3v2/pYES construct are tolerant to 40 μM triasulfuron (see Figure 12B) and sensitive to 60 μM triasulfuron. To demonstrate that this trisulfuron- tolerant phenotype was directly correlated with the functional expression of the CYP71C3v2 coding sequence, DBY2616 strains containing the CYP71C3v2/pYES construct and pYES vector were cured of their plasmids with a 5-fluoroorotic acid (5-FOA) treatment and replated on minimal media containing triasulfuron. As shown in Figure 12C, these 5-FOA-cured DBY2616 strains are sensitive to 40 μM triasulfuron.

For analysis of the herbicide tolerance levels in another yeast strain, CYP71C3v2/pYES construct and the pYES vector were transformed into a W(R) LEU2 strain (ura^~, ade^", his^", trp^") and plated on minimal media containing triasulfuron. For this analysis, the engineered W(R) strain that overexpresses yeast NADPH P450 reductase was converted to a leucine auxotroph by transformation with a LEU2 gene. As shown in Figure 12A, galactose-induced W(R) LEU2 transformants expressing the CYP71C3v2/pYES construct are tolerant to 60 μM triasulfuron. As in the case of the 5-FOA-cured DBY2616 strains, 5-FOA-cured W(R) LEU2 strains are sensitive to 60 μM triasulfuron (see Figure 12D) .

The present invention also contemplates a method for controlling undesired vegetation in a location containing an agronomically useful plant that is being cultivated. Preferably, the plant has been transformed with an isolated polynucleotide having SEQ ID NO:l and, as a result, is resistant to one or more herbicides. More specifically, the method involves applying an effective amount of one or more herbicides to a location containing the hereinbefore described transformed plant to control all undesired vegetation. As used herein, the term "undesired vegetation" refers to those plants which compete with other plants for food and nutrients and/or are harmful to other agronomically useful plants growing m the same location. For example, weeds, such as pigweed, velvet leaf, lambs quarters, Chenopodium album and quack grass, are considered to be undesired vegetation. The location to which the herbicide is applied may be a field, garden or container containing the hereinbefore described transformed plant.

An effective amount of herbicide to be applied to the location can be easily determined by one of ordinary skill in the art. Typically, an effective amount is that amount of herbicide required to inhibit the metabolism, growth or replication of the undesired vegetation. An effective amount of herbicide may be applied to a location m one application or a number of applications depending upon what the conditions of the location dictate.

The present invention also contemplates a method for identifying compounds having herbicidal activity. More specifically, the method involves treating an organism, such as yeast, bacteria or plants transformed with an isolated polynucleotide having SEQ ID N0.1 and is resistant to heroicides one or more compounds. The organism may be treated with the compound (s) once or more that once depending upon what the experimental conditions dictate. After the organism has been treated, it is examined over a period of time to determine if any of the tested compounds has any effect on the metabolism, growth and/or replication of the organism. Any compound or combination of compounds which effects the metabolism, growth or replication of the organism is then identified as having herbicidal activity and selected. Such selected compound and/or combination of compounds can be further tested and/or developed as a herbicide.

By way of example, and not of limitation, examples of the present invention will now be given.

Example 1: METHODS

Example la: Seed growth and herbicide application

Approximately 100 grams (g) of Captan-treated corn seeds { Zea mays mbred B73) were soaked overnight m a one liter flask with flowing tap water. Seeds (not treated with Captan) were sterilized using a 30% (v/v) bleach solution containing 0.05% (v/v) Tween-20 for 45 minutes and washed four times with 400 ml of sterile distilled water. For induction with naphthalic anhydride (NA) , 100 g of seeds were coated with the inducer by shaking the seeds vigorously in a 100 ml bottle with 1 g dry powdered NA. Seeds were then arranged in rows on sterile white teri towels and moistened with sterile distilled water. The ten towels were subsequently sandwiched between cafeteria trays and placed upright in a tub of distilled water. The seeds were grown at 25-30°C for 2.5 or 6.5 days. Approximately 16 hours before harvesting, the seedlings were triasulfuron-treated by soaking the teri towel with 25 ml of a solution containing ( 0.052% (w/v) ) of a 75% (w/w) powder of the commercial form of triasulfuron (Amber, provided by CIBA-Geigy, Greensboro, NC) corresponding to a final concentration of 1 mM triasulfuron. For RNA extraction, 5-10 grams of maize coleoptiles were collected, frozen in liquid nitrogen and stored at -80°C.

Example lb : Pathogen treatments

Six-day-old maize coleoptiles grown as outlined above in Example la, were pricked with a 25 gauge needle and injected at the base, half way along the length, and at the tip with a sterile nutrient broth solution. The sterile nutrient broth solution injected contained either no bacteria or a saturated culture of the maize pathogenic bacteria Erwinia stuartn or the maize pathogenic bacteria Acidovorax avenae . The plants were allowed to grow for an additional 12-16 hours after treatment before harvesting for mRNA analysis.

Example lc: RNA Extractions and RT-PCR

Total RNA was isolated according to the method of Puissant and Houdeb e (1990), herein incorporated by reference. Approximately 5-10 g of liquid nitrogen frozen rraize coleoptiles were ground to a fine powder in a chilled mortar and pestle. The ground tissue was placed in four to eight Falcon 2059 certπfuge tubes (Becton Dickinson, Lincoln Park, NJ) each containing 5 ml of GuISCN extraction buffer (4 M guanid ium isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% (w/v) N-lauroyl sarkosme, 0. IM β-mercaptoethanol) , gently mixed by inverting the tubes and allowed to thaw at 4°C. The samples were mixed with 0.5 ml 2 M sodium acetate (pH 4.0) and 5 ml water-saturated phenol, and 1 ml of chloroform was added, the samples were mixed and centrifuged at 7000xg for 15 minutes at 4°C. The aqueous phase (~7 ml) was precipitated at 4°C with an equal volume of isopropanol. The nucleic acids were collected by centrifugation 3300xg for 10 minutes at 4°C, and each pellet was resuspended m 2 ml of 4 M LiCl, vortexed, and centrifuged at 3300xg for 10 minutes at 4°C. Each pellet was resuspended m 2 ml of TE buffer containing 0.5% (w/v) SDS, samples were combined and an equal volume of chloroform was added. After vortexmg and centrifugation at 3300xg for 10 minutes at 4°C, nucleic acids were precipitated at 4°C after adding 0.1 volume of 2 M sodium acetate (pH 5.0⁾ and an equal volume of isopropanol. The nucleic acids were pelleted at 3300xg for 15 minutes at 4°C and washed with 70% ethanol and 100% ethanol. The samples were air dried for 15 minutes and resuspended in 300 μl of sterile water and frozen at -80°C. Typically, 1 mg total RNA was recovered from 10 g of coleoptile tissue .

