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Baixe genoma xylela e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! NATURE | VOL 406 | 13 JULY 2000 | www.nature.com 151 articles The genome sequence of the plant pathogen Xylella fastidiosa The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis*, São Paulo, Brazil * A full list of authors appears at the end of this paper ............................................................................................................................................................................................................................................................................ Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis—a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium–bacterium and bacterium–host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer. Citrus variegated chlorosis (CVC), which was first recorded in Brazil in 1987, affects all commercial sweet orange varieties1. Symptoms include conspicuous variegations on older leaves, with chlorotic areas on the upper side and corresponding light brown lesions, with gum-like material on the lower side. Affected fruits are small, hardened and of no commercial value. A strain of Xylella fastidiosa was first identified as the causal bacterium in 1993 (ref. 2) and found to be transmitted by sharpshooter leafhoppers in 1996 (ref. 3). CVC control is at present limited to removing infected shoots by pruning, the application of insecticides and the use of healthy plants for new orchards. In addition to CVC, other strains of X. fastidiosa cause a range of economically important plant diseases including Pierce’s disease of grapevine, alfalfa dwarf, phony peach disease, periwinkle wilt and leaf scorch of plum, and are also associated with diseases in mulberry, pear, almond, elm, sycamore, oak, maple, pecan and coffee4. The triply cloned X. fastidiosa 9a5c, sequenced here, was derived from the pathogenic culture 8.1b obtained in 1992 in Bordeaux (France) from CVC-affected Valencia sweet orange twigs collected in Macaubal (São Paulo, Brazil) on May 21, 1992 (ref. 2). Strain 9a5c produces typical CVC symptoms on inoculation into experimental citrus plants5, and into Nicotiana tabacum (S. A. Lopes, personal communication) and Catharantus roseus (P. Brant- Monteiro, personal communication)—two novel experimental hosts. General features of the genome The basic features of the genome are listed in Table 1 and a detailed map is shown in Fig. 1. The conserved origin of replication of the large chromosome has been identified in a region between the putative 50S ribosomal protein L34 and gyrB genes containing dnaA, dnaN and recF6. The Escherichia coli DnaA box consensus sequence TTATCCACA is found on both DNA strands close to dnaA. In addition, there are typical 13-nucleotide (ACCACCAC- CACCA) and 9-nucleotide (two TTTCATTGG and two TTTTA- TATT) sequences in other intergenic sequences of this region. This region is coincident with the calculated GC-skew signal inversion7. We have designated base 1 of the X. fastidiosa genome as the first Tof the only TTTTAT sequence found between the ribosomal protein L34 gene and dnaA. The overall percentage of open reading frames (ORFs) for which a putative biological function could be assigned (47%) was slightly below that for other sequenced genomes such as Thermotoga maritima8 (54%), Deinococcus radiodurans9 (52.5%) and Neisseria meningitidis10 (53.7%). This may reflect the lack of previous complete genome sequences from phytopathogenic bacteria. Plas- mid pXF1.3 contains only two ORFs, one of which encodes a replication-associated protein. Plasmid pXF51 contains 64 ORFs, of which 5 encode proteins involved in replication or plasmid stability and 20 encode proteins potentially involved in conjugative transfer. One ORF encodes a protein similar to the virulence- associated protein D (VapD), found in many other bacterial pathogens11. Four regions of pXF51 present significant DNA simi- larity to parts of transposons found in plasmids from other bacteria, suggesting interspecific horizontal exchange of genetic material. The principal paralogous families are summarized in Table 2. The complete list of ORFs with assigned function is shown in Table 3. Seventy-five proteins present in the 21 completely sequenced genomes in the COG database12 (as of 15th March 2000) were also found in X. fastidiosa. Each of these sequences was used to Table 1 General features of the Xylella fastidiosa 9a5c genome Main chromosome Length (bp) 2,679,305 G+C ratio 52.7% Open reading frames (ORFs) 2,782 Coding region (% of chromosome size) 88.0% Average ORF length (bp) 799 ORFs with functional assignment 1,283 ORFs with matches to conserved hypothetical proteins 310 ORFs without significant data base match 1,083 Ribosomal RNA operons 2 (16SrRNA-Ala-TGC-tRNA-Ile-GAT- tRNA-23SrRNA-5SrRNA) tRNAs 49 (46 different sequences corresponding to all 20 amino acids) tmRNA 1 ............................................................................................................................................................................. Plasmid pXF51 Length (bp) 51,158 G+C ratio 49.6% Open reading frames (ORFs) 64 Protein coding region (% of plasmid size) 86.9% ORFs with functional assignment 30 ORFs with matches to conserved hypothetical proteins 8 ORFs without significant data base match 24 ............................................................................................................................................................................. Plasmid pXF1.3 Length (bp) 1,285 G+C ratio 55.6% Open reading frames (ORFs) 2 ORFs with functional assignment 1 ............................................................................................................................................................................. © 2000 Macmillan Magazines Ltd generate a phylogenetic tree of the 22 organisms. In 69% of such trees, X. fastidiosa was grouped with Haemophilus influenzae and E. coli, consistent with a phylogenetic analysis undertaken with the 16S rRNA gene13. One ORF, a cytosine methyltransferase (XF1774), is interrupted by a Group II intron. The intron was identified on the basis of the presence of a reverse transcriptase-like gene (as in other Group II introns), conserved splice sites, conserved sequence in structure V and conserved elements of secondary structure14. Group II introns are rare in prokaryotes, but have been found in different evolutive lineages including E. coli, cyanobacteria and proteobacteria15. Transcription, translation and repair The basic transcriptional and translational machinery of X. fasti- diosa is similar to that of E. coli16. Recombinational repair, nucleo- tide and base-excision repair, and transcription-coupled repair are present with some noteworthy features. For example, no photolyase was found, indicating exclusively dark repair. Although the main genes of the SOS pathway, recA and lexA, are present, ORFs corresponding to the three DNA polymerases induced by SOS in E. coli (DNA polymerases II, IVand V)17 are missing, indicating that the mutational pathway itself may be distinct. Energy metabolism Even though X. fastidiosa is, as its name suggests, a fastidious organism, energy production is apparently efficient. In addition to all the genes for the glycolytic pathway, all genes for the tricarboxylic acid cycle and oxidative and electron transport chains are present. ATP synthesis is driven by the resulting chemi- osmotic proton gradient and occurs by an F-type ATP synthase. Fructose, mannose and glycerol can be utilized in addition to glucose in the glycolytic pathway. There is a complete pathway for hydrolysis of cellulose to glucose, consisting of 1,4-b-cellobiosidase, endo-1,4-b-glucanase and b-glucosidase, suggesting that cellulose breakdown may supplement the often low concentrations of mono- saccharides in the xylem18. Two lipases are encoded in the genome, but there is no b-oxidation pathway for the hydrolysis of fatty acids, presumably precluding their utilization as an alternative carbon and energy source. Likewise, although enzymes required for the break- down of threonine, serine, glycine, alanine, aspartate and glutamate are present, pathways for the catabolism of the other naturally occurring amino acids are incomplete or absent. The gluconeogenesis pathway appears to be incomplete. Phos- phoenolpyruvate carboxykinase and the gluconeogenic enzyme fructose-1,6-bisphosphatase, which are required to bypass the irreversible step in glycolysis, are not present. The absence of the first is compensated by the presence of phosphoenolpyruvate synthase and malate oxidoreductase, which together can generate phosphoenolpyruvate from malate. There appears, however, to be no known compensating pathway for the absence of fructose-1,6- bisphosphatase. It is possible that among the large number of unidentified X. fastidiosa genes there are non-homologous genes that compensate for steps in such critical pathways. Barring this possibility, however, the absence of a functional gluconeogenesis pathway implies a strict dependence on carbohydrates both as a source of energy and anabolic precursors. The glyoxylate cycle is absent and the pentose phosphate pathway is incomplete. In the latter pathway, genes