MXPA01011894A - Use of genes of the deoxy-d-xylulose phosphate biosynthetic pathway for altering the concentration of isoprenoid. - Google Patents

Abstract

The invention relates to the use of DNA sequences from bacteria and parasites, namely the genes gcpE and yfgB for integration in the genome of viruses, eukaryotes, and prokaryotes and thereby altering the concentration of isoprenoid. The invention also relates to methods for identifying substances that exhibit a herbicidal, antiparasitic, antiviral, and fungicidal activity in plants and an antimycotic, antiparasitic and antiviral activity in humans and animals.

Description

USE OF GENES OF BIOSINTETIC PATHS OF PHOSPHATE OF DESOXI-D-XILULOSA TO ALTER THE CONCENTRATION OF ISOPRENOIDS The present invention relates to the use of DNA sequences (SEQ: 1,3,5,7), which encode the protein gcpE or yfgB of bacteria and parasites, and which modify the content of isoprenoids after their integration into the genome of viruses, eukaryotes and prokaryotes, as well as a process to measure the activity of the gcpE gene in the synthesis of isoprenoids. This is related to processes for the identification of substances that have herbicidal, antiparasitic, antiviral, fungicidal action in plants, and antiparasitic, antifungal and antiviral action in humans and animals. The trajectory of biosynthesis for the formation of isoprenoids by means of the classical path of acetate / mevalonate, and a trajectory of independent biosynthesis of alternative mevalonate, the trajectory of deoxy-D-xylulose phosphate (Rohtier, M., Knani, M., Simonin, P., Sutter, B., and Sahm, H. (1993): Biochem. J. 295: 517-524). In US Pat. No. 5,858,367 the use of aarC oligonucleotides for the idejitification of antibacterial substances is described.

Surprisingly, it has been found that the gcpE protein additionally has a kinase function in the alternative metabolic pathway of isoprenoid biosynthesis, and catalyzes the phosphorylation of a sugar or a 5 precursor of isoprenoid biosynthesis, in particular 2-C-methyl-D-erythritol, 2-C-methyl-D-erythritol phosphate, in particular 2-C-methyl-D-erythritol-4, 2-C phosphate -methyl-D-erythrose, 2-C-methyl-D-erythrose phosphate, in particular 2-C-methyl-D-erythrose-4 phosphate, CH2 (OH) -C (CH3) = C (OH) - CH2-0- 10 PO (OH) 2, CH2 (OH) -C (CH3) = C (OH) -CH2-OH, CH2 (OH) -CH (CH3) -CO-CH2-0- P0 (0H) 2, CH2 (0H) -CH (CH3) -C0-CH2-0H, CH2 = C (CH3) -CO-CH2-0-PO (OH) 2, • CH2 = C (CH3) -CO-CH2-OH, CH2 = C (CH3) -CH (OH) -CH2-0-PO (OH) 2, CH2 = C (CH3) -CH (OH) -CH2- 0H, CH2 (OH) -C (= CH2) -C (OH) -CH2-0-P0 (OH) 2, CH2 (OH) -C (= CH2) -C (OH) -CH2-OH, CHO- CH (CH3) -CH (OH) -CH2-0-PO (OH) 2, 15 CHO-CH (CH3) -CH (OH) -CH2-OH, CH2 (OH) -C (OH) (CH3) -CH = CH-0-PO (OH) 2, CH2 (OH) -C (OH ) (CH3) -CH = CH-OH, CH (OH) = C (CH3) -CH (OH) -CH2-0-PO (OH) 2, CH (OH) = C (CH3) -CH (OH) -CH2-OH, CH3-C (CH3) = CH-CH2-0- PO (OH) 2, CH3-C (CH3) = CH-CH2-OH, CH2 = C (CH3) -CH2-CH2-0- PO (OH) 2, CH 2 = C (CH 3) -CH 2 -CH 2 -OH. Accordingly, the invention relates to the use of DNA sequences, which encode the gcpE or yfgB protein of bacteria and parasites [of bacteria, or gcpE or yfgB protein of parasites], or DNA sequences that encode an analog or derivative of this protein, in which it has 25 suppressed, added or replaced with other amino acids one or more amino acids, without substantially reducing the enzymatic action of the polypeptide. In particular, this relates to the use of DNA sequences SEQ 1,3,5,7. The initial ^ origin of the sequences listed SEQ 1 and 5, as well as 5 proteins 2 and 6, is the organism Escherichia coli, strain K12. The initial origin of the sequences listed SEQ 3 and 7, as well as proteins 4 and 8, is the organism Plasmodium Falciparum, strain 3D7. These DNA sequences are described in the Patent of 10 the United States of America Number 5 858 367, and they will also be found under the access numbers that follow, • through the Internet address: htt: // www3. ncbi. nlm. nih gov / Entrez / protein. html: AAD07695, AAD18517, AAC75568, AAC67648, AAC65433, P36979, 15 CAA15530, CAA98356, CAA98355, AAC24056, AAC07467, P54482, P44667, P27434, P27433, BAA17717, BAA20919, BAA16402, S23058, AAB51469, 1819264A, CAA45783, CAA45782, AAA21360, AAA21359, BAA02549, 139486, 2113330A. The sequences according to the invention are Suitable for the expression of genes in viruses, eukaryotes and prokaryotes, which are responsible for the isoprenoid biosynthesis of the 1-deoxy-D-xylulose pathway. In accordance with the invention eukaryotes or eukaryotic cells include animal cells, Plant, algae, yeast, fungi, and prokaryotes or prokaryotic cells include bacteria, archaebacteria, and eubacteria. When a DNA sequence is incorporated into a genome in which the DNA sequence declared above is located, the expression of the genes described above in viruses, eukaryotes and prokaryotes is enabled. The viruses, eukaryotes and prokaryotes transformed according to the invention are cultured in a manner known per se, and the isoprenoid formed during that culture is isolated and optionally purified. Not all isoprenoids need to be isolated, since in some cases isoprenoids are released directly into ambient air. The preparation of the transgenic viruses, eukaryotes and prokaryotes used, for the purpose of modifying the isoprenoid content, can be carried out by means of the following steps: a) The production of a DNA sequence having the following subsequences i) promoter that is active in viruses, eukaryotes and prokaryotes, and which assures the formation of an RNA in the target tissue or target target cells, ii) DNA sequence encoding a polypeptide having the amino acid sequence of the gcpE or yfgB protein of bacteria or parasites, or an analog or derivative of this polypeptide, iii) 3 'untranslated sequence leading to an addition of poly-A residues to the 3' end of the RNA in viruses, eukaryotic and prokaryotic flB, 5 b) transfer and incorporation of the DNA sequence within the genome of viruses, prokaryotic or eukaryotic cells, with or without the use of a vector (eg, viral DNA, plasmid). Whole plants, intact, can be regenerated 10 from plant cells transformed in this way. The sequences encoding the gcpE or yfgB proteins or their analogs or derivatives can be provided with a promoter that assures transcription in certain organs or cells, promoter that couples in the sense orientation 15 (3 'end of the promoter to the 5' end of the coding sequence) to the sequence encoding the protein to be formed. A termination signal that determines the termination of the mRNA synthesis is attached to the 3 'end of the coding sequence. For the purpose of directing the protein 20 to be expressed, to certain subcellular compartments, such as chloroplasts, amyloplasts, mitochondria, vacuoles, cytosol or intercellular spaces, an additional sequence coding for a so-called signal sequence or a transit peptide can be inserted between the promoter and the coding sequence. The sequence must be in the same reading frame as the coding sequence of the protein. A large number of cloning vectors are available for the purpose of preparing the introduction of the DNA sequences according to the invention, within higher levels, vectors containing a replication signal for E. coli. and a marker that allows the selection of the transformed cells. Examples for vectors are pBR 322, pUC series, M13mp series, pACYC 184, EMBL 3, and so on. Depending on the method by which the genes are introduced If desired within the plant, additional DNA sequences may be required. If, for example, the Ti plasmid is used or • Ri to transform the plant cells, at least one right border must be inserted, but frequently the right border and the left border of the T-DNA of the Ti plasmid and 15 Ri, as a flanking region inside the genes that are going to be introduced. The use of T-DNA to transform plant cells has been intensively investigated, and is described comprehensively in EP 120516; Hoekama, in "The Binary Plant Vector System, Offset-drukkeril Kanters B.V. • 20 Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4,1-46 and An et al. (1985) EMBO J. 4, 277-287. Once the introduced 7DNA has been incorporated into the genome, it is generally stable and also retained in the descendants of the originally transformed cells. This normally contains a selection marker, which imparts to the transformed plant cells, resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin or phosphinothricin and others. Gk In this way it is intended that the particular marker that is 5 use allows the selection of transformed cells from cells lacking the inserted DNA. Many techniques are available to introduce DNA into a plant. These techniques include the transformation with the help of agrobacteria, for example, Agrobacterium termefaciens, protoplast fusion, DNA microinjection, electroporation, as well as ballistic methods and infection. • by virus. Afterwards whole plants can be regenerated from the transformed plant material, in a suitable medium which can contain antibiotics or biocides for the purposes of 15 selection. No particular requirement is placed on the plasmids for injection and electroporation. However, if entire plants are to be regenerated from those transformed cells, a selectable marker gene must be present. The transformed cells grow in the plants of • 20 in the conventional manner (McCormick et al. (1986), Plant Cell Reports 5, 81-84). Plants can be grown normally, and crossed with plants that have the same transformed genome or other genomes. The resulting individuals have the corresponding phenotypic properties. Expression vectors containing one or more of the DNA sequences, according to the invention, are suitable for incorporation of DNA into host organisms. These expression vectors are obtained by means of B to provide the DNA sequences, according to the invention, with suitable functional regulation signals. These regulatory signals are DNA sequences that are responsible for the expression, for example, of promoters, operators, enhancers, ribosomal binding sites, and are recognized by host cells. 10 Other regulatory signals that control, for example, the replication or recombination of recombinant DNA in • the host organism, optionally can also be a constituent part of the expression vector. The cells and host organisms suitable for Expressing the enzymes according to the invention are those comprising non-intrinsic enzymes with the function of DOXP synthase, DOXP reductoisomerase or gcpE kinase. This is the case in archaebacteria, animals, fungi, slime molds and some eubacteria. The absence of that The activity of intrinsic enzymes substantially facilitates the detection and purification of recombinant enzymes. As a consequence, it is also possible for the first time to measure the activity with little effort, in crude extracts of the host cells, and in particular the inhibition of The activity of the recombinant enzymes, according to the invention, by means of different chemical and pharmaceutical products. The enzymes according to the invention are then conveniently expressed in eukaryotic cells, if post-translational modifications and the native fold of the polypeptide chain are to be achieved. On the other hand, depending on the expression system, it is ensured that, when the genomic DNA sequences are expressed, the introns are eliminated by splicing the DNA, and the enzymes are produced in the polypeptide sequences characteristic of the parasites. Using recombinant DNA techniques, sequences encoding introns can be removed from, or inserted for experimental purposes within the DNA sequences to be expressed. The protein can be isolated from the host cell or from the culture supernatant of the host cell, using methods known to the person skilled in the art. In vitro reactivation of the enzymes may also be required. For the purpose of facilitating purification, the enzymes according to the invention, or the subsequences of the enzymes can be expressed as fusion proteins with different peptide chains. Oligohistidine sequences and sequences derived from glutathione S-transferase, thioredoxin or peptides from Fixation to calmodulin are particularly suitable for this purpose. The fusions with sequences derived from thioredoxin are especially suitable for prokaryotic expression, because consequently the solubility of the recombinant enzymes increases. The enzymes according to the invention or the subsequences of the enzymes can be further expressed as fusion proteins with such peptide chains, known to the person skilled in the art that the recombinant enzymes are transported within the extracellular medium or within certain compartments of the host cells.