Poly (A) ⁺ mRNA was isolated from approximately 1 mg of total RNA using the rapid mRNA purification kit (Amresco, Solon, OH) as outlined in the manufacturer's directions. The mRNA was resuspended in sterile water and stored at -80°C. Typically, 10 μg mRNA were recovered per mg total RNA. 100 ng mRNA isolated from naphthalic anhydride-treated 6.5-day-old seedlings were reverse transcribed at 50°C for 30 minutes in a 50 μl reaction containing 4 U AMV reverse transcriptase (Promega, Madison, WI) and 100 pmol PC-1 oligo (dT) primer (Table 1) in IxPCR buffer (50 mM KC1, 10 mM Tris-HCl (pH 8.4), 200 μM dNTPs, 50 μg/ml gelatin). The first strand cDNA products were PCR amplified in a 50 μl reaction containing 2.5 U Taq polymerase (Gibco BRL, Gaithersburg, MD) , 100 pmol of the degenerate PN-3 and nondegenerate PC-1 oligo (dT) primers (See Figure 5). Twenty five cycles of PCR amplification were performed with each consisting of: 95°C denaturation for 1 minutes, 42°C or 60°C annealing for 2 minutes and 72°C extension for 2 minutes. A final 5 minutes 72°C extension step was done to complete synthesis of all DNA strands.

For cloning, 100 ng of pBluescript SK^" vector (Stratagene, LaJolla, CA) and half of the RT-PCR products derived from a single amplification reaction were mixed, extracted with phenol : chloroform (1:1), ethanol precipitated, resuspended in sterile water and restriction digested with 10 U EcoRI and 10 U BamHI for 2 hours at 37°C. The restriction cut products were reextracted with phenol: chloroform, ethanol precipitated, and ligated using 1 U T4 DNA ligase. The EcoRI-BamHI inserts of 800 ampicillin-resistant transformants were sized on 2.2% agarose gels and those in the 300-500 bp range (90 clones) were sequenced using T3 and T7 primers complementary to the Bluescript SK^" vector (Stratagene) and a Sequenase 2.0 kit (U.S. Biochemicals, Cleveland, OH) . RT-PCR clones containing the conserved F—G-R-C- G P450 sequence were sequenced in their entirety.

A maize CYP73A 7 (t-CAH ) RT-PCR clone was obtained by RT-PCR amplification using the conditions outlined above and the degenerate tCAH 5' and tCAH 3' primers (See Figure 5) complementary/identical to conserved amino acids 320-326 and 463- 469 in the pea t-CAH ( CYP73A9; Frank et al . , (1996) Plan t Physiol . 110:1035-1046, herein incorporated by reference) sequence. These sequences are also conserved in the maize t-CAH sequence ( CYP73A7; Potter et al., (1995) Drug Metabol . Drug Interact . 12:317-327, herein incorporated by reference).

Example Id: Northern Analysis

1 μg of mRNA was electrophoresed on 1.2% (w/v) agarose- formaldehyde gels and capillary-blotted to Hybond N nylon membranes (Amersham, Arlington Heights, IL) overnight using lOx SSC. Membranes were UV-crosslinked using a stratalinker (Stratagene) and prehybridized in 200 mM Na₂P0₄ (pH 7.2), 5% SDS, 1 mM EDTA, 10 mg/ml BSA, 0.1 mg/ml sheared salmon sperm DNA for at least 2 hours at about 65°C. Blots were probed with denatured ³²P-labeled probes added directly to the prehybridization solution at 60-65°C for 12-16 hours. Blots were washed twice for 15 minutes at 60-65°C with 40 mM of Na₂P0₄ (pH 7.2), 5% SDS, 1 mM EDTA, 5 mg/ml BSA, washed once for 5-30 minutes at 60 to 65°C in 40 mM of Na₂P0₄ (pH 7.2), 1% SDS, 1 mM EDTA and autoradiographed at -80°C. For quantification, hybridization signals were quantified by Phosphorimagery (Molecular Dynamics, Sunnyvale, CA) and compared after normalization to the level of constitutive maize 1055 mRNA (Sachs, M. , (1991) Molecular Response to Anoxi c Stress in Maize . In Plant Life Under Oxygen Deprivation. M.B. Jackson, D.D. Davies, and H. Lambers, eds. (Netherlands, Academic Publishing), pp. 129-139).

Example le : cDNA Library Construction and Screening

3 μg of mRNA from naphthalic anhydride/triasulfuron-treated 2.5-day-old maize seedlings were reverse transcribed at 42°C for 1.5 hours using 33 U AMV reverse transcriptase (Promega) in a 50 μl reaction containing lx RT buffer (50 mM Tris-HCl (pH S.3), 50 mM KC1, 10 mM MgCl₂, 10 mM DTT, 0.5 mM spermidine) , 500 _\ιY. dNTPs, 20 U RNAsin (Promega) and 5.7 μg Notl oligo (dT) primer (See Figure 5) . After reverse transcription, 72 μl of 5x second strand buffer (94 mM Tris-HCl (pH 8.0), 453 mM KC1, 23 mM MgCl₂, 18.75 mM DTT, 200 μM dNTPs), 3 U RNase H (Boehringer Mannheim, Indianapolis, IN) , 90 U DNA polymerase I (Boehringer Mannheim) were added and the second strand reaction was incubated at 15°C for 2.5 hours. The cDNA was phenol : chloroform (1:1) extracted, ethanol precipitated, dried and resuspended in 38 μl sterile water, 10 μl 5x end-polishing buffer (90 mM (NH₄)₂S0₄, 330 mM Tris-HCl (pH 8.3), 33 mM MgCl₂, 50 mM β-mercaptoethanol, 200 μM dNTPs) . The repair reaction was carried out with 10 U T4 DNA polymerase (Gibco BRL) for 1 hour at 37°C, phenol : chloroform (1:1) extracted and ethanol precipitated. A 30 μl adaptor ligation reaction was set up containing 2 μg BstXI adaptors (Invitrogen, Carlsbad, CA) and 2.5 U T4 DNA ligase. After an overnight 15°C ligation, excess adaptors were removed by centrifugation through a 0.5 ml Sephacryl S-400 (Promega) column. Test ligations containing 1,2 or 3 μl of the cDNA insert and 100 ng of BstXI-cut pYES vector (Invitrogen) in 20 μl ligation reactions were incubated overnight at 4°C and 5-10 μl of each ligation reaction were electroporated into electrocompetent TOPP10F' cells (Invitrogen). Approximately 40,000 primary transformants were screened by colony hybridization using inserts from the NA PCR 1, 3, 4 and 5 clones and the conditions previously described for Northern analysis. For the primary and secondary screens, equivalent counts of the NA PCR 1, 3, 4 and 5 probes were combined. For the tertiary screen, M-labeled probes representing individual NA PCR clones were used to screen restriction fragments derived from clones identified in the secondary screen.

