WO2000028053A2 - Homologues de glutamine amidotransferases vegetales - Google Patents
Homologues de glutamine amidotransferases vegetales Download PDFInfo
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- WO2000028053A2 WO2000028053A2 PCT/US1999/025950 US9925950W WO0028053A2 WO 2000028053 A2 WO2000028053 A2 WO 2000028053A2 US 9925950 W US9925950 W US 9925950W WO 0028053 A2 WO0028053 A2 WO 0028053A2
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- isolated polynucleotide
- nucleic acid
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- nucleotide sequence
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Definitions
- An isolated polynucleotide of the present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides, of the nucleic acid sequence of the SEQ ID NOs:l, 3, 5, 7, 9, 11, and 13.
- nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell.
- the skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
- Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
- Native gene refers to a gene as found in nature with its own regulatory sequences.
- Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
- Endogenous gene refers to a native gene in its natural location in the genome of an organism.
- Coding sequence refers to a nucleotide sequence that codes for a specific amino acid sequence.
- Regulatory sequences refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
- promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 75:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
- “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
- Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
- a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
- any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1621-1632).
- Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms.
- Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:211) and particle-accelerated or "gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Patent No. 4,945,050, incorporated herein by reference).
- nucleic acid fragments encoding at least a portion of several histidine biosynthetic enzymes have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art.
- the nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art.
- two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
- the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.
- the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl.
- a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.
- the present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide of a gene (such as a glutamine amidotransferase homolog) preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 1, 9, 11, 13, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer.
- the amplified nucleic acid fragment preferably will encode a portion of a polypeptide.
- Plasmid vectors comprising the instant chimeric gene can then be constructed.
- the choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBOJ. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:18-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern.
- tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
- This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded histidine biosynthetic protein.
- An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6).
- the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers.
- Southern blots Maniatis
- the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map.
- the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 52:314-331).
- Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
- nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
- FISH direct fluorescence in situ hybridization
- nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 77:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 76:325-332), allele- specific ligation (Landegren et al. (1988) Science 241:1011-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:361 ⁇ ), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet.
- Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 5(5:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84).
- the latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which
- Chemicals used included okadaic acid, cyclosporin A, calyculin A, cypermethrin.
- Chemicals used included valinomycin, bafilomycin Al, oligomycin, ionomycin.
- d A23187 is commercially available from several vendors including Calbiochem. e Corn developmental stages are explained in the publication "How a corn plant develops" from the Iowa State University Coop. Ext. Service Special Report No. 48 reprinted June 1993. f This clone was mislabeled during processing. It belongs to the cen3n library even though it is labeled as a rlr48 clone.
- a chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed.
- the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below.
- Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (SequenaseTM DNA Sequencing Kit; U.S. Biochemical).
- the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3' region.
- Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos.
- the embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
- the particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells.
- gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
- Ten ⁇ g of plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
- Calcium chloride 50 ⁇ L of a 2.5 M solution
- spermidine free base (20 ⁇ L of a 1.0 M solution) are added to the particles.
- the suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 ⁇ L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 ⁇ L of ethanol. An aliquot (5 ⁇ L) of the DNA-coated gold particles can be placed in the center of a KaptonTM flying disc (Bio-Rad Labs).
- a seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J Biol. Chem. 2(51:9228-9238) can be used for expression of the instant polypeptides in transformed soybean.
- the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Neo I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.
- the cDNA fragment of this gene may be generated by polymerase chain reaction
- the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly.
- green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
- the cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430.
- This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system.
- Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector.
- Nde I site at the position of translation initiation was converted to an Neo I site using oligonucleotide-directed mutagenesis.
- Cells are then harvested by centrifugation and re-suspended in 50 ⁇ L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride.
- a small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator.
- the mixture is centrifuged and the protein concentration of the supernatant determined.
- One ⁇ g of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
- EXAMPLE 7 Evaluating Compounds for Their ability to Inhibit the Activity of a Histidine Biosynthetic Enzyme
- the polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 6, or expression in eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines.
- the instant polypeptides may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags.
- Common fusion protein partners include glutathione S-transferase ("GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide ("(His ⁇ ").
- GST glutathione S-transferase
- Trx thioredoxin
- (His ⁇ ) C- and/or N-terminal hexahistidine polypeptide
- the fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme.
- proteases include thrombin, enterokinase and factor Xa.
- any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.
- Purification of the instant polypeptides may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor.
- the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme.
- the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin.
- a (His)6 peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification.
- Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B.
- a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include ⁇ -mercaptoethanol or other reduced thiol.
- the eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired.
- Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBondTM affinity resin or other resin.
- Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activation of the instant polypeptides disclosed herein.
- Measurement of the optimal activity of histidine biosynthetic enzymes may be accomplished by suppression of the His auxotrophy of corresponding Escherichia coli hisH and hisF mutants.
- An assay was developed for in vitro analysis of HisHF activity.