• Consequently, both the purification and the investigation of the biological activity of the enzymes can be facilitated. When expressing the enzymes according to the invention, it may be convenient to modify the individual codons. At present it may also be advisable to purposefully substitute the bases in the coding region, if the codons used in the parasites are different from the codons that are used in the heterologous expression system, in order to ensure the optimal synthesis of the protein. In addition, deletion of the untranslated 5 'and 3' regions is often advisable, for example, if many sequence motifs 25 of ATTTA destabilizing sequence are present in the 3 'region of the to DNA. Then these must be deleted in the preferred expression in eukaryotes. Variations of this kind are deletions, additions, or replacement of the bases, and are also subject of the present invention. The enzymes according to the invention can also be obtained under standardized conditions, by means of in vitro translation, by means of methods known to a person skilled in the art. The systems suitable for this purpose are rabbit reticulito and extracts of wheat germ and bacterial lysates. The mRNA transcribed in vitro can also be transferred to Xenopus oocyten. The oligo- and polypeptides, whose sequences are derived from the peptide sequence of the enzymes according to the invention, can be obtained by chemical synthesis. Given the proper selection of the sequences, those peptides have properties that are characteristic of the enzymes according to the invention. These peptides can be produced in large quantities and are particularly suitable for investigating the kinetics of enzyme activity, the regulation of enzyme activity, the three-dimensional structure of enzymes, the inhibition of enzyme activity by means of different chemical and pharmaceutical products, and the binding geometry and binding affinity of different ligands. Another object of this invention are methods for determining the enzymatic activity of the gcpE kinase. That activity can be determined using known methods. The determination is carried out by detecting the flfc phosphorylation of a sugar or a phosphorous sugar, or of a precursor of isoprenoid biosynthesis, in particular the phosphorylation of 2-C-methyl-D-erythritol, 2-C phosphate. -methyl- D-erythritol, in particular 2-C-methyl-D-erythritol-4,2-C-methyl-D-erythrose phosphate, 2-C-methyl-D-erythrose phosphate, in particular phosphate 2; -C-methyl-D-erythrose-4, CH2 (OH) -10 C (CH3) = C (0H) -CH2-0-PO (OH) 2, CH2 (OH) -C (CH3) = C (OH ) -CH2-0H, CH2 (OH) -CH (CH3) -CO-CH2-0-PO (OH) 2, CH2 (OH) -CH (CH3) -CO-CH2-OH, CH2 = C • (CH3) -CO-CH2-0-PO (OH) 2, CH2 = C (CH3) -C0-CH2-0H, CH2 = C (CH3) -CH (OH) -CH2-0-PO (OH) 2, CH2 = C (CH3) -CH (OH) -CH2-0H, CH2 (OH) -C (= CH2) -C (OH) -CH2-0-PO (OH) 2, CH2 (OH) -C (= CH2) -C (OH) -CH2-OH, CHO-CH (CH3) -CH15 (OH) -CH2-0-PO (OH) 2, CHO-CH (CH3) -CH (OH) -CH2 -OH, CH2 (OH) -C (OH) (CH3) -CH = CH-0-PO (OH) 2, CH2 (OH) -C (OH) (CH3) -CH = CH-OH, CH (OH) ) = C (CH3) -CH (OH) -CH2-0-PO (OH) 2, CH (OH) = C (CH3) -CH (OH) -CH2-OH, CH3-C (CH3) = CH- CH2-0-PO (OH) 2, CH3-C (CH3) = CH-CH2-OH, CH2 = C (CH3) -CH2-? CH2-0-P0 (0H) 2, CH2 = C (CH3) -CH2-CH2-OH. The present invention also provides the use of this measurement method to identify substances that inhibit the activity of particular enzymes. It has been found that the metabolic pathway of deoxy-D-xylulose phosphate is also present in many parasites, viruses and fungi.