Example If: RACE Cloning

The 5' end of the CYP71C3v2 cDNA was cloned by RACE amplification of DNA extracted from the NA/T 2.5-day-old seedling cDNA library using the 3 ' Sal2 primer specific for the CYP71 C3v2 transcript (See Figure 5) and the T7 vector primer (Stratagene) or 3'pYES SEQ primer (See Figure 5) specific for the pYES vector. 30 PCR cycles were performed, each consisting of a 95°C denaturation for 1 minute, 55-65°C annealing for 2 minutes and 72°C extensions for 2 minutes. The resulting PCR products were cloned into pBluescnpt SK⁺ and six clones were sequenced using T3 and T7 vector primers. The longest of the six RACE clones obtained by this strategy contained 6 nucleotides preceding the translation initiation site.

Example Ig: Genomic DNA Isolation

0.5-1.0 g of 6.5 day old etiolated maize coleoptiles were placed in a 15 ml Falcon 2059 centrifuge tube, frozen with liquid nitrogen and ground to a fine powder with a glass rod. 700 μl of urea extraction buffer, which contains 7 M urea, 312 mM NaCl, 20 mM EDTA, 1% N-lauroyl sarkosme, 50 mM Tris-HCl (pH 8.0), was added to the tube and the tissue was thawed at room temperature with frequent gentle mixing. The sample was mixed with 500 μl of phenol : chloroform (1:1) and the tubes were incubated at 37 °C for 15 minutes a rotary shaker. After incubation, the contents of the Falcon 2059 tube were transferred to a 1.5 ml microfuge tube and centrifuged at 14,000xg for 10 minutes. The aqueous phase (-500 μl) was transferred to a fresh 1.5 ml tube containing 50 μl of 4.4 M NH₄OAc, mixed with 700 μl isopropanol and centrifuged at 14,000xg for 1 minute. The DNA pellet was resuspended in 360 μl of sterile water, reprecipitated with 50 μl 4.4 NH₄OAc and 700 μl isopropanol and centrifuged at 14,000xg for 1 minute. The final DNA pellet was washed once with 70% ethanol, dried for 10-

15 mm by inverting the tubes on a kimwipe and resuspended m 50- 100 μl of sterile water. Typically, 50 μg of genomic DNA were recovered per gram of coleoptile tissue.

Example lh: Genomic Southern Analysis

40 μg of genomic DNA was digested with 60 U of each restriction enzyme for 6-8 hours at 37°C, electrophoresed on 0.8% agarose gels containing lx TBE and capillary-blotted to Hybond N nylon membranes overnight using lOx SSC. The membranes were UV- crosslinked and prehybndized m 200 mM of Na₂HP0, (pH 7.2), 5% SDS, 1 mM EDTA, 10 mg/ml BSA, 0.1 mg/ml sheared salmon sperm DNA for at least 2 hours at 65°C. The blots were hybridized for 12-

16 hours at 60-65°C with ³²P-labeled randomly primed DNA probes added directly to the prehybridization solution. The blots were washed twice for 15 minutes at 60-65°C with 40 mM Na₂HP0₄ (pH 7.2), 5% SDS, 1 mM EDTA, 5 mg/ml BSA and once for 30 minutes at 60-65°C with 40 mM of Na₂HP0₄ (pH 7.2), 1% SDS, 1 mM EDTA) and autoradiographed at -80°C.

Example li : Yeast Strains

The yeast strains used were DBY2616 (MATa; his 4-539am , lys 2-801am, ura 3-52 , sue 2-437) (Kaiser and Botste , (1986) Mol . Cell Biol . 6:2382-2391, herein incorporated by reference) and three derivatives of W303-1B (MATα; ade 2-1 , his-3-11 , -15 , trp 1 -1 , leu 2-3, -112, ura 3-1 ; can^R ; cyr⁺) (Pompon et al . , (1996) Meth . Enzymol . 272:51-64, herein incorporated by reference) designated W(R), WATll and WAT21. DBY2616 expresses constitutive, levels of yeast NADPH-dependent P450 reductase. W(R) overexpresses yeast NADPH-dependent P450 reductase, WATll overexpresses Arabidopsis NADPH-dependent P450 reductase isoform 1 and WAT21 overexpresses Arabidopsis NADPH-dependent P450 reductase isoform 2 ( Id. ) .

Example 1j : Construction of CYP71C3v2 Yeast Expression Vector

For subcloning of the CYP71C3v2 cDNA into the pYES2 yeast expression vector (Invitrogen) , 50 ng of plasmid DNA were amplified using the 5' ENG and 3' ENG primers (See Figure 5) in 50 μl of lx PCR buffer (50 mM KC1, 10 mM Tris-HCl (pH 8.4), 200 μM dNTPs, 50 μg/ml gelatin) containing 5 U of Vent DNA polymerase (New England Biolabs, Beverly, MA) . Twenty five cycles of PCR amplification were performed consisting of: 95°C denaturation for 1 minute, 65°C annealing for 2 minute and a 72°C extension for 2 min. A final 5 minutes at 72°C extension step was included to complete the synthesis of all DNA strands. The PCR products were phenol: chloroform (1:1) extracted, ethanol precipitated, resuspended in 20 μl of sterile water, digested with 10 U of Hindlll and Xbal and subcloned into Hindlll-Xbal digested pYES2 vector.

