Herbicides test method

Abstract

An isolated DNA molecule encoding a protein from a plant, which protein has pantothenate synthetase activity; a non-naturally occuring chimeric gene comprising a promoter operably linked to a DNA molecule encoding a protein from a plant having pantothenate synthetase activity; a recombinant vector comprising the chimeric gene wherein the vector is capable of being stably transformed into host cell, a host cell stably transformed with a vector wherein the host cell is capable of expressing the DNA molecule; a method for assaying a protein having pantothenate synthetase activity; the use as herbicides of compounds which inhibit pantothenate synthetase, and a herbicidal composition, comprising one or more active ingredients which show significant pantothenate synthetase inhibition in an assay, are disclosed.

1 . An isolated DNA molecule encoding a protein from a plant, which protein has pantothenate synthetase activity.  
     
     
         2 . An isolated DNA molecule according to  claim 1 , wherein the plant is  Lotus japonicus.    
     
     
         3 . An isolated DNA molecule according to  claim 2 , wherein the DNA molecule encodes a protein comprising the amino acid sequence set forth in FIG. 1. 2 .  
     
     
         4 . An isolated DNA molecule according to  claim 3 , wherein the DNA molecule comprises the nucleotide sequence set forth in FIG. 1. 2 .  
     
     
         5 . An isolated DNA molecule according to  claim 1 , wherein the plant is  Oryza sativa.    
     
     
         6 . An isolated DNA molecule according to  claim 5 , wherein the DNA molecule encodes a protein comprising the amino acid sequence set forth in FIG. 2. 2 .  
     
     
         7 . An isolated DNA molecule according to  claim 6 , wherein the DNA molecule comprises the nucleotide sequence set forth in FIG. 2. 2 .  
     
     
         8 . A non-naturally occuring chimeric gene comprising a promoter operably linked to a DNA molecule encoding a protein from a plant having pantothenate synthetase activity.  
     
     
         9 . A non-naturally occuring chimeric gene according to  claim 8  wherein the protein comprises an amino acid sequence selected from the group set forth in FIG. 1. 2  and FIG. 2. 2 .  
     
     
         10 . A recombinant vector comprising the chimeric gene of any of  claims 8  to  9  wherein the vector is capable of being stably transformed into a host cell.  
     
     
         11 . A host cell stably transformed with the vector of  claim 10  wherein the host cell is capable of expressing the DNA molecule.  
     
     
         12 . A host cell according to  claim 11  selected from the group consisting of a bacterial cell, a yeast cell and an insect cell.  
     
     
         13 . A method of producing a protein having pantothenate synthetase activity in a host organism comprising, 
 a) inserting a DNA sequence encoding a protein having antothenate synthetase activity into an expression casette designed for the chosen host;    b) inserting the resultant molecule, containing the individual lements linked in proper reading frame, into a vector capable of being transformed into the host cell;    c) growing the thus transformed host cell in a suitable culture medium; and    d) isolating the protein product either from the transformed cell or the culture medium or both and purifying it.    
     
     
         14 . A method for assaying a protein having pantothenate synthetase activity comprising, 
 a) incubating the protein in a suitable reaction mixture in which the protein is capable of catalyzing the conversion of pantoate, β-alanine and ATP to pantothenate, AMP and pyrophosphate; and    b) determining the amount of pyrophosphate generated by the catalytic activity of pantothenate synthetase by a calorimetric technique.    
     
     
         15 . A method for assaying a protein having pantothenate synthetase activity comprising, incubating the protein in a suitable reaction mixture in which the protein is capable of catalyzing the conversion of pantoate, 
 a) β-alanine and ATP to pantothenate, AMP and pyrophosphate; and    b) converting the pyrophosphate formed by the catalytic activity of pantothenate synthetase into inorganic phosphate by the catalytic activity of an inorganic pyrophosphatase; and    c) determining the amount of inorganic phosphate generated by the catalytic activity of the inorganic pyrophosphatase by a colorimetric technique.    
     
     
         16 . A method according to  claim 15  wherein the inorganic pyrophosphatase is yeast pyrophosphatase.  
     
     
         17 . A method for assaying a chemical for the ability to inhibit the activity of a pantothenate synthetase enzyme from a plant comprising, 
 a) incubating the pantothenate synthetase enzyme in a first reaction mixture under conditions specified in  claim 14  or  claim 15;     b) combining the chemical and the pantothenate synthetase enzyme in a second reaction mixture under the same conditions as in the first reaction mixture; and    c) measuring the amount of either pyrophosphate or inorganic phosphate in the first and second reaction mixtures; wherein the chemical is capable of inhibiting the activity of the pantothenate synthetase enzyme if the amount of either pyrophosphate formed or inorganic phosphate formed in the second reaction mixture is significantly less than the amount of either pyrophosphate formed or inorganic phosphate formed in the first reaction mixture.    
     
     
         18 . The use as herbicides of compounds which inhibit pantothenate synthetase.  
     
     
         19 . Herbicidal composition, comprising one or more active ingredients which show significant panthotenate synthetase inhibition in an assay according to  claim 17 .  
     
     
         20 . A nucleotide probe capable of specifically hybridizing to plant pantothenate synthetase gene or mRNA, wherein the probe comprises a contiguous portion of the coding sequence for a pantothenate synthetase from a plant at least 10 nucleotides in length.  
     
     
         21 . A method of producing a DNA molecule comprising a DNA portion encoding a protein having pantothenate synthetase activity comprising, 
 a) preparing a nucleotide probe capable of specifically hybridizing to a plant pantothenate synthetase gene or mRNA, wherein the probe comprises a contiguous portion of the coding sequence for a pantothenate synthetase from a plant at least 10-nucleotides in length;    b) probing for other pantothenate synthetase coding sequences in populations of cloned genomic DNA fragments or cDNA fragments from a chosen organism using the nucleotide probe prepared according to step a); and    c) isolating a DNA molecule comprising a DNA portion encoding a protein having pantothenate synthetase activity.    
     
     
         22 . The use of a nucleotide probe according to  claim 19  to amplify pantothenate synthetase coding sequences from a chosen organism via the process of polymeras chain reaction.