'V 11? -? I? T? Aaa = ja The invention, consequently, is also related to a process for classifying a compound. In accordance with this process, a host organism is provided which fl comprises a recombinant expression vector, wherein the The vector comprises at least a part of the oligonucleotide sequence, which encodes the gcpE or yfgB protein, or variants or homologs of this sequence, and furthermore a compound, which is suspected of having an antimicrobial, antiparasitic, antiviral and antifungal action in 10 humans and animals, or a bactericidal, antimicrobial action, ? - > herbicide or fungicide in plants. Subsequently, the host organism is contacted with the compound and the dead activity of the compound is determined.

Example 1 Increased accumulation = carotenoids in E. coli. by overexpression of scpe e yfsB. The first pAC-LYC plasmid was constructed in accordance with published protocols (Cunningham, F X Jr. and 20 collaborators, 1996, Plant Cell 8: 1613-1626). The plasmid supports the genes, which are necessary for the synthesis of Lycopen of carotenoids of IPP and DMAPP. Consequently, E. coli cells, which have been transformed with pAC-LYC, form pink colonies. Yes 25 the availability of the educts for the synthesis of the carotenoids increases, the carotenoids accumulate incrementally, and the colonies look dark pink. An improved formation of educts ^ fc can be realized by overexpression of genes of the DOXP path. Consequently, the gcpe and yfgB genes of E. coli are cloned into suitable expression vectors. The gcpe gene was amplified from E. coli DNA with the 5 'primers -CCA TGG GCC ATA ACC AGG CTC CAA TCC AA-3' and 5 '-GGA TCC TTT TTC AAC CTG CTG AAC GTC AAT-3' , by PCR, and cloned inside 10 of the vector pCR2.1-TOPO. The insert was cloned by means of the Neo I and Bam H 1 restriction interfaces within the expression vector pQE60. The yfgB gene was amplified with the primers 5 '-GGA TCC ATG TCT GAA CAA TTA GTC ACA-3' and 5 '-AAG CTT TCA GAC CGC TTT AAT GTC GAT GGC-3', and was cloned into the 15 vector pCRT7 / NT TOPO. The insert was cloned by means of the restriction interfaces Bam H 1 and Hind III in the expression vector pQE30. The bacteria, which had been transformed with pAC-LYC and one of the two expression constructs, show a remarkably darker coloration than • 20 bacteria that, as a control, had been transformed only with the empty pQE30 vector. Photometric quantification of carotenoid enrichment produced 210 percent gcpe and 173 percent yfgB, compared with the control. Example 2 Increased accumulation of carotenoids = n EL, falciparum, by overexpression of scpe e yfsB. (ß Analogous experiments were carried out by using 5 of the gcpe e yfgB gene of P. falciparum. The gcpe gene was amplified with the primers 5 '-CTG ATT CTT TTT ATG TTA CTG TTT TAT TCT CAT GTA-3' and 5 '-CTA CCC TTT TTT ATT TGT AAG AAC ATC ATT AGT TAC GTT AAC-3', and the yfgB gene was amplified with the GAA AAG TCA AAA AGG TAC ATA AGC primers 10 CTG ATT AAG and 5 '-TAA CAT GTC CTC TAT GCC TAT ATA TTT ATA TAA TG-3? Genomic DNA from strain P. falciparum 3D7 was used • as a template, and the DNA-P or thermoformed polymerase was used for PCR. The PCR products were phosphorylated by T4-Polynucleotide kinase and cloned in the vectors 15 pQE32, which had been linearized by Sma I and dephosphorylated by basic phosphatase. The orientation of the inserts was verified by restriction analysis. The colonies of bacteria obtained with these constructions do not show marked changes of color. However the • 20 photometric analysis produced a carotenoid accumulation of 117 percent (gcpe) and 113 percent (yfgB). The comparatively minor accumulation of carotenoids with P. falciparum genes appears to be due to the lower expression of frequently observed P. falciparum genes 25 in E. coli, due to a high content of A / T.