Example Ik: Yeast Transformation

50 μl of electrocompetent DBY2616 and W303-1B (W(R), WATll, WAT21) yeast were transformed with 1 μg of recombinant CYP71C3v2/pYES plasmid or the pYES vector by electroporation using a BIO-RAD Gene Pulser set at 1.5 kV, 25 μF and 200 Ω. One milliliter of cold 1 M sorbitol was added to the electroporation cuvette and the transformants were recovered by plating on CSM- URA GLU plus 1 M sorbitol plates (Bio 101 complete supplemented media containing 2% glucose, 1 M sorbitol and lacking uracil) . DNA was isolated from individual colonies and PCR amplified with the INT PR3 and 3' ENG primers (See Figure 5) to confirm the presence of the pYES vector containing CYP71 C3v2 cDNA inserts.

Example 11: Yeast Growth Conditions for P450 Expression

The W(R), WATll and WAT21 yeast strains were grown 2 ml of 2X CSM-URA GLU (Bio 101 complete supplemented media containing 2% glucose, and lacking uracil) m a 15 ml Falcon 2059 tube shaken overnight at 200 rpm and 30°C. 1 ml of the culture was added to 50 ml of 2X CSM-URA GLU and shaken in a 125 ml erlenmeyer flask at 150 rpm for -8 hrs at 30°C to an O.D. of 1.2- 1.3 (~7-8xl0⁷cells/ml) and induced by adding sterile 20% galactose (glucose-free grade Sigma, St. Louis, MO), to a 2% final concentration, 100 mg/ml δ-ammolevilunic acid to a final concentration of 50 mg/L and 100 mM FeEDTA to a final concentration of 50 μM. At this point additional histidme, tryptophan, leucme and lysine ammo acid supplements were added to final concentrations of 20 mg/L each and adenine was added to a final concentration of 50 mg/L. The cultures were further incubated at 30°C for 8.5 hours prior to isolating microsomal protein or mRNA.

Example lm: Yeast RNA Extractions

Total RNA was isolated according to the method of Puissant and Houdebme (1990) BioTechmques 8 : 148-149, herein incorporated by reference, by pelleting yeast cells from a 50 ml culture at 2,000xg for 6 minutes, washing them with 50 ml of DEPC-treated sterile water and repelleting them at 2,000xg for 6 minutes. The yeast pellet was resuspended in 2 ml of GuISCN extraction buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate (pK 7.0), 0.5% (w/v) N-lauroyl sarkosine, 0.1 M β-mercaptoethanol) , 0.2 ml 2 M sodium acetate (pH 4.0), 2 ml water-saturated phenol and 0.4 ml chloroform, split into four equal volumes, and bead-beater, with a Mini-Beadbeater three times for 20 seconds at 5,000 rpm at 4°C using 0.5 mm glass beads in 2 ml tubes. The samples were centrifuged at 13,000xg for 10 minutes and the supernatants were transferred to 1.5 ml Eppendorf tubes. The aqueous phase was precipitated at 4°C with an equal volume of isopropanol. The nucleic acids were collected by centrifugation at 14,000xg for 10 minutes at 4°C, and each pellet was resuspended in 0.5 ml of 4 M LiCl, vortexed, and centrifuged at 14,000xg for 10 minutes at 4°C. Each pellet was resuspended in 0.5 ml TE buffer containing 0.5% SDS and an equal volume of chloroform was added. After vortexing and centrifuging at 14,000xg for 10 minutes at 4°C, nucleic acids were reprecipitated at 4°C after adding one-tenth volume of 2 M sodium acetate (pH 5.0) and an equal volume of isopropanol. The nucleic acids were pelleted at 14,000xg for 15 minutes at 4°C and washed once with 70% ethanol and once with 100% ethanol. The samples were air dried for 15 minutes, resuspended in 100 μl sterile water and frozen at -80°C. Typically, 200 μg total RNA was recovered per 50 ml yeast culture.

Poly (A) + mRNA was isolated from approximately 1 mg of total RNA using the rapid mRNA purification kit (Amresco, Solon, OH) as outlined in the manufacturer's directions. The mRNA was resuspended in sterile water and stored at -80°C. Typically, 10 μg mRNA were recovered per mg total yeast RNA. Example In. Microsomal Protein Isolation and CO Difference

Spectra

Microsomes were isolated by pelleting yeast cells from each 50 ml culture at 2,000xg for 6 minutes, washing with 50 ml of sterile water and repelletmg them at 2,000xg for 6 minutes. The yeast cells were resuspended in 1.0-1.5 ml of microsome isolation buffer (MIB) containing 100 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M sucrose, 10% glycerol, 1 mM DTT, 50 μg/ml aprotinin, leupeptm and pepstatm, and bead-beaten with a Mmi-Beadbeater (Biospec Products, Bartlesville, OK) three times for 20 sec at 5,000 rpm at 4°C using 0.5 mm glass beads in a 2 ml tube. The samples were centrifuged at 13,000xg for 1 minute and the supernatants transferred to 1.5 ml Eppendorf tubes. An additional 1 ml of MIB was added to the 2 ml tube and the yeast were bead-beaten twice for 20 seconds at 5,000 rpm at 4°C. The samples were centrifuged at 13,000xg for 1 minute and the supernatants combined. Microsomes were pelleted by centrifugation at 150,000xg for 1 hour and 15 minutes and resuspended in 1 ml of microsomal storage buffer (MSB) containing 10 mM KP0₄ (pH 7.5), 1 mM EDTA, 1 mM DTT, 20% glycerol, to a final protein concentration of 2-4 mg/ml. CO difference spectra and total P450 quantifications were performed on samples containing 2-4 mg/ml microsomal protein using the method of Omura and Sato, (1964) J. Biol . Chem . 239:2370-2378, herein incorporated by reference. An extinction coefficient of 91 mMMm^"1 was used for P450 quantification (450 and 490 nm wavelength pair) and an extinction coefficient of 111 mM^"1cm^"1 for P420 quantification (420 and 490 nm wavelength pair) .