for neither 6-phosphogluconic dehydrogenase nor transaldolase were identified. Small molecule metabolism X. fastidiosa exhibits extensive biosynthetic capabilities, pre- sumably an absolute requirement for a xylem-dwelling bacterium. Most of the genes found in E. coli necessary for the synthesis of all amino acids from chorismate, pyruvate, 3-phosphoglycerate, glu- tamate and oxaloacetic acid16 were identified. However, some genes in X. fastidiosa are bi-functional, such as phosphoribosyl-AMP cyclohydrolase/phosphoribosyl-ATP pyrophosphatase (XF2213), aspartokinase/homoserine dehydrogenase I (XF2225), imidazole- glycerolphosphate dehydratase/histidinol-phosphate phosphatase (XF2217) and a new diaminopimelate decarboxylase/aspartate kinase (XF1116) that would catalyse the first and the last steps of lysine biosynthesis. In addition, the gene for acetylglutamate kinase (XF1001) has an acetyltransferase domain at its carboxy-terminal end that would compensate for the missing acetyltransferase in the arginine biosynthesis pathway. Other missing genes include phos- phoserine phosphatase, cystathionine b-lyase, homoserine O-suc- cinyltransferase and 2,4,5-methyltetrahydrofolate-homocysteine methyltransferase. The first two enzymes are also absent in the Bacillus subtilis genome, the third is absent in Haemophilus influ- enzae and the fourth is missing in both genomes12. We thus presume that alternative, unidentified enzymes complete the biosynthetic pathways in these organisms and in X. fastidiosa. The pathways for the synthesis of purines, pyrimidines and nucleotides are all complete. X. fastidiosa is also apparently capable of both synthesizing and elongating fatty acids from acetate. Again, however, some E. coli enzymes were not found, such as holo acyl- carrier-protein synthase (also absent in Synechocystis sp., H. influenzae and Mycoplasma genitalium) and enoyl-ACP reductase (NADPH) (FabI) (also absent from M. genitalium, Borrelia burgdorferi and Treponema pallidum)12. X. fastidiosa appears to be capable of synthesizing an extensive variety of enzyme cofactors and prosthetic groups, including biotin, folic acid, pantothenate and coenzyme A, ubiquinone, glutathione, thioredoxin, glutaredoxin, riboflavin, FMN, FAD, pyrimidine nucleotides, porphyrin, thiamin, pyridoxal 59-phosphate and lipo- ate. In a number of the synthetic pathways, one or more of the enzymes present in E. coli are absent, but this is also true for at least one other sequenced Gram-negative bacterial genome in each case12. We therefore again infer that the missing enzymes are either not essential or replaced by unknown proteins with novel structures. Transport-related proteins A total of 140 genes encoding transport-related proteins were identified, representing 4.8% of all ORFs. For comparison, E. coli, B. subtilis and M. genitalium have around 10% of genes encoding transport proteins, whereas Helicobacter pylori, Synechocystis sp. and Methanococcus jannaschii have 3.5–5.4% (ref. 19). Transport sys- tems are central components of the host–pathogen relationship (Fig. 2). There are a number of ion transporters and transporters for the uptake of carbohydrates, amino acids, peptides, nitrate/nitrite, sulphate, phosphate and vitamin B12. Many different transport articles 152 NATURE | VOL 406 | 13 JULY 2000 | www.nature.com Table 2 Largest families of paralogous genes Family (total number of families = 312) Number of genes (total number of genes = 853) ............................................................................................................................................................................. ATP-binding subunits of ABC transporters 23 Reductases/dehydrogenases 12 Two-component system, regulatory proteins 12 Hypothetical proteins 10 Transcriptional regulators 9 Fimbrial proteins 9 Two-component system, sensor proteins 9 ............................................................................................................................................................................. Figure 1 Linear representation of the main chromosome and plasmids pXF51 and pXF1.3 of the Xylella fastidiosa genome. Genes are coloured according to their biological role. Arrows indicate the direction of transcription. Genes with frameshift and point mutations are indicated with an X. Ribosomal RNA genes, the tmRNA, the principal repeats, prophages and the group II intron are indicated by coloured lines. Transfer RNAs are identified by a single letter identifying the amino acid. Pie chart represents the distribution of the number of genes according to biological role. The numbers below protein-producing