MXPA99000939A - Hppd gene and inhibitors - Google Patents

Abstract

The nucleic acid sequence encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD) from Arabidopsis thaliana is disclosed. Also, a vector containing the DNA coding for HPPD, and transformed cells are disclosed. In addition, the description teaches of methods for the identification herbicide resistant HPPD, and herbicides which are inhibitors of HPPD as well as a method of conferring herbicide resistant on plants. Furthermore, the description teaches of a method for weed control.

Description

HPPD GENES AND INHIBITORS Field of the Invention This invention pertains to DNA encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD), herbicides that inhibit HPPD and methods for screening compounds to identify herbicides that inhibit HPPD. The invention also pertains to HPPD variants which are resistant to the herbicide inhibiting action, methods for screening HPPD variants and plants comprising HPPD resistant to herbicides. Background of the Invention In plants, 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13,11.27) is a key enzyme in the biosynthesis of plastoquinones and tocopherols. The 4-hydroxyphenylpyruvate acid (derived from corysmic acid via the Shikimate pathway) is oxidized and decarboxylated by HPPD to homogentisic acid (Fiedler and Schuitz, Dev. Plaru Biol. 8: 537, 1982, Fiedler et al., Planta 155: 511 1982). The subsequent proliferation and decarboxylation of homogentisic acid results in an arrangement of plastoquinones and tocopherols. In animals, HPPD is involved in tyrosine catabolism. A genetic deficiency of this route in humans and mice leads to a hereditary tyrosinemia type 1. This disease can be treated by NTBC (2- (2-nitro-4-trifluoromethyl-Ibenzoyl) -1,3-cyclohexanedione, an inhibitor of HPPD, which prevents the accumulation of intermediates and tyrosine catabolism that are hepatotoxic (Ellis et al., Tax. And Appl. Pharm., 133: 12, 1995.) Since plastoquinones and tocopherols are essential compounds for plants, inhibitors of this Enzyme are potential herbicides A class of H PPD inhibitors, the tricetones, have recently been shown to possess herbicidal activity (Prisbylia et al., Brighton Crop Protection Conference: Weeds, British Crop Protection Council, Surrey, UK, pp. 731-738, 1993; Schuiz et al., FEBS Letts., 318: 162, 1993). Selective herb bleaching (2- (2-chloro-4-methanos ulf onylbenzoyl) -1- 3-cyclohexanedione) of corn causes strong bleaching in plants. susceptible accompanied by a per of carotenoids and chlorophyll with an increase in phytoene and tyrosine (Barta et al., Pest. Sci. 45: 286, 1995; Soeda and others, Pestic. Biochem. Physiol. 29: 35, 1987; Mayonado and others, Pestic. Biochem. Physiol, 35: 138, 1989). The treatment of Lemna with sulcotrione severely inhibited the growth and the effect of herbicide could be abolished with homogentisic acid. It was shown that the partially purified enzyme extracted from corn was severely inhibited by sulcotrione with a calculated ICSo of 45 mM (Schuiz et al., 1993, supra). HPPD analysis of partially purified weed (Echinochloa crus-galli L.) showed that sulcotrione is a potent competitive inhibitor of the enzyme with a 9.8 mM KI (Secor, Plant Physiol 106: 1429, 1994). Canadian Patent Application No. 2,116,421 describes the identification of HPPD inhibitors derived from 2-benzoylcyclohexamine 1,3-diones. An albino mutant (psdl) isolated from a T-DNA labeled as an Arabidopsis population was originally selected by virtue of a severe pigment deficiency, which was thought to be due to a defect in carotenoid biosynthetic genes (Norris et al., Plant Cell 7: 2139, 1995). When the albino psdl mutant was germinated in an MS2 medium and subsequently transferred to an MS2 medium supplemented with 4-hydroxyphenylpyruvate (OHPP) or homogentisic acid (HGA), the plants turned green on HGA but not on OHPP. Further analysis of this mutant indicated that the defect that causes the albino phenotype is not due to a mutation in a carotenoid biosynthesis enzyme directly, but results from a mutation in H PPD that prevents the biosynthesis of a plastoquinone essential for biosynthesis of carotenoid. Despite the importance of this route in plants, the genes encoding plant enzymes for the biosynthesis of plastoquinone and tocopherol have not been previously isolated. Thus, there is a need in the art for methods and compositions that provide H PPD genes, H PPD inhibitors useful as herbicides and HPPD variants resistant to herbicides. The inventors of the present invention have isolated the gene encoding H PPD from plants, have expressed it in E. coli, and have shown that the H PPD of bacterially expressed plants is enzymatically active and that its enzymatic activity is inhibited by tricetone herbicides. Brief Description of the Drawings Figure 1. is an illustration of the amino acid sequence of 4-hydroxyphenylpyruvate dioxygenase (H PPD) from Arabidopsis thaliana (AtH PPD) and shows the alignment of this sequence with related sequences of mice, humans, pigs and Streptomyces avermítílis (S.A. ver.). Figure 2 is a graphic illustration of the production of brown pigment by E. coli transformed with the H PPD gene of Arabidopsis ("Arabidopsis") compared to E. coli transformed with a control vector ("plasmid"). The effect on pigment formation is shown by adding increasing concentrations of tyrosine to the culture medium. Figure 3A is an illustration of the elution profile of CLAR on the E. coli medium transformed with a control vector. The Figure 3B is an elution illustration of a CLAR profile of the E. coli medium transformed with the H PPD gene of Arabidopsis.