Example lo: Complementation of Yeast with CYP71C3v2

Prior to complementation analysis, the W(R) yeast strain was made auxotrophic for leucme by transformation with the LEU2 gene contained on the pADNS plasmid. For complementation analysis the yeast strains DBY2616 and W(R) containing the CYP71C3v2/pYES plasmid or the empty pYES vector were grown at 30°C in 50 ml of YGIM (a yeast nitrogen base media containing 5g/L ammonium sulfate and lacking amino acids, 4% galactose, 20 mg/L histidine, 20 mg/L tryptophan, 20 mg/L lysine) with shaking at 150 rpm for 36-48 hours to 1.0-1.5xl0⁸ cells/ml. An aliquot from this culture was diluted with sterile water to contain approximately 150-250 cells/100 ml and plated on YGIM plates containing 50 mg/L δ- aminolevulinic acid, 50 μM FeEDTA, and 40 μM triasulfuron (DBY2616) or 60 μM triasulfuron (W(R)LEU2). The plates were incubated for 6 days at 30°C and growth comparisons were made.

Example lp : Curing of Yeast with 5-fluoroorotic acid (5-FOA)

The yeast used in the complementation assay above were cured of their pYES plasmids by 5-FOA treatment. For this, the yeast were grown overnight in 2x CSM-LEU GLU (Bio 101 complete supplemented media containing 2% glucose and lacking leucine) plus 20 mg/L uracil, diluted to approximately l.OxlO⁴ cells/ml and plated on 2x CSM-LEU GLU plus 20 mg/L uracil plates containing 4 mM 5-FOA. The plates were incubated at 30°C for 3-4 days and the 5-FOA resistant colonies were restreaked on 2x CSM-LEU GLU plates containing or lacking 20 mg/L uracil to confirm the ura phenotype and pYES plasmid loss.

Example 2 : Yeast Growth Conditions for CYP71C3v2 Expression and Type I Binding Spectra with the Herbicide Triasulfuron

W(R) strains containing the pYES vector plasmid or the CYP71C3v2/pYES plasmid were grown in 2 ml of 2x CSM-URA GLU (see Example 11 above) in a 15 ml Falcon 2059 tube shaken overnight at 200 rpm at 30°C. 1 ml of the culture was added to 50 ml of 2x CSM-URA GLU and shaken in a 125 ml Erlenmeyer flask at 150 rpm for 8.5 hrs at 30°C and induced by adding sterile 20% galactose to a final 2% concentration, 100 mg/ml δ-aminolevulinic acid to a final concentration of 50 mg/L and 100 mM FeEDTA to a final concentration of 50 μM. At this point, histidine, tryptophan, leucine and lysine amino acid supplements were added to final concentrations of 20 mg/L each and adenine was added to a final concentration of 50 mg/L. The cultures were further incubated at 30°C for 15 hrs prior to isolating microsomes.

Yeast microsomes were isolated as described above in Example In. Specifically, microsomes were isolated by pelleting yeast cells from each 50 ml culture at 2,000xg for 6 minutes, washing with 50 ml of sterile water and repelleting them at 2,000xg for 6 minutes. The yeast cells were resuspended in 1.0-1.5 ml of microsome isolation buffer (MIB) containing 100 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M sucrose, 10% glycerol, 1 mM DTT, 50 μg/ml aprotinin, leupeptin and pepstatin, and bead-beaten with a Mini- Beadbeater (Biospec Products, Bartlesville, OK) three times for 20 sec at 5,000 rpm at 4°C using 0.5 mm glass beads in a 2 ml tube. The samples were centrifuged at 13,000xg for 1 minute and the supernatants transferred to 1.5 ml Eppendorf tubes. An additional 1 ml of MIB was added to the 2 ml tube and the yeast were bead-beaten twice for 20 seconds at 5,000 rpm at 4°C. The samples were centrifuged at 13,000xg for 1 minute and the supernatants combined. Microsomes were pelleted by centrifugation at 150,000xg for 1 hour and 15 minutes and resuspended in 1 ml of microsomal storage buffer (MSB) containing 10 mM KPO. (pH 7.5), 1 mM EDTA, 1 mM DTT, 20% glycerol, to a final protein concentration of 2-4 mg/ml. CO difference spectra and total P450 quantifications were performed on samples containing

2-4 mg/ml microsomal protein using the method of Omura and Sato,

(1964) J. Biol . Chem . 239:2370-2378. Type I binding spectra were performed as described by Jefcoate (C.R. Jefcoate, ( 1978 ) Meth . Enzmol . , 52:258-279, herein incorporated by reference) using 100 μl of microsomal protein in 900 μl microsomal storage buffer and 25 μl of a 1 mM triasulfuron stock solution (50 μM final concentration) . Samples were analyzed in split cuvettes with the reference cuvette containing microsomal storage buffer plus herbicide in one chamber and protein alone in the other chamber. The sample cuvette contained buffer alone in one chamber and protein plus herbicide in the other chamber. The samples were scanned from 450 nm to 350 nm using a Hitachi spectrophotometer . Figure 14 shows that the CYP71C3v2 protein expressed in the W(R) yeast strain binds to the herbicide triasulfuron.

Example 3 : Analysis of Triasulfuron Tolerance in Transgenic Arabidopsis Seedlings Expressing CYP71C3v2 Protein

Transgenic Arabidopsis T₃ seeds carrying pKYLX vector sequences (designated vector line #1 and vector line #2) (described in Maita et al . PNAS, (1993) 90:6110-6114, herein incorporated by reference) or CYP71C3v2 cDNA sequences under constitutive expression from the 35S promoter (designated 35S:CYP71C3v2 line #2) or CYP71C3v2 cDNA sequences not expressing from the 35S promoter (designated 35S : CYP71C2v2 line #1) were plated on agar plates containing MS salts (Sigma Chem. Co.) and 0.167 μM triasulfuron. Seedlings were incubated for 4 weeks at 25°C under constant light and photographed (See Figure 15) . Reverse transcription-PCR analysis with primers directed against CYP71C3v2 indicated that 35S:CYP71C3v2 line #2 expresses RNA transcripts from this transgene and that 35S: CYP71C3v2 line #1 does not express detectable RNA transcript. The data shown in Figure 15 indicates the transgenic Arabidopsis plants carrying a 35S : CYP71C3v2 cDNA construct expressing the CYP71C3v2 transcript under control of the constitutive 35S promoter (designated 35S : CYP71C3v2 line #2) are capable of growing and flowering in the presence of 0.167 μM triasulfuron. Flowering plants are shown with arrows in Figure 15. At this level of herbicide, transgenic plants transformed with the vector alone (designated vector lines #1 and #2) and transgenic plants not expressing the cDNA at a discernible level (designated 35S : CYP71C3v2 line #1) grow poorly and start dying before any flowering has occurred. This data indicates that expression of the CYP71C3v2 polypeptide has the ability to confer triasulfuron tolerance on transgenic plants. At this level of RNA expression and herbicide tested, it shows that the flowering process is the most sensitive designator of triasulfuron tolerance .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: Schuler, Mary A. Persans, Michael