LIST OF SEQUENCES < 110 > Jomaa, Hassan < 120 > Use of genes from the biosynthetic pathway of deoxy-D-xylulose phosphate to alter the concentration of isoprenoids < 130 > 15904 < 140 > < 141 > < 150 > 19923567.8 < 151 > 1999-05-21 < 150 > 19923568.6 < 151 > 1999-05-21 < 160 > 8 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 1119 < 212 > DNA < 213 > Escherichia coli < 220 > < 221 > CDS < 222 > (1) .. (1119) < 400 > 1 atg cat aac cag gct cea att caá cgt aga aaa tea here cgt att tac 48 Met His Asn Gln Wing Pro lie Gln Arg Arg Lys Ser Thr Arg lie Tyr 1 5 10 15 gtt ggg aat gtg ccg att ggc gat ggt gct ccc ate gcc gta cag tcc 96 Val Gly Asn Val Pro lie Gly Asp Gly Wing Pro lie Wing Val Gln Ser 20 25 30 atg acc aat acg cgt acg here gac gtc gaa gca acg gtc aat ca a ate 144 Met Thr Asn Thr Arg Thr Thr Asp Val Glu Wing Thr Val Asn Gln lie 35 40 45 aag gcg ctg gaa cgc gtt ggc gct gat ate gtc cgt gta tc gta ccg 192 Lys Wing Leu Glu Arg Val Gly Wing Asp He Val Val Val Ser Val 55 55 60 acg atg gac gcg gca gaa gcg ttc aaa etc aaa cag cag gtt aac 240 Thr Met Asp Wing Ala Glu Wing Phe Lys Leu He Lys Gln Gln Val Asn 65 70 75 80 gtg ccg ctg gtg gct gac ate drops ttc gac tat cgc att gcg ctg aaa 288 Val Pro Leu Val Wing Asp He His Phe Asp Tyr Arg He Wing Leu Lys 85 90 95 gta gcg gaa tac ggc gtc gat tgt ctg cgt att aac ect ggc aat ate 336 Val Ala Glu Tyr Gly Val Asp Cys Leu Arg He Asn Pro Gly Asn I have 100 105 110 ggt a at gaa gag cgt att cgc atg gtg gtt gac tgt gcg cgc gat aaa 384 Gly Asn Glu Glu Arg He Arg Met Val Val Asp Cys Ala Arg Asp Lys 115 120 125 aac att ccg ate cgt att ggc gtt aac gcc gga tcg ctg gaa aaa gat 432 Asn He Pro He Arg He Gly Val Asn Wing Gly Ser Leu Glu Lys Asp 130 135 140 ctg caa gaa aag tat ggc gaa ccg acg ccg cag gcg ttg ctg gaa tct 480 Leu Gln Glu Lys Tyr Gly Glu Pro Thr Pro Gln Ala Leu Leu Glu Ser 145 150 155 160 gcc atg cgt cat gtt gat cat etc gat cgc ctg aac ttc gat cag ttc 528 wing Met Arg His Val Asp His Leu Asp Arg Leu Asn Phe Asp Gln Phe 165 170 175 • aaa gtc age gtg aaa gcg tct gac gtc gtc etc gct gtt gag tct tat 576 Lys Val Ser Val Lys Wing Ser Asp Val Phe Leu Wing Val Glu Ser Tyr 180 185 190 cgt ttg ctg gca aaa cag ate gat cag ccg ttg cat ctg ggg ate acc 624 Arg Leu Leu Ala Lys Gln He Asp Gln Pro Leu His Leu Gly He Thr 195 200 205 gaa gcc ggt ggt gcg cgc age ggg gca gta aaa tcc gcc att ggt tta 672 Glu Wing Gly Gly Wing Arg Ser Gly Wing Val Lys Ser Wing He Gly Leu 210 215 220 ggt ctg ctg ctg tct gaa ggc ate ggc gac acg ctg cgc gta tcg ctg 720 Gly Leu Leu Ser Glu Gly He Gly Asp Thr Leu Arg Val Ser Leu 225 230 235 240 • gcg gcc gat ccg gtc gaa gag ate aaa gtc gtt gt ttc gat att ttg aaa 768 Wing Wing Asp Pro Val Glu Glu He Lys Val Gly Phe Asp He Leu Lys 245 250 255 tcg ctg ct tcg cgt tcg cga ggg ate aac tcc ate gcc tgc ccg acc 816 Ser Leu Arg He Arg Ser Arg Gly He Asn Phe He Wing Cys Pro Thr 260 265 270 tgt tcg cgt cag gaa ttt gat gtt ate ggt acg gtt aac gcg ctg gag 864 Cys Ser Arg Gln Glu Phe Asp Val He Gly Thr Val Asn Ala Leu Glu 275 280 285 cac cgc ctg gaa gat ate ate act ccg atg gac gtt tcg att ate ggc 912 Gln Arg Leu Glu Asp He He Thr Pro Met Asp Val Ser He He Gly 290 295 300 tgc gtg gtg aat ggc cea ggt gag gcg ctg gtt tct here etc ggc gtc 960 Cys Val Val Asn Gly Pro Gly Glu Ala Leu Val Ser Thr Leu Gly Val 305 310 315 320 ggc aac aac aag aaa age ggc etc tat gaa gat ggc gtg cgc aaa 1008 Thr Gly Gly Asn Lys Ly s Ser Gly Leu Tyr Glu Asp Gly Val Arg Lys 325 330 335 gac cgt ctg gac aac aac a gat a g ate gac cag ctg gaa gca cgc att 1056 Asp Arg Leu Asp Asn Asn Asp Met He Asp Gln Leu Glu Ala Arg He 340 345 350 . cgt gcg aaa gcc agt cag ctg gac gaa gcg cgt cga att gac gtt cag 1104 Arg Ala Lys Ala Ser Gln Leu Asp Glu Ala Arg Arg He Asp Val Gln 355 360 365 cag gtt gaa aaa taa 1119 Gln Val Glu Lys 370 < 210 > 2 < 211 > 372 < 212 > PRT < 213 > Escherichia coli < 400 > 2 Met His Asn Gln Ala Pro He Gln Arg Arg Lys Ser Thr Arg He Tyr 1 5 10 15 Val Gly Asn Val Pro He Gly Asp Gly Ala Pro He Wing Val Gln Ser 20 25 30 Met Thr Asn Thr Arg Thr Thr Asp Val Glu Ala Thr Val Asn Gln He 35 40 45 Lys Wing Leu Glu Arg Val Gly Wing Asp He Val Val Val Ser Val 55 50 60 Thr Met Asp Wing Ala Glu Wing Phe Lys Leu He Lys Gln Gln Val Asn 65 70 75 80 Val Pro Leu Val Ala Asp He His Phe Asp Tyr Arg He Ala Leu Lys 85 90 95 Val Wing Glu Tyr Gly Val Asp Cys Leu Arg He Asn Pro Gly Asn He 100 105 110 Gly Asn Glu Glu Arg He Arg Met Val Val Asp Cys Ala Arg Asp Lys 115 120 125 Asn He Pro He Arg He Gly Val Asn Wing Gly Ser Leu Glu Lys Asp 130 135 140 Leu Gln Glu Lys Tyr Gly Glu Pro Thr Pro Gln Wing Leu Leu Glu Ser 145 150 155 160 Wing Met Arg His Val Asp His Leu Asp Arg Leu Asn Phe Asp Gln Phe 165 170 175 Lys Val Ser Val Lys Wing Ser Asp Val Phe Leu Wing Val Glu Ser Tyr 180 185 190 Arg Leu Leu Wing Lys Gln He Asp Gln Pro Leu His Leu Gly He Thr 195 200 205 • Glu Ala G and Gly Wing Arg Ser Gly Wing Val Lys Ser Wing He Gly Leu 210 215 220 Gly Leu Leu Ser Glu Gly He Gly Asp Thr Leu Arg Val Ser Leu 225 230 235 240 Wing Wing Asp Pro Val Glu Glu He Lys Val Gly Phe Asp He Leu Lys 245 250 255 Being Leu Arg He Arg Being Arg Gly He Asn Phe He Wing Cys Pro Thr 260 265 270 Cys Ser Arg Gln Glu Phe Asp Val He Gly Thr Val Asn Wing Leu Glu 275 280 285 Gln Arg Leu Glu Asp He He Thr Pro Met Asp Val Ser He He Gly 290 295 300 Cys Val Val Asn Gly Pro Gly Glu Ala Leu Val Ser Thr Leu Gly Val 305 310 315 320 Thr Gly Gly Asn Lys Lys Ser Gly Leu Tyr Glu Asp Gly Val Arg Lys 325 330 335 Asp Arg Leu Asp Asn Asn Asp Met He Asp Gln Leu Glu Ala Arg He 340 345 350 Arg Ala Lys Ala Ser Gln Leu Asp Glu Ala Arg Arg He Asp Val Gln 355 360 365 Gln Val Glu Lys • 370 < 210 > 3 < 211 > 2109 < 212 > DNA < 213 > Plasmodium falciparum LL < 220 > < 221 > CDS < 222 > (70) .. (2109) • < 400 > 3 cagcctataa atattattat ttattattat tttttttttt ttttttttt atgcctgaat 60 aaccacaaa atg agt tat ata aaa aga ctg att ctt ttt atg tta ctg ttt 111 Met Ser Tyr He Lys Arg Leu He Leu Phe Met Leu Leu Phe 1 5 10 tat tct cat gta aaa att aaa aaa tta ttt att aaa att tct aat gta 159 Tyr Ser His Val Lys He Lys Lys Leu Phe He Lys He Ser Asn Val 15 20 25 30 aac ata ttt ttt gca gaa gca aag aaa aat gga aaa aag gaa ttc ttt 207 • Asn He Phe Phe Wing