The elution position of the normal homogentisic acid normal is indicated by an arrow. The insert in Figure 3B is an illustration of the absorption spectrum of the homogentisic acid peak. Figure 4 is a graphic illustration of the effect of increasing concentrations of sulcotrione on the enzymatic activity of HPPD of extracts derived from E. coli cells transformed with the HPPD gene of Arabidopsis. SUMMARY OF THE INVENTION The present invention provides isolated purified nucleic acids encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD) from plants, in particular HPPD derived from Arabidopsis thaliana, as well as conservative sequence variants and conservative variants of their function; DNA vectors comprising nucleic acid encoding HPPD operably linked to a transcription regulatory element; and cells comprising HPPD vectors, including, without limitation, bacterial, fungal, plant, insect and mammalian cells. In one embodiment, a bacterial cell that expresses high levels of HPPD of plants is provided. Also encompassed are HPPD polypeptides and enzymatically active fragments derived therefrom. In another aspect, the invention provides methods for identifying herbicide / HPPD inhibitors, which are carried out by: (a) providing a microbacterial cell expressing HPPD from plants; (b) incubating the cells in the presence of a test compound to form a test culture and in the absence of a test compound to form a control culture; (c) monitor the level of homogentisic acid or oxidation products thereof, in the test and control cultures; and (d) identifying as a compound that inhibits H PPD and compound that reduces the level of homogentisic acid or oxidation products thereof, in the test culture in relation to the control culture. In the above methods, the monitoring step can be achieved, for example, by measuring the absorbance of the cultures at 450 nm or by visually detecting the detection of a brown pigment. Alternatively, an inhibitor is identified as a compound that inhibits the growth of the test culture, wherein the inhibition can be reversed by the addition of homogentisic acid to the culture. In a further aspect, the invention provides methods for identifying variants of H PPD resistant to herbicides, which are carried out by: (a) providing a population of cells expressing H PPD; (b) mutagenizing the cell population; (c) contacting the mutagenized population of cells with a herbicide, under inhibitory conditions for the growth of non-mutagenized cells; (d) recovering cells resistant to the inhibitory effects of the herbicide by the growth and / or formation of the pigment; and (e) sequencing the nucleic acid encoding HPPD from the recovered cells. Alternatively, the HPPD encoding DNA is subjected to site-directed random mutagenesis in vitro, followed by expression in a heterologous cell and screening or screening for cells that inhibit herbicide resistance.