(n) TITLE OF INVENTION: Maize Cytochrome P450 Monooxygenase cDNA

(m) NUMBER OF SEQUENCES: 19

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Rockey, Milnamow & Katz, Ltd.

(B) STREET: 180 N. Stetson, 2 Prudential Plaza, Suite

4700

(C) CITY: Chicago

(D) STATE: IL

(E) COUNTRY: U.S.A.

(F) ZIP: 60601

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(vm) ATTORNEY/AGENT INFORMATION:

(A) NAME: Mueller, Lisa V.

(B) REGISTRATION NUMBER: 38,978

(C) REFERENCE/DOCKET NUMBER: UICU 95047

(IX) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 312-616-5400

(B) TELEFAX: 312-616-5460

(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1847 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 7..1608

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

TCGACA ATG GCA CTC CAG GCA GCC TAC GAG TAC CTG CAG CAG GCC GTC 48

Met Ala Leu Gin Ala Ala Tyr Glu Tyr Leu Gin Gin Ala Val 1 5 10

GGC CAT GGC GCG TGG TCG TCC ACG CAG ACG CTG ACG CTG CTG CTC ATC 96 Gly His Gly Ala Trp Ser Ser Thr Gin Thr Leu Thr Leu Leu Leu He 15 20 25 30

GCC GTA CCC ACC GTA CTA CTG CTG CTA GCG TCC CTC GCC AAG AGC ACG 144 Ala Val Pro Thr Val Leu Leu Leu Leu Ala Ser Leu Ala Lys Ser Thr 35 40 45

TCG TCG TCC GGT AGG GGC AAG CCG CCG CTC CCT CCC TCG CCG CCG GGC 192 Ser Ser Ser Gly Arg Gly Lys Pro Pro Leu Pro Pro Ser Pro Pro Gly 50 55 60

ACC CTC CCC ATC GTG GGG CAC CTA CAC CAC ATC GGG CCC CAG ACC CAC 240 Thr Leu Pro He Val Gly His Leu His His He Gly Pro Gin Thr His 65 70 75

ATC TCG CTC CAG GAG CTG GTG GCC AAG TAC GGG CAC AAC GGG TTC CTG 288 He Ser Leu Gin Glu Leu Val Ala Lys Tyr Gly His Asn Gly Phe Leu 80 85 90

TTC CTC CGC GCC GGC GCC GTG CCC ACC CTG ATC GTG TCG TCG CCC AGC 336 Phe Leu Arg Ala Gly Ala Val Pro Thr Leu He Val Ser Ser Pro Ser 95 100 105 110

GCC GCC GAG GCC GTG ATG CGC ACC CAC GAC CAC ATC TTC GCG TCC CGG 384 Ala Ala Glu Ala Val Met Arg Thr His Asp His He Phe Ala Ser Arg 115 120 125

CCG TGG TCC ATG GCC TCC CAC ATC CTC CGC TAC AAC ACC TGC GAC GTG 432 Pro Trp Ser Met Ala Ser His He Leu Arg Tyr Asn Thr Cys Asp Val 130 135 140

GCC TTC TCG CCG CTC GGC GAA TAC TGG CAG CAG ACC AGG AAG CTG ATG 480 Ala Phe Ser Pro Leu Gly Glu Tyr Trp Gin Gin Thr Arg Lys Leu Met 145 150 155

AAC ACG CAC CTG CTC AGC AAC AAG AAG GTC TAC TCC TTC CGC CAT GGC 528 Asn Thr His Leu Leu Ser Asn Lys Lys Val Tyr Ser Phe Arg His Gly 160 165 170

CGC GAG GAA GAG GTG TGC CTC GTC GTG GAC AAC CTC CGC GAG GCG GCC 576 Arg Glu Glu Glu Val Cys Leu Val Val Asp Asn Leu Arg Glu Ala Ala 175 180 185 190

GCC AAG TCG CCG TCG ACG GCC GTG GAC ATG AGC GAG GTG CTG GCG GCG 624 Ala Lys Ser Pro Ser Thr Ala Val Asp Met Ser Glu Val Leu Ala Ala 195 200 205