- the substrate for HisHF PRFAR [(N ⁇ IXS'-phosphoribulosyl) formimino]-5-aminoimidazole-4- carboxamide ribonucleotide)]
- PRFAR phosphoribosyl pyrophosphate
- PRFAR was purified by Q-Sepharose chromatography followed by HPLC using a Cj column. Purified PRFAR has the expected UV spectrum with major peak at OD290-
- the HisHF catalyzed conversion of PRFAR to AICAR and IGP is monitored at O.D.300 for the decrease of PRFAR level.
- the optimized buffer condition was determined to be 15 mM KPi, pH 7.5, 5 mM Glutamine, 100 uM PRFAR.
- the steady state kinetic constants were determined for HisHF by varying the concentration of PRFAR or glutamine, the two substrates for the reaction.
- EXAMPLE 9 Determination of Amino Acids Essential for Histidine Biosynthetic Enzyme Activity Site-directed mutagenesis of the HisHF enzyme allows the determination of the amino acids essential for activity and catalysis.
- Site-directed mutagenesis was carried out for, both, the hisH domain (glutamine amidotransferase) and the hisF domain (cyclase). Based on sequence homology with other trpG-type glutamine amidotransferases and the cysteine protease family, three conserved residues in the hisH domain that constitute the putative "catalytic triad" for the glutamine amidotransfer step were mutagenized.
- the mutants (based on the Arabidopsis thaliana sequence, SEQ ID NO : 15) include Cys 141 - Ala or Ser;
- Asp403 ⁇ Ala, Lys404 ⁇ Ala, Ser409 ⁇ Ala, Asp447 ⁇ Ala, Glu508 ⁇ Ala, Asp517 ⁇ Ala, and Glu553- ⁇ Aia are unable to complement the yeast strain ZXY72-1B suggesting that they are essential for enzyme activity.
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Abstract
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99956903A EP1127144A2 (fr) | 1998-11-05 | 1999-11-04 | Homologues de glutamine amidotransferases vegetales |
US09/831,233 US6855867B1 (en) | 1998-11-05 | 1999-11-04 | Plant glutamine amidotransferase homologs |
AU13413/00A AU1341300A (en) | 1998-11-05 | 1999-11-04 | Plant glutamine amidotransferase homologs |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10727598P | 1998-11-05 | 1998-11-05 | |
US60/107,275 | 1998-11-05 |
Publications (2)
Publication Number | Publication Date |
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WO2000028053A2 true WO2000028053A2 (fr) | 2000-05-18 |
WO2000028053A3 WO2000028053A3 (fr) | 2000-08-17 |
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ID=22315811
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1999/025950 WO2000028053A2 (fr) | 1998-11-05 | 1999-11-04 | Homologues de glutamine amidotransferases vegetales |
Country Status (3)
Country | Link |
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EP (1) | EP1127144A2 (fr) |
AU (1) | AU1341300A (fr) |
WO (1) | WO2000028053A2 (fr) |
-
1999
- 1999-11-04 AU AU13413/00A patent/AU1341300A/en not_active Abandoned
- 1999-11-04 WO PCT/US1999/025950 patent/WO2000028053A2/fr not_active Application Discontinuation
- 1999-11-04 EP EP99956903A patent/EP1127144A2/fr not_active Withdrawn
Non-Patent Citations (5)
Title |
---|
DATABASE EMEST14 [Online] EMBL Heidelberg, Germany AC/ID AI899863, 28 July 1999 (1999-07-28) SHOEMAKER R ET AL.: "Glycine max cDNA clone similar to: glutamine amidotransferase/cyclase" XP002136030 * |
DATABASE EMEST21 [Online] EMBL Heidelberg, Germany AC/ID AW066760, 18 October 1999 (1999-10-18) WALBOT V: "Maize ESTs from various cDNA libraries sequenced at Stanford University" XP002136029 * |
DATABASE NEW_TREMBL [Online] EMBL Heidelberg, Germany AC/ID CAB36536, 17 June 1999 (1999-06-17) BEVAN M ET AL.: "Glutamine amidotransferase/cyclase" XP002136028 * |
FUJIMORI K AND OHTA D: "An Arabidopsis cDNA encoding a bifunctional glutamine amidotransferase/cyclase suppresses the histidine auxotrophy of a Saccharomyces cerevisiae his7 mutant" FEBS LETTERS, vol. 428, no. 3, 29 May 1998 (1998-05-29), pages 229-234, XP002136027 * |
KLEM T J AND DAVISSON V J: "Imidazole glycerole phosphate synthase: the glutamine amidotransferase in histidine biosynthesis" BIOCHEMISTRY, vol. 32, 1993, pages 5177-5186, XP002136052 * |
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Publication number | Publication date |
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EP1127144A2 (fr) | 2001-08-29 |
AU1341300A (en) | 2000-05-29 |
WO2000028053A3 (fr) | 2000-08-17 |
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