In still another aspect, the invention encompasses variant proteins of H PPD that are resistant to herbicides. Preferably, a variant H-PPD herbicide-resistant protein, when expressed, in a cell that requires H PPD activity for availability, exhibits: (i) catalytic activity alone, sufficient to maintain the viability of a cell in which expresses; or catalytic activity in combination with any variant H-PPD herbicide-resistant protein also expressed in the cell, which may be the same as or different than the first variant protein of H PPD, sufficient to maintain the availability of a cell in which it is expressed; and (ii) catalytic activity that is more resistant to the herbicide than its wild-type PPD H. Also provided are nucleic acids encoding H PPD variants resistant to herbicides, DNA vectors comprising the nucleic acids and cells comprising the vectors encoding H PPD variants. Genes encoding H PPD variants resistant to herbicides can be used as genetic markers, such as, for example, in plasmids and methods for the introduction and selection of any other desired gene. In another aspect, the present invention provides a method for conferring resistance to herbicides on a cell or cells and particularly a cell or plant cells, such as, for example, a seed. A gene of H PPD, preferably the H PPD gene of Arabidopsis thaliana, is mutated to alter the ability of a herbicide to inhibit the enzymatic activity of the H PPD. The mutant gene is cloned into a compatible expression vector, and the gene is transformed into a herbicide-responsive cell under conditions in which it is expressed at levels sufficient to confer herbicide resistance on the cell. Methods for seed control are also contemplated, wherein a culture containing an H PPD gene of herbicide resistance, according to the present invention, is grown and treated with an effective amount of the herbicide for weed control. Detailed Description of the Invention The present invention encompasses purified, isolated nucleic acids encoding plant 4-hydroxyphenylpyruvate dioxygenase (H PPD), expression systems in which enzymatically active HPPD is produced and screening methods for identifying H inhibitors. PPD. The present invention also encompasses methods for screening and producing HPPD variants of plants that are resistant to the inhibitory action of herbicides, DNA encoding these variants, vectors including these DNAs, variant H PPD proteins and cells expressing these variants . Additionally, methods to produce resistance to herbicides in plants are provided by expressing these variants and weed control methods. Isolation and Characterization of the HPPD of Arabidopsis Encoding the Gene The present inventions have isolated and sequenced the gene encoding H PPD from Arabidopsis thaliana, using the methods described below. In short, a cDNA bank of Arabidopsis thaliana? Yes (The ledge and others, Proc. Nati. Acad. Sci. USA 8_8 .: 1731, 1991) was sieved using the method in RCP (Amaravadi et al., Bio Techniques 1_6: 98, 1994). Initiators: a forward primer, designated ATHPPD1 F (5'-CGTGCTCAGCGATGATCAGA-3 ') and an inverse primer, designated ATHPPD 1 R (5'-CGGCCTGTCACCTAGTGGTT-3') were synthesized based on an Arabidopsis EST sequence (GenBank ID No : T20952) that showed homology to the H PPD sequences of mammals. The primers were evaluated in a polymerase chain reaction (PCR) using as a DNA standard a 1 μl aliquot (containing 3 x 10 6 pfu / ml) from the cDNA phage bank. For PCR, 50 μl of reaction, contained 1 X of RCP buffer, 200 mM of each deoxynucleoside triphosphate, 1.25 units of AmpliTaq DNA Polymerase (all of Perkin Elmer) and 7.5 pmoles of each primer. The reaction mixture was heated at 95 ° C for 2 minutes and amplified using 35 cycles of: 95 ° C for 1 minute, 48 ° C for 2 minutes, 72 ° C for 1 minute, 30 seconds. This was followed by incubation at 72 ° C for 7 minutes. A fragment of predicted size of 12 bp was produced. This fragment was cloned into the pCRI I vector (TA Cloning Kit, Invitrogen) and sequenced and found to be identical to the Arabidopsis EST sequences (with the addition of 3 residues that were not determined in the reported EST sequence) . Bank screening: the cDNA library was plated on 13 plates containing NZCYM agar at a density of 40, 000 pfu / plate. The phage of each plate was eluted in SM and the aliquots of 13 individual phage combinations were used as standards for PCR with the pair of primers ATH PPD 1 F and ATH PPD 1 R. The CPR conditions were as described above. (In the first round, 1 μl of each eluted phage combination was used as a standard and 5 μl was used in the subsequent turns). In the first round, ten of the thirteen phage combinations were positive for CPR. One of the positive combinations was selected for additional sieving. In the second round, eluates of 10 plates of 5,000 pfu / plate gave 1 positive combination. In the third round, 10 plates of approximately 20 pfu / plate gave 2 positive combinations. The positive combinations of the third round were seeded into plates and 36 individual plates were picked and sifted to find a single positive plate of H PPD. The plasmid having the insert was excised from this phage via the properties of automatic subcloning of the vector. Restriction analysis indicated that this plasmid contained a 1.5 kb insert. Sequence analysis: the template DNA to be sequenced was prepared using the Wizard DNA Purification System (Promega). The sequencing reactions were carried out using the DNA Sequencing System fmoles (Promega) and the sequence gels were run on Hydrolink Long Ranger gels (AT Biochem). The insert of the plasmid containing H PPD isolated from the cDNA library was sequenced using two primers that hybridize to the vector? Yes on opposite sides of the Xhol cloning site in addition to a series of internal primers: ATH PPD1 F ATH PPD 1 R as before; and ATHPPD2F (5'-CTTCTACCGATTAACGAGCCAGTG-3 '); ATHPPD2R (5'-CACTGGCTCGTTAATCGGTAGAAG-3 '); ATHPPD3F (5'-TCCATCACATCGAGTTCTGGTGCG-3 '); ATHPPD3R (5'-AAAAGGAATCGGAGGTCACCGGA-3 '); ATHPPD4F (5'-CTGAGGTTAAACTATACGGCGA-3 '); and ATH PPD4R (5'TCGCCGTATAGTTTAACCTCAG-3 '). All the sequence information was confirmed by sequencing both threads. The translation of the nucleotide sequence of H P PD, sequence comparisons and multiple sequence alignments were carried out using The Wisconsin Package software, Version 8.0 (Genetics Computer Group, Madison, Wisconsin). The results indicated that the 1.5 kb insert contains an open reading frame of 445 amino acids (Figure 1). A TFASTA investigation of the GenEMBL database identified five sequences known to have partial homology: Streptomyces H PPD (U 1 1864); F rat alloantigen (M 18405), H PPD of mouse (D29987); H PPD of pig (D 13390) and H PPD of human (X72389). Comparisons in the form of direct pairs of the Arabidopsis sequence with those mentioned above; showed an average similarity of 56% and average identity of 37%. Additionally, a number of conserved tyrosine and histidine residues have been observed, which have been proposed as metal binding sites in H PPD of mammals (Ruetschi et al., Eur.J. Biochem. 205: 459, 1992; Denoya et al. , J. Bacterio !. 176: 5312, 1994), in the sequence of Arabidopsis. Genomic organization of the H PPD gene in Arabidopsis: Southern blot analysis was performed using genomic DNA prepared from Arabidopsis seedlings according to the Dellaporta method (Dellaporta et al., Plant Mol. Biol. Rep. 1: 19, 1983). 10μG of DNA were digested with the restriction enzymes Bam HI, EcoRI and Hindl ll, after which the digestions were separated on a 0.9% agarose gel, transferred to a Duralon-UV Membrane (Stratagene) using the System of VacuGene plot (Pharmacy) and were interlaced using the Stratalinker UV interlayer (Stratagene). The H PPD probe was prepared by: (i) gel purifying (using the GeneClean, Bio 101, I nc.) Xhol / Sstl fragment of the DNA digestion of plasmid H PPD /? Yes. The fragment contains 50 upstream sequence bases over the ATG start codon and extends to a 55 base position upstream of the TFA retention codon; and (ii) marking the fragment using Fluorine Marking Fluorine Prime-lt (Stratagene). The labeled probe was hybridized to the membrane for 2 hours at 68 ° C using the QuikHyb Rapid Hybridization Solution (Stratagene). The membrane was washed with 0.1 X SSC / 0.1% SDS at room temperature and two times at 60 ° C, after said hybridization was visualized using the Non-Radioactive Detection System I luminator (Stratagene). Only one band hybridized to the probe under conditions of high restriction in both Bam H i and H l nd l l l digestions. Two bands were observed in the EcoRI digestions, reflecting the presence of an internal EcoRI site in the H PPD sequence. These results suggested that H PPD is encoded by a single copy gene in Arabidopsis. The entire coding sequence of H PPD is amplified from the Arabidopsis genomic DNA using ATH primers PPD5F (5'-CCATGGGCCACCAAAACG-3 ') and ATH PPD5R (5'-CTGCAGTCATCCCACTAACTGTTTG-3'). The resulting genomic H PPD fragment, which was slightly longer than the corresponding cDNA fragment, was cloned into the pCRI l vector (TA Cloning Kit, Invitrogen) and sequenced. A single intron of 107 bp was detected, located at the position of nucleotide 1 163-165 of the cDNA sequence. Nucleic Acids, Vectors, Expression Systems and Polypeptides In the practice of the present invention, many techniques were used in molecular biology, microbiology, recombinant DNA and protein biochemistry such as those fully explained in, for example, Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and I I, 1984 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Transcription and Translation, 1984 (Hames and Higgins eds); A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Pres, Inc.); and Protein Purificaction: Principles and Practice, Second Edition (Springer-Verlag, N .Y.). The present invention encompasses nucleic acid sequences encoding plant H PPD, enzymatically active fragments and derivatives thereof and related H PPD derived sequences from other plant species. As used herein, a nucleic acid that is "derived from" a sequence of H PPD, refers to a nucleic acid sequence corresponding to a region of the sequence, sequences that are homologous or complementary to the sequence and " conservative variants of sequences "and" conservative variants of function ". Conservative sequence variants are those in which a change of one or more nucleotides at a given codon position does not result in alteration in the amino acid encoded at that position. Conservative variants of function are those in which a given amino acid residue in H PPD has been changed without altering the overall conformation and function of the H PPD polypeptide, including, but not limited to, the replacement of an amino acid with one having properties similar physico-chemical (such as, for example, acidic, basic, hydrophobic and the like). Fragments of H PPD that retain enzymatic activity can be identified according to the methods described herein, e.g. , expression in E. coli followed by enzymatic analysis of the cell extract. H PPD sequences derived from plants other than Arabidopsis thaliana can be isolated by routine experimentation using the methods and compositions provided herein. For example, hybridization of a nucleic acid comprising all or part of the H PPD sequence of Arabidopsis under intermediate restriction conditions (such as, for example, aqueous 2X SSC solution at 65 ° C) to cDNA or genomic DNA derived from other plant species can be used to identify H PPD homologs. The cDNA libraries derived from different plant species are commercially available (Clontech, Palo Alto, CA, Stratagene, La Jolla, CA). Alternatively, PCR-based methods can be used to amplify related HPPD sequences from cDNA or genomic DNA derived from other plants. The expression of the sequence identified in, v. gr. , E. coli, using methods described in greater detail below, was carried out after the enzymatic activity of the polypeptide encoded by the sequence corresponds to that of H PPD. Consequently, HPPD sequences derived from dicotyledonous and monocotyledonous plants are within the scope of the invention.