TAC ACC AAC GAC GTG GTG AGC CGG TCG GTG CTG GGC TCG ACG CAC CGG 672

Tyr Thr Asn Asp Val Val Ser Arg Ser Val Leu Gly Ser Thr His Arg 210 215 220

AAG AAA GGC CGG AAC ACG CTG TTC AGG GAG ATG ACC ATG ACC AAC GTG 720

Lys Lys Gly Arg Asn Thr Leu Phe Arg Glu Met Thr Met Thr Asn Val

225 230 235

GAC CTC CTG GTG GGG TTC AAC CTG GAG TAC TAC ATC CCG CGG TGG CCG 768

Asp Leu Leu Val Gly Phe Asn Leu Glu Tyr Tyr He Pro Arg Trp Pro

240 245 250

CTG ACG GAC CTG CTC TTC AGG CTC GTG TGC TGG AAG GTC ACG CGC CAC 816

Leu Thr Asp Leu Leu Phe Arg Leu Val Cys Trp Lys Val Thr Arg His

255 260 265 270

CTC AAG CGC TGG GAC GCC CTG CTG GAG GAG GTG ATC CAC GAG CAC GTG 864

Leu Lys Arg Trp Asp Ala Leu Leu Glu Glu Val He His Glu His Val

275 280 285

GAG ATG AGG AAG CTG TCC GGC GAC AAG GAG AAG GAG TCG GAC GAC TTC 912

Glu Met Arg Lys Leu Ser Gly Asp Lys Glu Lys Glu Ser Asp Asp Phe 290 295 300

ATC GAC ATC TTC CTC TCC AGA TAC GAG GAG TAC GGC TTC ACC ATG GAT 960

He Asp He Phe Leu Ser Arg Tyr Glu Glu Tyr Gly Phe Thr Met Asp

305 310 315

AAC GTC AAG TCC CTC CTC ATG AAC GTG TTC GAG GCA GCC ATC GAG ACC 1008

Asn Val Lys Ser Leu Leu Met Asn Val Phe Glu Ala Ala He Glu Thr

320 325 330

TCA TAT CTG GTG CTG GAG TCC GCC ATG GCC GAG CTC ATG AAC CAC AGG 1056

Ser Tyr Leu Val Leu Glu Ser Ala Met Ala Glu Leu Met Asn His Arg

335 340 345 350

CGC GTC ATG AAG AAG CTG CAA GCG GAG GTA CGG GCG TAC GGA GCG GAG 1104

Arg Val Met Lys Lys Leu Gin Ala Glu Val Arg Ala Tyr Gly Ala Glu

355 360 365

AAG AAG CTG GAC ATG ATC AGG GAG GAC GAC CTG AGC AGC CTG CCG TAC 1152

Lys Lys Leu Asp Met He Arg Glu Asp Asp Leu Ser Ser Leu Pro Tyr 370 375 380

CTA AAG GCG TCC ATG AAG GAA GCG CTG CGG CTG CAC CCA CCG GGG CCC 1200

Leu Lys Ala Ser Met Lys Glu Ala Leu Arg Leu His Pro Pro Gly Pro

385 390 395

CTG CTG CTG CCG CAC TAC TCC ACC GCC GAC TGC CAG ATC GAC GGG TAC 1248

Leu Leu Leu Pro His Tyr Ser Thr Ala Asp Cys Gin He Asp Gly Tyr

400 405 410

CAC ATC CCC GCC AAC ACG CGC GTC CTC GTG AAC GGC TGG GCC ATC GGC 1296 His He Pro Ala Asn Thr Arg Val Leu Val Asn Gly Trp Ala He Gly 415 420 425 430

AGA GAC CCG GCG GTC TGG GAG AAG CCC GAG GAG TTC ATG CCG GAG AGG 1344 Arg Asp Pro Ala Val Trp Glu Lys Pro Glu Glu Phe Met Pro Glu Arg 435 440 445

TTC ATG CGG GAC GGC TGG GAC AAG TCC AAC AGC TAC AGC GGC CAG GAC 1392 Phe Met Arg Asp Gly Trp Asp Lys Ser Asn Ser Tyr Ser Gly Gin Asp 450 455 460

TTC AGG TAC CTG CCG TTC GGG TCT GGG CGC CGG ATC TGC CCC GGG GCC 1440 Phe Arg Tyr Leu Pro Phe Gly Ser Gly Arg Arg He Cys Pro Gly Ala 465 470 475

AAC TTC GCG CTC GCG ACC ATG GAG ATC ATG CTC GCC AAC CTC ATG TAC 1488 Asn Phe Ala Leu Ala Thr Met Glu He Met Leu Ala Asn Leu Met Tyr 480 485 490

CAT TTC GAC TGG GAG GTC CCC AAT GAG AAG GAA GAC GGT GGC GGG AAG 1536 His Phe Asp Trp Glu Val Pro Asn Glu Lys Glu Asp Gly Gly Gly Lys 495 500 505 510

GTG AGC ATG GAC GAG ACG TTC GGG CTG ATG CTT CGC AGG AAC GAG CCG 1584 Val Ser Met Asp Glu Thr Phe Gly Leu Met Leu Arg Arg Asn Glu Pro 515 520 525

CTC TAC CTT GTT CCT AGG GCC GTC TAGCTAGCTA GTTACTGTGC CGCCGCCGCC 1638 Leu Tyr Leu Val Pro Arg Ala Val 530

TCCTATTCAA GAATGCATGA GTCTTAGACG TGTGGCAGTA ATAAATTGCG TGGGGTGTTG 1698

TAAAATAATA AGCACGTTTG TTGAGTTGTA ATGGATTCAC ATTATGTGCA TTCGAGACAG 1758

GTCCAAGGAA TTGTATCTCT AAAGTGAAAA TTTCCTTTTT CACCACGATG AATGCATGTC 1818

TACTCTCTCA CCGGTAAAAA AAAAAAAAA 1847

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 534 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Ala Leu Gin Ala Ala Tyr Glu Tyr Leu Gin Gin Ala Val Gly His 1 5 10 15

Gly Ala Trp Ser Ser Thr Gin Thr Leu Thr Leu Leu Leu He Ala Val 20 25 30

Pro Thr Val Leu Leu Leu Leu Ala Ser Leu Ala Lys Ser Thr Ser Ser 35 40 45

Ser Gly Arg Gly Lys Pro Pro Leu Pro Pro Ser Pro Pro Gly Thr Leu 50 55 60

Pro He Val Gly His Leu His His He Gly Pro Gin Thr His He Ser 65 70 75 80

Leu Gin Glu Leu Val Ala Lys Tyr Gly His Asn Gly Phe Leu Phe Leu 85 90 95

Arg Ala Gly Ala Val Pro Thr Leu He Val Ser Ser Pro Ser Ala Ala 100 105 110

Glu Ala Val Met Arg Thr His Asp His He Phe Ala Ser Arg Pro Trp 115 120 125

Ser Met Ala Ser His He Leu Arg Tyr Asn Thr Cys Asp Val Ala Phe 130 135 140

Ser Pro Leu Gly Glu Tyr Trp Gin Gin Thr Arg Lys Leu Met Asn Thr 145 150 155 160

His Leu Leu Ser Asn Lys Lys Val Tyr Ser Phe Arg His Gly Arg Glu 165 170 175

Glu Glu Val Cys Leu Val Val Asp Asn Leu Arg Glu Ala Ala Ala Lys 180 185 190

Ser Pro Ser Thr Ala Val Asp Met Ser Glu Val Leu Ala Ala Tyr Thr 195 200 205

Asn Asp Val Val Ser Arg Ser Val Leu Gly Ser Thr His Arg Lys Lys 210 215 220

Gly Arg Asn Thr Leu Phe Arg Glu Met Thr Met Thr Asn Val Asp Leu 225 230 235 240

Leu Val Gly Phe Asn Leu Glu Tyr Tyr He Pro Arg Trp Pro Leu Thr 245 250 255

Asp Leu Leu Phe Arg Leu Val Cys Trp Lys Val Thr Arg His Leu Lys 260 265 270

Arg Trp Asp Ala Leu Leu Glu Glu Val He His Glu His Val Glu Met 275 280 285

Arg Lys Leu Ser Gly Asp Lys Glu Lys Glu Ser Asp Asp Phe He Asp 290 295 300

He Phe Leu Ser Arg Tyr Glu Glu Tyr Gly Phe Thr Met Asp Asn Val 305 310 315 320 Lys Ser Leu Leu Met Asn Val Phe Glu Ala Ala He Glu Thr Ser Tyr 325 330 335