Glu Wing Lys Lys Asn Gly Lys Lys Glu Phe Phe 35 40 45 ctt ttt tta tta cta aat ata aaa aat age caa cag aaa aaa act 255 Leu Phe Leu Leu Asn He Lys Lys Asn Ser Gln Gln Lys Lys Thr Tyr 50 55 60 cat att acc aaa agg aat acc ata aat aaa agt gat tta t tat tat 303 His He Thr Lys Arg Asn Thr He Asn Lys Ser Asp Phe Leu Tyr Ser 65 70 75 tta cta aat gaa gaa ggg aat tct tea aaa aag gaa tat aaa aat tta 351 Leu Leu Asn Glu Glu Gly Asn Ser Ser Lys Lys Glu Tyr Lys Asn Leu 80 85 90 aaa gat gaa gaa aaa tat aat ate ata ata ata ata ata ata aaa tat tgt 399 Lys Asp Glu Glu Lys Tyr Asn He He Gln Asn He Lys Lys Tyr Cys 95 100 105 110 gaa tgt act aaa aaa tat aaa agg etc cea here cga gaa gta gtt att 447 Glu Cys Thr Lys Lys Tyr Lys Arg Leu Pro Thr Arg Glu Val Val He 115 120 125 gga aat gtt aaa att gga gat aat aat aat ata gct att caat act atg 495 Gly Asn Val Lys He Gly Gly Asn Asn Lys He Wing He Gln Thr Met 130 135 140 gct age tgt gat aga aat gta gaa gag tgt gta ta t ca att att aga 543 Wing Ser Cys Asp Thr Arg Asn Val Glu Glu Cys Val Tyr Gln He Arg 145 150 155 aaa tgt aaa gat ttg ggt gct gac att gta agg ttg act gtt ca gga 591 Lys Cys Lys Asp Leu Gly Wing Asp He Val Arg Leu Thr Val Gln Gly 160 165 170 gtt caa gaa gca ca gct agt tat cat att aaa gaa aaa tta tta tct 639 Val Gln Glu Ala Gln Ala Ser Tyr His He Lys Glu Lys Leu Leu Ser 175 180 185 190 gaa aat gta aat ate cea tta gta here gat att cat ttt aat ect aaa 687 Glu Asn Val Asn He Pro Leu Val Thr Asp He His Phe Asn Pro Lys 195 200 205 ata gct tta atg gca gct gat gtg ttt gaa aaa att cga gtg aat cea 735 I have Ala Leu Met Wing Wing Asp Val Phe Glu Lys He Arg Val Asn Pro 210 215 220 gga aat tat gtt gat gga aga aaa aaa tgg ata gat aaa gtt tat aaa 783 Gly Asn Tyr Val Asp Gly Arg Lys Lys Trp He Asp Lys Val Tyr Lys 225 230 235 act aaa gaa gaa ttt gat gaa ggg aaa tta ttt ata aaa gaa aaa ttt 831 Thr Lys Glu Glu Phe Asp Glu Gly Lys Leu Phe He Lys Glu Lys Phe 240 245 250 • gta cea tta att gaa aaa tgt aaa aga tta aat aga gca ata aga att 879 Val Pro Leu He Glu Lys Cys Lys Arg Leu Asn Arg Wing He Arg He 255 260 265 270 ggt here aat cat gga ttc ctt tea tct cga gta tta tea tat tat gga 927 Gly Thr Asn His Gly Phe Leu Ser Being Arg Val Leu Being Tyr Tyr Gly 275 280 285 gat here cea tta gca tta gta gaa agt gct atg aga ttt tct gat tta 975 Asp Thr Pro Leu Wing Leu Val Glu Being Wing Met Arg Phe Ser Asp Leu 290 295 300 tgt aat gaa aac aat ttt aac aat ctt gtt ttt tct tct atg aaa gct tct 1023 Cys Asn Glu Asn Asn Phe Asn Asn Leu Val Phe Ser Met Lys Wing Ser 305 310 315 • aat gct tat gtt atg ata ata ata tta tat aga tta tta g tact aaa caa 1071 Asn Ala Tyr Val Met He Gln Ser Tyr Arg Leu Leu Val Ser Lys Gln 320 325 330 tat gaa aga aat atg att ttc ect ata cat tta gga gtt here gaa gca 1119 Tyr Glu Arg Asn Met Met Phe Pro He His Leu Gly Val Thr Glu Wing 335 340 345 350 gga ttt ggt gat aat gga aga ata aaa tct tat tta ggt ata gga tct 1167 Gly Phe Gly Asp Asn Gly Arg He Lys Ser Tyr Leu Gly He Gly Ser 355 360 365 tta tta tat gat ggt ata gga gat acc att cgt ata tcc tta here gaa 1215 Leu Leu Tyr Asp Gly He Gly Asp Thr He Arg He Ser Leu Thr Glu 370 375 380 gat ect tgg gaa gag tta act ect tgt aaa aaa tta gtt gaa aat tta 1263 Asp Pro Trp Glu Glu Leu Thr Pro Cys Lys Lys Leu Val Glu Asn Leu 385 390 395 aa aa aga ata ttt tat aat gaa aat ttt aaa gaa gat aat gaa tta 1311 Lys Lys Arg He Phe Tyr Asn Glu Asn Phe Lys Glu Asp Asn Glu Leu 400 405 410 aaa aat aat gaat atg gat acc aaa aattata tta aat ttt gaa gaa aat 1359 Lys Asn Asn Glu Met Asp Thr Lys Asn Leu Leu Asn Phe Glu Glu Asn 415 420 425 430 tat cga aat ttt aat aat ata aaa aaa aga aat gta gaa aaa aat aat 1407 Tyr Arg Asn Phe Asn Asn He Lys Lys Arg Asn Val Glu Lys Asn Asn 435 440 445 aat gta tta cat gaa gag tgc act ata ggt aat gta gta acc ata aaa 1455 Asn Val Leu His Glu Glu Cys Thr He Gly Asn Val Val Thr He Lys 450 455 460 gag tta gaa gat tct ctg ca ata att ttt aaa gat tta aat tta gaa gta 1503 Glu Leu Glu Asp Ser Leu Gln He Phe Lys Asp Leu Asn Leu Glu Val 465 470 475 gat tea aat gga aat ttg aaa aag gga gcc aaa here act gat atg gtt 1551 Asp Ser Asn Gly Asn Leu Lys Lys Gly Wing Lys Thr Thr Asp Met Val 480 485 490 att ata aat cat tat cat aat ata ata aat tta gga aaa aaa act gtg 1599 He He As Asp Phe His Asn He Thr Asn Leu Gly Lys Lys Thr Val 495 500 505 510 gat aaa tta atg ca gtg gga att aat ata gta gtt ca tat gaa cea 1647 Asp Lys Leu Met Gln Val Gly He Asn He Val Val Gln Tyr Glu Pro 515 520 525 cat aat ata gaa ttt ata gaa aaa atg gaa cea aat aat gat aat aat 1695 His Asn He Glu Phe He Glu Lys Met Glu Pro Asn Asn Asp Asn Asn 530 535 540 aat aat aat aat aat aat aat ata tta tat tat gtg gat ata aaa aat 1743 Asn Asn Asn Asn Asn Asn He Leu Phe Tyr Val Asp He Lys Asn 545 550 555 att atg aac agt tea gaa aaa aat att aaa tta agt aat tct aaa gga 1791 He Met Asn Ser Ser Glu Lys Asn He Lys Leu Ser Asn Ser Lys Gly 560 565 570 ^ iíAiMitia tat gga tta att tta aac gga aaa gaa gat ata ata ata ata aaa aaa 1839 Tyr Gly Leu He Leu Asn Gly Lys Glu Asp He Gln Thr He Lys Lys 575 580 585 590 ata aaa gaa tta aat cgt ect tta ttc att cta tta aaa tea gat 1887 He Lys Glu Leu Asn Arg Arg Pro Leu Phe He Leu Leu Lys Ser Asp 595 600 605 aac ata tat gaa cat gta tta ata acc aga aga att aat gaa ctt tta 1935 Asn He Tyr Glu His Val Leu He Thr Arg Arg He Asn Glu Leu Leu 610 615 620 cata tcc tta tat aat ata ata tat ata tat ata cat tat gtt gat att aat 1983 Gln Ser Leu Asn He Asn He Pro Tyr He His Tyr Val Asp He Asn 625 630 635 tea aat aat tat gat gat ata tta gtt aat tea here tta tat gca gga 2031 Ser Asn Asn Tyr Asp Asp He Leu Val Asn Ser Thr Leu Tyr Wing Gly 640 645 650 agt tgt ttg atg gat tta atg ggg gat ggt ctt att gtt aac gta act 2079 Be Cys Leu Met Asp Leu Met Gly Asp Gly Leu He Val Asn Val Thr 655 660 665 670 aat gat gtt ctt here aat aaa aaa ggg tag 2109 Asn Asp Val Leu Thr Asn Lys Lys Gly 675 680 < 210 > 4 < 211 > 679 < 212 > PRT < 213 > Plasmodium falciparum < 400 > 4 Met Ser Tyr He Lys Arg Leu He Leu Phe Met Leu Leu Phe Tyr Ser 1 5 10 15 His Val Lys He Lys Lys Leu Phe He Lys He Ser Asn Val Asn He 20 25 30 Phe Phe Wing Glu Wing Lys Lys Asn Gly Lys Lys Glu Phe Phe Leu Phe 35 40 45 Leu Leu Asn He Lys Lys Asn Ser Gln Gln Lys Lys Thr Tyr His He 50 55 60 - L-L.-.