The nucleic acids of the present invention include polymers containing purine and pyrimidine of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polyideoxyribo nucleotides. These include, single-stranded or double-stranded molecules, ie DNA-DNA, AD N-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (AN P) formed by conjugating bases of the structure of the base of amino acids. This also includes nucleic acids containing modified bases. The nucleic acids can be isolated directly from the cell. Alternatively, the PCR can be used to produce the nucleic acids of the invention, using chemically synthesized strands or genomic material as standards. The PCR initiators can be synthesized using the sequence information provided herein and can also be designed to introduce appropriate new restriction sites, if appropriate, to facilitate incorporation into the given vector for recombinant expression. The nucleic acids of the present invention can be flanked by natural Arabidopsis regulatory sequences, or can be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5 'and 3 regions. 'without coding and similar. The nucleic acids can also be modified by any means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the nucleotides present in nature with an analog and internucleotide modifications such as, for example, those with uncharged linkages (eg. methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged ligatures (e.g., phosphorothioates, phosphorodithioates, etc.). The nucleic acids may contain one or more of the covalently linked portions, such as, for example, proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (v. gr., acridine, psoralen, etc.) chelators (eg, metals, radioactive metals, iron, oxidative metals, etc.), and alkylating agents. The nucleic acid can be derived by the formation of a methyl or ethyl phosphotriester or an alkyl phosphoroamidate ligature. In addition, the nucleic acid sequences of the present invention can also be modified with a tag capable of providing a detectable signal, either directly or indirectly. Illustrative labels include radioisotopes, fluorescent molecules, biotin, and the like. The invention also provides nucleic acid vectors comprising the described H PPD sequences or derivatives or fragments thereof. A large number of vectors, including plasmid and fungal vectors, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Nonlimiting examples include pKK plasmids (Clontech), pUC plasmids, pET plasmids (Novagen, I nc Madison Wl), or pRSET or pREP (Invitrogen, San Diego, CA), and many appropriate host cells using methods described or cited herein or in some manner known to those skilled in the art of the disclosure. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. , antibiotic resistance and one more expression rolls. Suitable host cells can be transformed / transfected / infected as appropriate by any suitable method including electroporation, CaCI2 mediated DNA uptake, fungal infection, microinjection, micropoiectil or other established methods. Suitable host cells include, bacteria cells, archaebacteria, fungi, especially yeast and plant and animal, especially mammalian cells. Of particular interest are £. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carisbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora and CHO cells, COS cells, HeLa cells, and myeloid and lymphoid cell lines of immortalized mammals. Preferred replication systems include M 13, Col B 1, SV40, baculovirus, lambda, adenovirus and the like. A large number of transcription initiation regions and termination regulators have been isolated and shown to be effective in the transcription and translation of heterologous proteins in different hosts. Examples of these regions, alignment methods, manner of handling, etc. , are known in the art. Under appropriate expression conditions, host cells can be used as a source of produced recombinant H PPD-derived peptides and polypeptides. Advantageously, the vectors may also include a transcriptional regulatory element (i.e., a promoter) operably linked to the H-PPD portion. The promoter may optionally contain portions and / or ribosome binding sites. Non-limiting examples of bacterial promoters compatible with E. coli include, trc promoter, β-lactamase (penicillinase) promoter, lactose promoter; tryptophan (trp) promoter; BAD arabinose operon promoter, lambda derivative P 1 promoter and ribosome binding site of the N gene; and the hybrid tac promoter derived from sequences of the UV5 promoters trp and lac. Non-limiting examples of yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, gaiactokinase promoter (GALI), galactoepimerase promoter and alcohol dehydrogenase (ADH) promoter. Suitable promoters for mammalian cells include, without limitation, viral promoters such as Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV) and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences and poly A addition sequences and enhancer sequences that increase expression may also be included. Sequences that cause gene amplification may also be convenient. In addition, sequences that facilitate the secretion of the recombinant product from the cells, including, but not limited to, bacteria, yeast and animal cells such as secretory signal sequences and / or hormone region sequences, may also be included. Nucleic acids encoding the wild-type polypeptides or H H PD variants can also be introduced into cells from recombination events. For example, said sequence can be introduced into a cell and therefore can perform homologous recombination at the site of an exogenous gene or a sequence with substantial identity to the gene. Other methods based on recombination, such as non-homologous recombinations or deletion of endogenous genes by homologous recombination, can also be used. H-HPD-derived polypeptides according to the present invention, including H-HPD function-conserving variants, can be isolated from wild-type or mutant Arabidopsis cells or from heterologous cell organisms (including, but not limited to, bacteria, fungi, insects, plants and mammals) in which the coding sequence of proteins derived from HH PD has been introduced and expressed. In addition, the polypeptides can be part of recombinant fusion proteins. Alternatively, the polypeptides can be chemically synthesized by commercially available automatic methods, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. "Purification" of a H H PD polypeptide refers to the isolation of the H H PD polypeptide in a form that allows the enzymatic activity to be maintained without interference from other components of the cell in which the polypeptide is expressed. Methods for polypeptide purification are well known in the art, including, without limitation, preparative disk gel electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography, and counter-current distribution. . For some purposes, it is preferable to introduce the polypeptide into a recombinant system in which the H H PD protein contains an additional label sequence that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can be purified from a crude lysate of the host cell by chromatography on an appropriate solid phase matrix. Alternatively, antibodies raised against HHPD against peptides derived therefrom can be used as purification reagents. Other methods of purification are also possible. The present invention also encompasses derivatives and homologues of H H PD polypeptides. For some purposes, the nucleic acid sequences encoding the peptides can be altered by substitutions, additions or deletions that provide functionally equivalent molecules. That is, conservative variants of function. For example, one or more amino acid residues within the sequences can be replaced by another amino acid of similar properties, such as, for example, positively charged amino acids (arginine, lysine and histidine); negatively charged amino acids (aspartate and glutamate), polar neutral amino acids; and non-polar amino acids. The isolated polypeptides can be modified, for example, by phosphorylation, sulfonation, acylation or other protein modifications. They can also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds. Screening Methods for Identifying HPPD Inhibitors / Herbicides The methods and compositions of the present invention can be used to identify compounds that inhibit the function of HH PD and are therefore useful as herbicides or as major compounds for the development of useful herbicides. . This is achieved by providing a cell that expresses H H PD and therefore produces a homogentisic acid of 4-hydroxyphenylpyruvate (OH PP). Cell cultures expressing H H PD are incubated in the presence of test compounds to form test cultures and in the absence of test compounds to form control cultures. The incubation is allowed to proceed for a sufficient time and under appropriate conditions to allow interference with the function of H H PD. At a predetermined time after the start of incubation with the test compound, an analysis was carried out to monitor the enzymatic activity of H H PD. In a preferred embodiment, the activity of H HPD is monitored visually by the appearance of red-brown pigments produced by oxidation and / or polymerization of homogentisic acid (La Du et al., Ochronois, Pigments in Pathology, M. Wolman (ed.) , Academic Press, NY, 1969). Alternatively, the enzymatic activity of H H PD can be monitored in cell extracts, using conventional assays such as those described in the following Example 1. Additional controls, with respect to the culture samples and the test samples are also included, so that, for example, a host cell that does not express HH PD (e.g., a host cell transformed with an expression plasmid) containing the HH PD gene in a reverse orientation or without insert). The H H PD inhibitor compounds are identified as those that reduce the activity of H H PD in the test cultures relative to the control cultures. Host cells that can be used in the practice of the present invention include, without limitation, bacterial, fungal, insect, mammalian and plant cells. Preferably, bacterial cells are used. More preferably, the bacterial cell is a variant (such as, eg, the tmp mutant of E. coli) exhibiting increased membrane permeability for the test compounds relative to the wild-type host cell. Preferably, the methods of the present invention are adapted to a high pass screen, allowing a multiplicity of compounds to be approved in a single analysis. Said inhibitor compounds can be found, for example, in natural product banks, fermentation banks (including plants and microorganisms), combinatorial banks, compound files and libraries of synthetic compounds. For example, libraries of synthetic compounds are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, U K), Comgenex (Princeton, NJ), Brandon Associates (Merri mack, N H) and Mrosrosource (New Mylford, CT). A rare chemical bank is available from Aldrich Chemical Company, Inc. (Milwaukee, Wl). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC) or are readily producible. Additionally, natural and synthetically produced benches and compounds are easily modified by conventional, chemical, physical and biochemical conventional means (Blondelle et al., Tib Tech 14:60, 1996). HHPD inhibitory assays according to the present invention is advantageous for adapting many different types of solvents and therefore allowing the testing of compounds from many sources.