Leu Val Leu Glu Ser Ala Met Ala Glu Leu Met Asn His Arg Arg Val 340 345 350

Met Lys Lys Leu Gin Ala Glu Val Arg Ala Tyr Gly Ala Glu Lys Lys 355 360 365

Leu Asp Met He Arg Glu Asp Asp Leu Ser Ser Leu Pro Tyr Leu Lys 370 375 380

Ala Ser Met Lys Glu Ala Leu Arg Leu His Pro Pro Gly Pro Leu Leu 385 390 395 400

Leu Pro His Tyr Ser Thr Ala Asp Cys Gin He Asp Gly Tyr His He 405 410 415

Pro Ala Asn Thr Arg Val Leu Val Asn Gly Trp Ala He Gly Arg Asp 420 425 430

Pro Ala Val Trp Glu Lys Pro Glu Glu Phe Met Pro Glu Arg Phe Met 435 440 445

Arg Asp Gly Trp Asp Lys Ser Asn Ser Tyr Ser Gly Gin Asp Phe Arg 450 455 460

Tyr Leu Pro Phe Gly Ser Gly Arg Arg He Cys Pro Gly Ala Asn Phe 465 470 475 480

Ala Leu Ala Thr Met Glu He Met Leu Ala Asn Leu Met Tyr His Phe 485 490 495

Asp Trp Glu Val Pro Asn Glu Lys Glu Asp Gly Gly Gly Lys Val Ser 500 505 510

Met Asp Glu Thr Phe Gly Leu Met Leu Arg Arg Asn Glu Pro Leu Tyr 515 520 525

Leu Val Pro Arg Ala Val 530

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GTAATAAATT GATGAAACGA AAATTAAAGG AATATTAATC CTCCGTCCTC CAAGAAAATT 60 AAAGGAATAA TCCTGCATGA ATTAATTATA CGCGCAG 97

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 124 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : GTAATCGATT CCATCGTCGT CCTCCTCATT ATTATTTTGG GCTAGAATCC ATGCATATAA 60 ATAGGCTGAG GCTCTCAACG TAATTAACTT CCTTTCCATG TGGCCGGCCG GCTTGACAAA 120 ACAG 124

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : CGGGATCCGA RGARTTYMGN CCNGARMG 28

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CGGAATTCTT TTTTTTTTTT TTT 23 (2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : AATTCGCGGC CGCTTTTTTT TTTTTTTT 28

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACATGATGCG GCCCTCTAGA 20

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GCAAGCTTAT GGCACTCCAG GCAGCCTAC 29

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CAGGATCCGC ACCCACGACC ACATCTT 27

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTAAGCTTGC TCTTCAGGCT CGTGT 25

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CAGATCGACG GGTACCACA 19

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CGCTCGAGAC TCATGCATTC TTGGATAGG 29

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CTTCTAGAGT CGGCGGTGGA GTAGTGC 27

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CATCTAGAAC AGCTTCCTCA TCTCCAC 27

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGTCTAGAGG CACAGTAACT AGCTA 25

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TCATGTCCAC GGCCGTCGAC 20 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CAGGATCCAT CGCSGAGCTS GTSAACCAY 29

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GTGAATTCRA AGTTCTGSAC SAGSCGGCC 29

Claims

HAT IS CLAIMED IS:

1. An isolated and purified polypeptide of about 534 amino acids comprising the amino acid sequence of SEQ ID NO: 2.

2. An isolated and purified polynucleotide comprising a nucleotide sequence consisting essentially of a nucleotide sequence selected from the group consisting of: a) the sequence of SEQ ID NO:l; b) sequences that are complementary to the sequence of (a) ; c) sequences that, on expression, encode a polypeptide identical or similar to that encoded by the sequence of (a) .

3. The polynucleotide of claim 2 that is a DNA molecule.

4. The polynucleotide of claim 3 wherein the nucleotide sequence is SEQ ID NO:l.

5. The polynucleotide of claim 2 that is an RNA molecule.

6. An expression vector comprising the DNA molecule of claim 3.

7. The expression vector of claim 6 further comprising a promoter operatively linked to the polynucleotide.

8. The expression vector of claim 6 wherein the polynucleotide has the nucleotide sequence of SEQ ID N0:1.

9. An oligonucleotide of from about 15 to about 50 nucleotides containing a nucleotide sequence of at least 15 nucleotides that is identical or complementary to a contiguous sequence of the polynucleotide of claim 2.

10. A host cell transformed with the expression vector of claim 6.

11. The transformed host cell of claim 10 that is a yeast cell.

12. The transformed host cell of claim 10 that is a plant cell.

13. The transformed host cell of claim 10 that is a bacterial cell.

14. A transgenic plant containing an isolated polynucleotide having SEQ ID NO: 1 wherein said plant is resistant to herbicides.

15. A method for controlling undesired vegetation in a location where a plant that has been transformed with an isolated polynucleotide having SEQ ID NO: 1 and is resistant to one or more herbicides is being cultivated, the method comprising the step of applying to the location an effective amount of one or more herbicides .

16. The method of claim 15 wherein the herbicides are triasulfuron.

17. A method for identifying compounds having herbicidal activity, the method comprising the steps of: a. transforming an organism with an isolated polynucleotide having SEQ ID NO: 1; b. treating the transformed organism with one or more compounds; and c. identifying and selecting those compounds exhibiting herbicidal activity.