Thr Lys Arg Asn Thr He Asn Lys Ser Asp Phe Leu Tyr Ser Leu Leu 65 70 75 80 Asn Glu Glu Gly Asn Ser Ser Lys Lys Glu Tyr Lys Asn Leu Lys Asp 85 90 95 Glu Glu Lys Tyr Asn He He Gln Asn He Lys Lys Tyr Cys Glu Cys • 100 105 110 Thr Lys Lys Tyr Lys Arg Leu Pro Thr Arg Glu Val Val He Gly Asn 115 120 125 Val Lys He Gly Gly Asn Asn Lys He Wing He Gln Thr Met Wing Ser 130 135 140 Cys Asp Thr Arg Asn Val Glu Glu Cys Val Tyr Gln He Arg Lys Cys 145 150 155 160 Lys Asp Leu Gly Wing Asp He Val Arg Leu Thr Val Gln Gly Val Gln 165 170 175 Glu Ala Gln Ala Ser Tyr His He Lys Glu Lys Leu Leu Ser Glu Asn 180 185 190 Val Asn He Pro Leu Val Thr Asp He His Phe Asn Pro Lys He Wing 195 200 205 Leu Met Ala Wing Asp Val Phe Glu Lys He Arg Val Asn Pro Gly Asn 210 215 220 Tyr Val Asp Gly Arg Lys Lys Trp He Asp Lys Val Tyr Lys Thr Lys 225 230 235 240 Glu Glu Phe Asp Glu Gly Lys Leu Phe He Lys Glu Lys Phe Val Pro 245 250 255 Leu He Glu Lys Cys Lys Arg Leu Asn Arg Wing He Arg He Gly Thr 260 265 27 0 • Asn His Gly Phe Leu Ser Ser Arg Val Leu Ser Tyr Tyr Gly Asp Thr 275 280 285 Pro Leu Ala Leu Val Glu Ser Wing Met Arg Phe Ser Asp Leu Cys Asn 290 295 300 Glu Asn Asn Phe Asn Asn Leu Val Phe Ser Met Lys Wing Ser Asn Wing 305 310 315 320 Tyr Val Met He Gln Ser Tyr Arg Leu Leu Val Ser Lys Gln Tyr Glu 325 330 335 Arg Asn Met Met Phe Pro He His Leu Gly Val Thr Glu Ala Gly Phe 340 345 350 ¿G ^ maí Gly Asp Asn Gly Arg He Lys Ser Tyr Leu Gly He Gly Ser Leu Leu 355 360 365 Tyr Asp Gly He Gly Asp Thr He Arg He Ser Leu Thr Glu Asp Pro 370 375 380 Trp Glu Glu Leu Thr Pro Cys Lys Lys Leu Val Glu Asn Leu Lys Lys 385 390 395 400 Arg He Phe Tyr Asn Glu Asn Phe Lys Glu Asp Asn Glu Leu Lys Asn 405 410 415 Asn Glu Met Asp Thr Lys Asn Leu Leu Asn Phe Glu Glu Asn Tyr Arg 420 425 430 Asn Phe Asn Asn He Lys Lys Arg Asn Val Glu Lys Asn Asn Asn Val 435 440 445 Leu His Glu Glu Cys Thr He Gly Asn Val Val Thr He Lys Glu Leu 450 455 460 Glu Asp Ser Leu Gln He Phe Lys Asp Leu Asn Leu Glu Val Asp Ser 465 470 475 480 Asn Gly Asn Leu Lys Lys Gly Wing Lys Thr Thr Asp Met Val He He 485 490 495 Asn Asp Phe His Asn He Thr Asn Leu Gly Lys Lys Thr Val Asp Lys 500 505 510 Leu Met Gln Val Gly He Asn He Val Val Gln Tyr Glu Pro His Asn 515 520 525 He Glu Phe He Glu Lys Met Glu Pro Asn Asn Asp Asn Asn Asn Asn 530 535 540 Asn Asn Asn Asn Asn He Leu Phe Tyr Val Asp He Lys Asn He Met 545 550 555 560 Asn Ser Ser Glu Lys Asn He Lys Leu Ser Asn Ser Lys Gly Tyr Gly 565 570 575 Leu He Leu Asn Gly Lys Glu Asp He Gln Thr He Lys Lys He Lys 580 585 590 Glu Leu Asn Arg Arg Pro Leu Phe He Leu Leu Lys Ser Asp Asn He 595 600 605 Tyr Glu His Val Leu He Thr Arg Arg He Asn Glu Leu Leu Gln Ser 610 615 620 Leu Asn He Asn He Pro Tyr He His Tyr Val Asp He Asn Ser Asn 625 630 635 640 Asn Tyr Asp Asp He Leu Val Asn Ser Thr Leu Tyr Wing Gly Ser Cys 645 650 655 Leu Met Asp Leu Met Gly Asp Gly Leu He Val Asn Val Thr Asn Asp 660 665 670 Val Leu Thr Asn Lys Lys Gly • 675 < 210 > 5 < 211 > 1200 < 212 > DNA < 213 > Escherichia coli • < 400 > 5 aatggctatc agaccgcttt aatgtcgatg gcttcaccct gcatccgttt acgcagggta 60 cgtttcgtac ggtcgataac atcgcccgcc aactgaccac aggcagcatc gatatcatca 120 ccacgagttt tacgcacaat agtggtgaaa ccgtagctca tcagcacttt tgagaaacgg 180 tcgatacggc tgttcgagct gcgtccatac ggcgcacccg ggaacgggtt ccacgggatc 240 aggttgatct tacacggcgt atctttcagc agttccgcca gttggtgcgc gtgttcagtg 300 ccgtcgttaa cgtggtcaag catcacgtat tcaatagtga ctcggccctg attggcgttg 360 gataacggcg gatttctcca caccgcagca aggaacgttt cgatattgta ctttttgttg 420 atcggcacaa tttcgtcacg aatttcgtcg ttcggcgcgt gcagggaaat tgccagtgca 480 acgtcgatca tatcgcccag tttatccagc gccggaacta caccggaagt ggaaagcgtg 540 acgcgacgtt tagacaggcc aaaaccgaaa tcatcaagca tgatttccat cgccggaacg 600 acgttgttca ggttgagcag cggctcgccc atgcccatca tcactacgtt agtgatcgga 660 cgctgaccgg tgacttttgc tgcgccgacg attttcgccg cacgccacac ctggccgata 720 atttccgaca cccgcaggtt gcggttaaag ccctgctggg cggtggaaca gaatttacac 780 tccagcgcac accccacctg cgaagagacg cagagcgtgg cacggtcgtc ttccgggata 840 tacaccgttt cgacgcgctg atcgccaacg gcgatcgccc atttaatggt gccgtcagat 900 gaacgctgtt cttcaaccac ttccggtgcg cggatttccg ccacctcttt cagtttgccg 960 cgcaacactt tgttgatgtc ggtcatctca tcaaagttgt cgcagcaata gtgatacatc 1020 cacttcatca cctgatcggc gcggaagggt ttttcaccta aatctttaaa aaactcccgc 1080 atctgctgac ggttgagatc cagcaggttg atttttccat etttegtggt gacgttttca 1140 attgtteaga ggtgtgacta catatgetat tccggcctcg ttattacaeg ttatggcccc 1200 < 210 > 6 < 211 > 384 < 212 > PRT < 213 > Escherichia coli Met Ser Glu Gln Leu Val Thr Pro Glu Asn Val Thr Thr Lys Asp Gly 1 5 10 15 Lys He Asn Leu Leu Asp Leu Asn Arg Gln Gln Met Arg Glu Phe Phe 20 25 30 Lys Asp Leu Gly Glu Lys Pro Phe Arg Wing Asp Gln Val Met Lys Trp 35 40 45 Met Tyr His Tyr Cys Cys Asp Asn Phe Asp Glu Met Thr Asp He Asn 50 55 60 ^ - Lys Val Leu Arg Gly Lys Leu Lys Glu Val Ala Glu He Arg Ala Pro ^^ 65 70 75 80 Glu Val Val Glu Glu Gln Arg Ser As Asp Gly Thr He Lys Trp Wing 85 90 95 He Wing Val Gly Asp Gln Arg Val Glu Thr Val Tyr He Pro Glu Asp 100 105 110 Asp Arg Ala Thr Leu Cys Val Ser Ser Gln Val Gly Cys Ala Leu Glu 115 120 125 Cys Lys Phe Cys Ser Thr Wing Gln Gln Gly Phe Asn Arg Asn Leu Arg 130 135 140 Val Ser Glu He He Gly Gln Val Trp Arg Ala Wing Lys He Val Gly 145 150 155 160 Wing Wing Lys Val Thr Gly Gln Arg Pro He Thr Asn Val Val Met Met 165 170 175 Gly Met Gly Glu Pro Leu Leu Asn Leu Asn Asn Val Val Pro Wing Met 180 185 190 Glu He Met Leu Asp Asp Phe Gly Phe Gly Leu Ser Lys Arg Arg Val 195 200 205 Thr Leu Ser Thr Ser Gly Val Val Pro Ala Leu Asp Lys Leu Gly Asp 210 215 220 Met He Asp Val Ala Leu Ala He Ser Leu His Ala Pro Asn Asp Glu 225 230 235 240 He Arg Asp Glu He Val Pro He Asn Lys Lys Tyr Asn He Glu Thr 245 250 255 Phe Leu Wing Wing Val Arg Arg Tyr