Once a compound by the methods of the present invention has been identified as an inhibitor of H H PD, in-vivo and in vitro tests can be carried out to further characterize the nature and mechanism of the H H PD inhibitory activity. For example, the effect of a compound identified on the in vitro enzymatic activity of purified or partially purified H H PD can be determined as described in the following Example 1. Classical enzyme kinetic plots can be used to distinguish, eg, , competitive and non-competitive inhibitors. The compounds identified as H H PD inhibitors using the methods of the present invention can be modified to improve potency, efficacy, absorption, stability and suitability for use in commercial herbicide applications, etc. These modifications are achieved and tested using methods well known in the art. Isolation of Herbicide Resistant HPPD Variants The present invention encompasses the isolation of H H PD variants that are resistant to the action of inhibitors / herbicides of H H PD. H H PD variants can occur naturally or can be obtained by random or site-directed mutagenesis. In one embodiment, a population of cells or organisms expressing HH PD was mutagenized using methods well known in the art, after which the cells or organisms are screened or screened to identify those that are resistant to toxic effects. of an HHPD inhibitor. The variant HHPD gene is then isolated from the resistant cell or organism using; v.gr., CPR techniques. In another embodiment, an isolated HHPD gene is subjected to random or site-directed mutagenesis in vivo, after which the mutagenized versions of the gene are re-introduced into an appropriate cell such as, eg, E. coli. and the cells are subjected to a selection or sieving procedure as before. The HHPD genes of variants are expressed in an appropriate host cell and the enzymatic properties of the variant HHPD polypeptides are compared to wild-type HHPD. Preferably, a given mutation results in a variant HHPD polypeptide that retains in vitro enzymatic activity toward 4-hydroxyphenylpyruvic acid (OHPP), ie, the conversion of OHPP to homogentisic acid (and therefore is expected to be biologically active in vivo), while exhibiting catalytic activity that is relatively more resistant to the selected herbicides than its wild-type HHPD. Preferably, when expressed in a cell that requires HHPD activity for viability, the HHPD variant exhibits (i) catalytic activity alone sufficient to maintain the viability of a cell in which it is expressed; or catalytic activity in combination with any herbicide-resistant variant HHPD protein is also expressed in the cell, which may be the same or different as the first HHPD protein, sufficient to maintain the viability of a cell in which it is expressed; and (ii) catalytic activity that is more resistant to the herbicide than its wild type H H PD. Therefore, any specific HH PD variant protein does not need to have the total catalytic activity necessary to maintain the viability of the cell, but it must have some catalytic activity in an amount, alone or in combination with the catalytic activity of additional copies that the same variant of HH PD and / or the catalytic activity of other variant proteins of HH PD, sufficient to maintain the viability of a cell that requires HHPD activity for viability. For example, catalytic activity can be increased to minimum acceptable levels by introducing multiple copies of a variant encoding the gene into the cell or by introducing the gene which also includes a relatively strong promoter to increase the production of the variant. The more resistant media than the catalytic activity of the variant are decreased by the herbicide (s), if, to a lesser degree than the catalytic activity of wild type H H PD is decreased in the herbicide (s). The most resistant, preferred HH PD variant retains sufficient catalytic activity to maintain the viability of a cell, plant or organism where at the same concentration of the same herbicide, wild type HH PD could not retain sufficient catalytic activity to maintain viability of the cell, plant or organism. Preferably, the catalytic activity in the absence of herbicide (s) is at least about 5% and, more preferably, is greater than about 20% of the catalytic activity of the wild type H H PD in the absence of herbicide (s). In the case of the H H PD variant resistant to triacetone, it is preferred that the variant protein of H H PD have (i) catalytic activity in the absence of the herbicide of more than about 20% of the catalytic activity of the wild type H H PD.; and (ii) catalytic activity that is relatively more resistant to the presence of the triacetone herbicides compared to the wild type H H PD. Herbicide resistant H H PD variants can be used as genetic markers of any cell that is normally sensitive to the inhibitory effects of the herbicide on growth and / or pigment formation. In one embodiment, the DNA encoding a herbicide-resistant H H PD variant is incorporated into a plasmid under the control of a suitable promoter.

Any desired gene can be incorporated into the appropriate gene where the final recombinant plasmid can be introduced into a herbicide-responsive cell. Cells that have been transformed with the plasmid are screened or sieved by incubation in the presence of a sufficient concentration of herbicides to inhibit the growth and / or formation of pigments. Plants Resistant to Chemicals and Plants that Contain Genes of HPPD Variants The present invention encompasses transgenic cells, including, but not limited to, seeds, organisms and plants into which genes encoding herbicide-resistant variants of HHPD have been introduced. The non-limiting examples of suitable recipient plants are listed in the following Table 1: TABLE 1 RECEPTOR PLANTS The expression of the variant HH PD polypeptides in transgenic plants confers a high level of resistance to herbicides including, but not limited to, tricetone herbicides such as, for example, sulcotrione, allowing the use of these herbicides during the cultivation of transgenic plants . Methods for introducing foreign genes into plants are known in the art. Non-limiting examples of such methods include Agrobacterium infection, particle bombardment, protoplast polyethylene glycol (PEG) treatment, protoplast electroporation, microinjection, macroinjection, culture injection, pollen tube route, dry seed inhibition. , laser drilling and electrophoresis. These methods were described in, for example, B. Jenes et al., And S.W. Ritchie and others. I n Transgenic Plants, Vol. 1, Engineering and Utilization, ed.

S.-D. Kung, R. Wu, Academic Press, Inc. Harcourt Brace Jovanovich 1993; and L. Mannonen et al., Critical Reviews in Biotechnology, 14: 2873-310, 1994. In a preferred embodiment, the DNA encoding a variant HH PD is cloned into a DNA vector containing an antibiotic resistance marker gene. and the plasmid containing recombinant HH PD DNA is introduced into Agrobacterium tumefaciens containing a Ti plasmid. This "binary vector system" is described in, for example, the U.S. Patent. No. 4,490,838, and in An et al., Plant Mol. Biol. Manual A3_: 1 -19 (1988). The transformed Agrobacterium is then co-cultivated with leaf disks of the recipient plant to allow infection and transformation of plant cells. The cells of transformed plants are then cultured in the regeneration medium, which promotes block formation, first in the presence of an appropriate antibiotic to select the transformed cells, then in the presence of herbicides. In plant cells successfully transformed with DNA encoding H H PD for herbicide resistance, shoot formation occurs even in the presence of herbicide levels that inhibit the formation of outgrowths of untransformed cells. After confirming the presence of variant HHPD DNA using, for example, polymerase chain reaction (PCR) analysis, the plants transformed and tested for their ability to withstand the herbicide spray and for their abilities for seed germination and initiation of roots and proliferation in the presence of herbicides. The methods and compositions of the present invention can be used for the production of HH PD variants resistant to herbicides, which can be incorporated into plants to confer selective herbicide resistance on plants. Intermediate variants of HH PD (for example, variants that exhibit sub-optimal specific activity but high resistance to herbicides or the inverse) are useful as standards for the design of second generation HH PD variants that retain adequate and high specific activity resistance. Herbicide resistant H H PD genes can be transformed into single or multiple copy crop species to confer herbicide resistance. Genetic engineering of crop species with reduced susceptibility to herbicides can: (1) Increase the spectrum and flexibility of application of effective and environmentally benign specific herbicides; (2) Increase the commercial value of these herbicides; (3) Reduce the weed pressure in crop fields by the effective use of herbicide on herbicide-resistant crop species in a corresponding increase in crop yield; (4) Increase sales of seeds for herbicide-resistant plants; (5) Increase resistance to crop damage by carrying the applied herbicides in a previous plantation; (6) Decrease susceptibility to changes in herbicide characteristics due to adverse weather conditions; and (7) Increase the tolerance of non-uniform or poorly applied herbicides. For example, the transgenic HPPD variant protein containing plants can be cultured. The culture can be treated with an effective amount to control the weed of the herbicide to which the transgenic plant of the HPPD variant is resistant, resulting in weed control in the crop without deleteriously affecting the crop. DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples are intended to illustrate the present invention without limitation. Example 1: Expression of HPPD of Arabidopsis in £. coli The following experiments were carried out to demonstrate the production of high levels of HPPD from enzymatically active Arabidopsis in £. coli A. Cloning and Bacterial Transformation: The HPPD coding sequence is cloned into the expression vector pKK233-2 (Clontech) so that the ATP initiation codon of HPPD was in frame with the trc promoter using the method based on RCP A designated initiator ATHPPD6F (5'GAAATCCATGGCACCAAAACG-3 '). which hybridizes in the region of the start codon of H PPD (in bold) include a single base change (C of A, in italics) to generate a Ncol site (underlined). The initiator ATH PPD6R (5'-CTTCTCCATGGTCATCCCACTAACTGT-3 '), which hybridizes in the region of the H PPD retention codon (in bold), includes an Ncol site outside the coding region (underlined). A PCR reaction was carried out using the above primers and, like the template DNA, the HPPD sequence was isolated from the cDNA library screen described above. The reaction mixture (100 μl) contained the following components: 2 ng of plasmid DNA; pH regulator solution of 1 X PCR, 200 mM of each deoxynucleotide triphosphate; 2.5 units of AmpliTaq DNA Polymerase (Perkin Elmer); 13 pmol initiator ATH PPD6F; and 11 pmol of initiator ATHPPD6F. The reaction mixture was heated at 95 ° C for 2 minutes, and then amplified 30 cycles of: 95 ° C, 1 min; 55 ° C, 2 min; 72 ° C, 1.5 minutes. This was followed by incubation at 72 ° C for 7 minutes. A PCR product of 1.3 kb was amplified. The fragment was resolved on a GTG gel of 1.0 Nm Sieve (FMC) and purified (GeneClean, Bio 101). The purified fragment was digested with Ncol and ligated into the vector pKK233-2 treated with alkaline phosphatase digested with Ncol (Clontech). The ligation mixture was transformed into Competent Cells of Efficiency of Bank DH5a (GibcoBRL). Transformants expressing H PPD were identified by the red-brown color produced when cultured overnight in LB with ampicillin. The transformants were also prepared by transforming DH5a cells with the empty vector pKK233-2 for use as a control in the enzyme analyzes. B. Production of Pigment Coffee and Homogentisic Acid in E. coli The formation of brown pigment was observed in colonies grown on solid medium and in liquid cultures of E. coli transformed with the H PPD gene of Arabidopsis. No similar brown pigmentation was associated with the £. coli not transformed with the £. coli transformed with the control vector. The formation of brown pigment (which exhibited a characteristic absorption at 450 nm) was increased by supplementing the medium with tyrosine (Figure 2). It is known that homogentisic acid changes to coffee when it is at rest or when it is alkalinized and exposed to oxygen, due to the formation of an ochronotic pigment (La Du et al., In Ocrhornosis.