Leu Glu Lys Ser Asn Wing Asn Gln 260 265 270 Gly Arg Val Thr He Glu Tyr Val Met Leu Asp His Val Asn Ace p Gly 275 280 285 Thr Glu His Wing His Gln Leu Wing Glu Leu Leu Lys Asp Thr Pro Cys 290 295 300 Lys He Asn Leu He Pro Trp Asn Pro Phe Pro Gly Wing Pro Tyr Gly 305 310 315 320 Arg Ser Ser Asn Ser Arg He Asp Arg Phe Ser Lys Val Leu Met Ser 325 330 335 Tyr Gly Phe Thr Thr He Val Arg Lys Thr Arg Gly Asp Asp He Asp 340 345 350 Wing Wing Cys Gly Gln Leu Wing Gly Asp Val He Asp Arg Thr Lys Arg 355 360 365 Thr Leu Arg Lys Arg Met Gln Gly Glu Wing He Asp He Lys Wing Val 370 375 380 < 210 > 7 < 211 > 1320 < 212 > DNA fl < 213 > Plasmodium falciparum < 220 > < 221 > CDS < 222 > (163). . (1173) < 400 > 7 taaataaata aattataaat ctttcaagaa tatatttttt ataaaaacat aaaatataaa 60 atatacatat atatatatat atatatttta tattactttt aaaattattt atttatacaa 120 atggaaattt aatgtgaaga atagaaaaaa cattttgtca at atga gaa aag tea 174 Met Glu Lys Ser • 1 aaa agg tac ata age ctg att aag atg gag agg aaa aaa ttt gag 222 Lys Arg Tyr He Ser Leu He Lys Met Met Glu Arg Lys Lys Phe Glu 5 10 15 20 aag tat aga tta aaa ca ata ata ata gat aat tat aaa gga aaa ata 270 Lys Tyr Arg Leu Lys Gln He Met Asp Asn He Tyr Lys Gly Lys He 25 30 35 att gaa ata aat aaa atg aaa aat att cea act gaa ata aga aga gaa 318 He Glu He Asn Lys Met Lys Asn He Pro Thr Glu He Arg Arg Glu 40 45 50 tta aaa aat ata ttt cat aat aat att tta agt ata aaa ccg ate aaa 366 # Leu Lys Asn He Phe His Asn Asn He Leu Ser He Lys Pro He Lys 55 60 65 tta gaa aaa tat gat aga gca tat gta aaa tta ttt cag tgt aaa gat 414 Glu Leu Lys Tyr Asp Arg Ala Tyr Lys Val Leu Phe Gln Cys Lys Asp 70 75 80 gaa aat att aag gca gaa here tea tta gat ttt ggt tcg cat aaa tct 462 Asn Glu Lys He Glu Ala Thr Ser Leu Asp Phe Gly Ser His Lys Ser 85 90 95 100 tta tgt ata tct age caa ata ggt tgt tct ttt gga tgt aag ttt tgt 510 Leu Cys He Ser Ser Gln He Gly Cys Ser Phe Gly Cys Lys Phe Cys 105 110 115 gct act ggt caa att ggt ata aaa tta caa aga gat ata gat gaa ata 558 Ala Thr Gly Gln He Gly He Lys Arg Gln Leu Asp He Asp Glu He 120 125 130 act gat cata ctt tta tat ttt caa tea aaa gga gtt gat ata aaa aat 606 Thr Asp Gln Leu Leu Tyr Phe Gln Ser Lys Gly Val Asp He Lys Asn 135 140 145 ata tct ttt atg ggt atg gga gaa ect tta gct aat cea tat gtt ttt 654 He Ser Phe Met Gly Met Gly Glu Pro Leu Ala Asn Pro Tyr Val Phe 150 155 160 gat tct ata caa ttt ttt aat gat aat aat tta ttt t ct ata tct aat 702 Asp Ser He Gln Phe Phe Asn Asp Asn Asn Leu Phe Be He Be Asn 165 170 175 180 aga cgt att aat ata tct act gtt ggt ctt tta cea gga att aaa aaa 750 Arg Arg He Asn He Ser Thr Val Gly Leu Leu Pro Gly He Lys Lys 185 190 195 tta aat aac ate ttt ect ca gtt aat tta gct t tc tea tta cat tct 798 Leu Asn Asn He Phe Pro Gln Val Asn Leu Ala Phe Ser Leu His Ser 200 205 210 cea ttt act gaa gaa agg gat caa ctt cea gta att aat aaa ttg ttt 846 Pro Phe Thr Glu Glu Arg Asp Gln Leu Val Pro He Asn Lys Leu Phe 215 220 225 ccg ttt aat gaa gtt ttt gat tta tta gat gaa aga ata gca aaa act 894 Pro Phe Asn Glu Val Phe Asp Leu Leu Asp Glu Arg He Wing Lys Thr 230 235 240 ggt aga aga gtt tgg ata agt tat att tta att aaa aat ctt aat gac 942 Gly Arg Arg Val Trp He Ser Tyr He Leu He Lys Asn Leu Asn Asp 245 250 255 260 aka aaa gat cat gca gaa gct ttg tct gat cat ata tgt aaa aga cea 990 Ser Lys Asp His Ala Glu Ala Leu Ser Asp His He Cys Lys Arg Pro 265 270 275 aat aac ata aga tac tta tat aat gta t gt tta ata ect tat aat aaa 1038 Asn Asn He Arg Tyr Leu Tyr Asn Val Cys Leu He Pro Tyr Asn Lys 280 285 290 ggt aat aga att tat ata ata ata ttt gaa tat ata ata tat ata ata 1086 Gly Asn Arg He Tyr Asn He Is Phe Glu Tyr He Tyr He Tyr He 295 300 305 tat tta cta ata ata aaa aaa ata ata tta tgt aaa tat att atg ttt 1134 Tyr Leu Leu He He Lys Lys Lys He Leu Cys Lys Tyr He Met Phe 310 315 320 falls here tta tat aaa tat ata ggc ata gag gac atg tta taaaaaagtg 1183 His Thr Leu Tyr Lys Tyr He Gly He Glu Asp Met Leu 325 330 335 caacatatat atatatatat atatatatat atatatatat acattttttt tatatttata 1243 ttatcttttt aatacattta ttecattaca ttgeagecaa aaatgttgac gaaaattttc 1303 atcgtttgga egatget 1320 < 210 > 8 < 211 > 337 < 212 > PRT < 213 > Plasmodium falciparum < 400 > 8 Met Glu Lys Ser Lys Arg Tyr He Ser Leu He Lys Met Met Glu Arg 1 5 10 15 Lys Lys Phe Glu Lys Tyr Arg Leu Lys Gln He Met Asp Asn He Tyr 20 25 30 Lys Gly Lys He He Glu He Asn Lys Met Lys Asn He Pro Thr Glu 35 40 45 He Arg Arg Glu Leu Lys Asn He Phe His Asn Asn He Leu Ser He 50 55 60 Lys Pro He Lys Glu Leu Lys Tyr Asp Arg Ala Tyr Lys Val Leu Phe 65 70 75 80 Gln Cys Lys Asp Asn Glu Lys He Glu Wing Thr Ser Leu Asp Phe Gly 85 90 95 Ser His Lys Ser Leu Cys He Ser Ser Gln He Gly Cys Ser Phe Gly 100 105 110 Cys Lys Phe Cys Wing Thr Gly Gln He Gly He Lys Arg Gln Leu Asp 115 120 125 He Asp Glu He Thr Asp Gln Leu Leu Tyr Phe Gln Ser Lys Gly Val 130 135 140 Asp He Lys Asn He Be Phe Met Gly Met Gly Glu Pro Leu Ala Asn 145 150 155 160 Pro Tyr Val Phe Asp Ser He Gln Phe Phe Asn Asp Asn Asn Leu Phe 165 170 175 Be He Be Asn Arg Arg He Asn He Be Thr Val Gly Leu Leu Pro 180 185 190 Gly He Lys Lys Leu Asn Asn He Phe Pro Gln Val Asn Leu Wing Phe 195 200 205 Ser Leu His Ser Pro Phe Thr Glu Glu Arg Asp Gln Leu Val Pro He 210 215 220 Asn Lys Leu Phe Pro Phe Asn Glu Val Phe Asp Leu Leu Asp Glu Arg 225 230 235 240 He Wing Lys Thr Gly Arg Arg Val Trp He Ser Tyr He Leu He Lys 245 250 255 Asn Leu Asn Asp Ser Lys Asp His Wing Glu Wing Leu Being Asp His He 260 265 270 Cys Lys Arg Pro Asn Asn He Arg Tyr Leu Tyr Asn Val Cys Leu He 275 280 285 Pro Tyr Asn Lys Gly Asn Arg He Tyr Asn He Ser Phe Glu Tyr He 290 295 300 Tyr He Tyr He Tyr Leu Leu He He Lys Lys Lys He Leu Cys Lys 305 310 315 320 Tyr He Met Phe His Thr Leu Tyr Lys Tyr He Gly He Glu Asp Met 325 330 335 Leu

Claims (12)

1. The use of the DNA sequence of the gcpE gene or ^ - yfgB of bacteria or parasites, for incorporation into the 5 genome of virus, eukaryotic and prokaryotic cells.

2. The use of DNA sequences, which hybridize the DNA sequence of the gcpE or yfgB proteins of bacteria or parasites, or their analogues or derivatives, derived from the sequence by insertion, deletion or replacement, and 10 which encode a plastid protein, which has the biological activity of the gcpE or yfgB protein, for incorporation into the genome of viruses, eukaryotic and prokaryotic cells.

3. The use of sequence SEQ 1, 3, 5, or 7, according to one of claims 1 or 2.

4. The use according to claim 1, 2 or 3, characterized in that these sequences are linked to controlling elements, the elements ensuring transcription and translation in the cell, and lead to • 20 expression of a DNA within the gcpE or yfgB protein.

5. Plant cells comprising the DNA sequence or DNA sequences, which hybridize the DNA sequence of the gcpE or yfgB genes of bacteria or parasites, or their analogs or their derivatives, which are derived from that sequence 25 by insertion, deletion or replacement, and which encode a plastid protein, which has the biological activity of the gcpE or yfgB proteins.

6. Transformed plant cells and transgenic plants regenerated from those plant cells, comprising the DNA sequence or DNA sequences, which hybridize the DNA sequence of the gcpE or yfgB gene of bacteria or parasites, or their analogs or their derivatives, which are derived from this sequence by insertion, deletion or replacement, and which encode a plastid protein, which has the biological activity of the gcpE or yfgB protein. The use according to one of claims 1 to 4 to correct, in particular increase the content of isoprenoids in viruses, eukaryotic and prokaryotic cells. The use according to one of claims 1 to 4 for the determination of the enzymatic activity of the gcpE or yfgB protein. The use according to one of claims 1 to 4 to identify substances that have an action of inhibition to the enzymatic activity of the gcpE protein. 10. A process for determining the enzymatic activity of the gcpE protein of bacteria or parasites, characterized in that the phosphorylation of a sugar or a phosphorous sugar is detected, or of a precursor of isoprenoid biosynthesis, in particular the phosphorylation of 2-C -methyl-D-erythritol, 2-C-methyl-D-erythritol phosphate, in particular 2-C-methyl-D-erythritol-4,2-C-methyl-D-erythrose phosphate, 2-C-phosphate -methyl-D-erythrose, in particular 2- ^ tw 5 C-methyl-D-erythrose-4, CH 2 (OH) -C (CH 3) = C (OH) -CH 2-0-PO (OH) 2 phosphate , CH2 (OH) -C (CH3) = C (OH) -CH2-OH, CH2 (OH) -CH (CH3) -CO-CH2-0-PO (OH) 2, CH2 (OH) -CH (CH3 ) -CO-CH2-OH, CH2 = C (CH3) -CO-CH2-0-PO (OH) 2, CH2 = C (CH3) - CO-CH2-OH, CH2 = C (CH3) -CH (OH ) -CH2-0-PO (OH) 2, CH2 = C (CH3) -CH (OH) -CH2-OH, CH2 (OH) -C (= CH2) -C (OH) -CH2-0-PO ( OH) 2, CH2 (OH) -C (= CH2) - 10 C (OH) -CH2-OH, CHO-CH (CH3) -CH (OH) -CH2-0-PO (OH) 2, CHO-CH (CH3) - CH (OH) - CH2 - OH, CH2 (OH) - C (OH) (CH3) - CH = CH - 0 - PO (OH) 2, CH2 (OH) - C (OH) (CH3) -CH = CH-OH, CH (O H) = C (CH3) -CH (OH) -CH2-0-PO (OH) 2, CH (OH) = C (CH3) -CH (OH) -CH2-OH, CH3-C (CH3) = CH -CH2-0-PO (OH) 2, CH3-C (CH3) = CH-CH2-OH, CH2 = C (CH3) -CH2-CH2-0-PO (OH) 2, CH2 = C (CH3) - CH2-CH2-OH. 11. A process for classifying a compound, the process comprising: a) the provision of a host cell, which contains a recombinant expression vector, wherein the vector comprises at least one fragment of the sequence of DNA encoding a gcpE or yfgB protein of bacteria or parasites, or an analog or derivative of the polypeptide, wherein one or more amino acids are deleted, added or replaced by other amino acids, without substantially reducing the enzymatic action of the polypeptide, and furthermore a composed of the one 25 suspects that it has an antifungal, antiparasitic, or antiviral action in humans and animals, b) puts the host cell in contact with the compound and c) determines the antifungal, antiparasitic, or antiviral action of the compound. 12. A process for classifying a compound, the process comprising: a) the provision of a host cell, which contains a recombinant expression vector, wherein the The vector comprises at least one fragment of the DNA sequence encoding a gcpE or yfgB protein of bacteria or parasites, or an analog or derivative of the polypeptide, wherein one or more amino acids are deleted, added or replaced by other amino acids, without reducing substantially the action 15 enzyme of the polypeptide, and in addition a compound suspected of having antiviral, antiparasitic, fungicidal or herbicidal action on plants, b) putting the host cell in contact with the compound and c) determining the antiviral, antiparasitic, fungicidal action or compound herbicide. 25