Pigments in Pathology, M. Wolman (ed.), Academic Press, NY, 1969).

Similar pigments are formed from the secretion that occurs naturally and the oxidation of homogentisic acid in certain bacteria (Trias et al., Can J. Microbiol 3_5: 1037, 1989; Goodwin and others, Can. J. Microbiol. 40:28, 1995). Therefore, the presentation of the brown pigment suggested that the cells of £. Coli transformed with the H PPD gene of Arabidopsis as described above produce large amounts of homogentisic acid. In addition, because tyrosine is metabolized by hydroxyphenylpyruvate (thus providing additional substrate for H PPD), the increased color development in the presence of increased tyrosine supports the conclusion that the brown pigment results from the activity of H PPD. This was confirmed by measuring the homogentisic acid directly using a CLAR-based method. The conditions of CLAR for the determination of homogentisic acid was identical to those described by Denoya et al. (J. Bacterio !. 179: 5312, 1994). The CLAR system consisted of a supply module of Waters 510 (Waters Assoc, Milford, MA), Waters 996 photodiode array detector, a WISP 710B automatic sampler and a Waters 840 data integration system. Reverse phase C18 Phenomenex Sperisorb 5 ODS (particle size 5mm, 250 X 4.6 mm id.), which was connected to a protective stainless steel column packed with C18 resin. The mobile phase (10 mM acetic acid: methanol: 85: 15 v / v) was run at a flow rate of 1 ml / min. The wavelength was set at 292 nM. The culture broth samples (1 ml) were acidified by mixing with 100 ml of glacial acetic acid and rinsed by centrifugation. 50 ml of the mixture was injected into the column. The peak corresponding to homogentisic acid was compared with a normal homogentisic acid for identification and quantification. The culture medium derived from overnight cultures of the cells of £. Control coli showed no trace of homogentic acid (Figure 3A). In contrast, the £. Coli transformed by HPPD produced a high level of homogentisic acid (Figure 3B). The peak eluting at 8 minutes co-migrated with authentic homogentisic acid and had an identical absorption spectrum with authentic homogentisic acid (insert). C. Analysis of HPPD activity The E. coli transformants were treated with 0.1 mg / ml lysosim in 50 mM potassium phosphate buffer (pH 7.3) at 30 ° C for 10 minutes. The cells were treated with sound (3 times, 5 seconds each, using an apparatus to deal with sound VibraCell, Sonics and Material, I nc., Danbury, CT) and the extract was subjected to centrifugation. The supernatant was desalted on an Econo-Pac 10DG column (Bio-Rad, Richmond, CA) which was equilibrated with 50 mM phosphate buffer (pH 7.3). The extract containing H desalted PPD was used for the analysis of H PPD. The enzymatic activity of H PPD was determined by the capture of 14CO2 released from hydroxyphenyl pyruvate 14C (Schuiz et al., FEBS Letts 318: 162. 1993, Secor, Plant Physiol. 106: 1429. 1994). The reactions were carried out in 20 ml scintillation flasks, each capped with a serum retentate through which a polypropylene well containing 50 μl of benzethonium hydroxide was suspended. Each 450 μl of the reaction mixture contained: 50 mM of potassium phosphate buffer (pH 7.3); 50 μl of a freshly prepared 1: 1 (v / v) mixture of 150 mM reduced glutathione and 3 mM dichlorophenyl indophenol; 2500 units of catalase; and bacterial extract (source of H PPD). Enzyme inhibitors were added when indicated. Hydroxyphenylpyruvate 14C (50 μl of a 2 mM solution), prepared according to the method of Secor (1994, supra), was added to initiate the reaction, which proceeded at 30 ° C for 30 minutes. The reaction was stopped by adding 100 μl of 4 N sulfuric acid and the mixture was incubated for an additional 30 minutes. The radioactivity trapped in benzethonium hydroxide was counted in a scintillation counter. The results indicated that the E. coli cells transformed with the H PPD gene of Arabidopsis expressed very high levels of H PPD activity, ie 2.7 μmoles / mg protein / hour. In contrast, the activity of HPPD was undetectable in cells of £. non-transformed or control coli. In addition, the activated H PPD was sensitive to inhibition by sulcotrione (Figure 4). The almost complete inhibition of activity was observed in more than 1 μM sulcotione. The concentration of sulcotrione required to cause 50% inhibition of activity was 100 nM. Example 2: High Step Screening of Test Compounds to Identify H PPD inhibitors The following method was used in a high pass mode to identify inhibitors of H PPD. The E. coli transformed with the H PPD gene of Arabidopsis as described in Example 1 above was grown overnight at 37 ° C in Luria Broth with 100 μg / ml ampicillin.

One liter of molten LB agar containing 100 μg / ml ampicillin and 1 μM tyrosine was cooled to 50 ° C. 0.1 ml of the culture of £. coli overnight was then added, 140 ml of the mixture was poured into each 9 x 9 sterile Sumilon biocharola (Vangard International, Neptune, NJ). The plates were allowed to solidify and dry for 30 minutes.

The test compounds (up to 25 μl) were applied to the test plate in sample wells (144 wells / plate, 5 cm diameter in a 12 x 12 arrangement) or in points (6 x 96 compounds / plate). Plates were incubated overnight at 37 ° C. The plates were classified by monitoring: (i) growth of £. coli and (i) intensity of the brown pigment. The areas in which the bacterial cells are viable but the pigment is reduced are classified as positive for H PPD inhibitors. All patents, applications, articles, publications and test methods mentioned above are incorporated herein by reference. Many variations of the present invention will be suggested by themselves to those skilled in the art in view of the above detailed description. Said obvious variations are within the intended full scope of the appended claims.

Claims (25)

1. CLAIMS 1. A purified isolated nucleic acid encoding HPPD from plants.
2. 2. A nucleic acid as defined in claim 1, derived from Arabidopsis thaliana.
3. 3. A nucleic acid as defined in claim 2, wherein the nucleic acid is selected from the nucleic acid of SEQ ID NO: 1, conservative sequence variants thereof and conservative variants of function thereof.
4. 4. A DNA vector comprising the nucleic acid sequence of claim 3, operably linked to a transcription regulatory element.
5. 5. A cell comprising a DNA vector as defined in claim 4, wherein the cell is selected from the group consisting of bacterial, fungal, plant, insect and mammalian cells.
6. 6. A cell as defined in claim 5, wherein the cell is a bacterial cell.
7. 7. A cell as defined in claim 5, wherein the cell is a plant cell.
8. 8. A seed comprising a cell as defined in claim 7.
9. 9. An HPPD protein comprising a protein encoded by a DNA as defined in claim 2.
10. 10. A method for identifying herbicide / H PPD inhibitors, the method comprising: (a) providing a microbial cell that expresses the H PPD of plants; (b) incubating the cell in the presence of a test compound to form a test culture and in the absence of a test compound to form a control culture; (c) monitor the level of homogentisic acid or oxidation products thereof, in the test and control cultures, and (d) identify as a compound that inhibits H PPD from any compound that reduces the level of homogentisic acid or oxidation products , in the test culture in relation to the control culture. eleven .
11. A method as defined in claim 10, wherein the microbial cell is £. coli
12. 12. A method as defined in claim 10, wherein the monitoring comprises measuring the absorbance of the cultures at 450 nm.
13. 13. A method as defined in claim 10, wherein the monitoring comprises detecting the formation of a brown pigment.
14. 14. A method for identifying variants of H PPD resistant to herbicides, the method comprising: (a) providing a population of cells expressing H PPD from plants; (b) mutagenizing the cell population; (c) contacting the mutagenized population of cells with a herbicide, under conditions inhibiting the growth or pigment production of non-mutagenized cells; (d) recovering cells resistant to the inhibitory effects of the herbicide on the growth and / or formation of the pigment; and (e) sequencing the nucleic acid encoding H PPD from the recovered cells to identify H PPD variants resistant to herbicides.
15. 15. A variant H PPD protein, where the protein is resistant to herbicides.
16. 16. A variant H PPD protein as defined in claim 15, wherein the variant H PPD protein, when expressed in a cell that requires H PPD activity for viability, exhibits: (i) catalytic activity alone sufficient to maintain the viability of a cell in which it is expressed; or catalytic activity in combination with any variant H-PPD herbicide-resistant protein also expressed in the cell, which may be the same as or different than the first variant H-PPD protein, sufficient to maintain the viability of a cell in which it is expressed; and (ii) catalytic activity that is very resistant to the herbicide than its wild type H PPD.
17. 17. A variant HPPD protein as defined in claim 15, wherein the protein is derived from Arabidopsis thaliana.
18. 18. A nucleic acid encoding a variant HPPD protein as defined in claim 15.
19. 19. A DNA vector comprising a nucleic acid as defined in claim 18.
20. 20. A cell comprising a DNA vector as was defined in claim 19, wherein the cell is selected from the group consisting of bacterial, fungal, plant, insect and mammalian cells.
21. 21. A cell as defined in claim 20, wherein the cell is a bacterial cell.
22. 22. A cell as defined in claim 20, wherein the cell is a plant cell.
23. 23. A seed comprising a cell as defined in claim 22.
24. 24. A method for imparting resistance to herbicides on a plant, the method comprising introducing into the plant a nucleic acid encoding a variant of HPPD resistant to herbicides as defined in claim 16, under conditions in which the nucleic acid is expressed in the plant.
25. 25. A method for seed control comprising culturing a culture containing a HPPD gene resistant to herbicides in the presence of an effective weed control amount of the herbicide.