genes correspond to gene IDs. Q © 2000 Macmillan Magazines Ltd include many more enzymes, which we did not find; however, some of the genes listed lie close to ORFs without significant database matches, suggesting that at least one (as yet undiscovered) poly- ketide pathway may be functional. Prophages Bacteriophages can mediate the evolution and transfer of virulence factors and occasional acquisition of new traits by the bacterial host. Because as much as 7% of the X. fastidiosa genome sequenced corresponds to double-stranded (ds) DNA phage sequences, mostly from the Lambda group, we suspect that this route may have been of particular importance for this bacterium. It is noteworthy that a very high percentage of phage-related sequences has also been detected in a second vascular-restricted plant pathogen, Spiroplasma citri33. We identified four regions, with a high density of ORFs homologous to phage sequences, that we considered to be prophages, in addition to isolated phage sequences dispersed throughout the genome. Two of these prophages (each ,42 kbp, designated XfP1 and XfP2) are similar to each other, lie in opposite orientations in distinct regions and appear to belong to the dsDNA, tailed-phage group. Both appear to contain most of the genes responsible for particle assembly, although we know of no reports of phage particle release from X. fastidiosa cultures. In prophage XfP1, we found two ORFs between tail genes V and W that are similar to ORF118 and vapA from the virulence-associated region of the animal pathogen Dichelobacter nodosus, which by homology encode a killer and a suppressor protein34. Interestingly, in prophage XfP2, we found two other ORFs also between tail genes V and W that are similar to hypothetical ORFs of Ralstonia eutropha trans- poson Tn4371 (ref. 35). The other two identified prophages, XfP3 and XfP4, are also similar in sequence to each other and to the H. influenzae cryptic prophage fflu (ref. 36). They both contain a 14,317-bp exact repeat. Few particle-assembly genes were found in these regions, suggesting that these prophages are defective. An ORF similar to hicB from H. influenzae, a component of the major pilus gene cluster in some isolates, was found in XfP4 (ref. 37). The presence of virulence-associated genes from other organisms within the prophage sequences is strong evidence for a direct role for bacteriophage-mediated horizontal gene transfer in the definition of the bacterial phenotype. Absence of avirulence genes Phytopathogenic bacteria generally have a limited host range, often confined to members of a single species or genus. This specificity is defined by the products of the so-called avirulence (avr) genes present in the pathogen, which are injected directly into host cells, on infection, through a type III secretory system38–40. BLAST41 searches with all known avr and type III secretory system sequences failed to identify genes encoding proteins with significant simi- larities in the genome of X. fastidiosa. Although the variability of avr genes amongst bacteria might account for this apparent lack, the high level of similarity of some components of the type III secretory system argues against this. We suspect that these genes are, in fact, not required because of the insect-mediated transmission and vascular restriction of the bacterium that obviates the necessity of host cell infection. Furthermore, if the differing host ranges of X. fastidiosa are molecularly defined, this may be by a quite different mechanism not involving avr proteins. Conclusions Before the elucidation of its complete genome sequence, very little was known of the molecular mechanisms of X. fastidiosa pathogeni- city. Indeed, this bacterium was probably the least characterized of all organisms that have been fully sequenced. Our complete genetic analysis has determined not only the basic metabolic and replicative characteristics of the bacterium, but also a number of potential pathogenicity mechanisms. Some of these have not previously been postulated to occur in phytopathogens, providing new insights into the generality of these processes. Indeed, the availability of this first complete plant pathogen genome sequence will now allow the initiation of the detailed comparison of animal and plant pathogens at the whole-genome level. In addition, the information contained in the sequence should provide the basis for an accelerated and rational experimental dissection of the interactions between X. fastidiosa and its hosts that might lead to fresh insights into potential approaches to the control of CVC. M Methods The sequencing and analysis in this project were carried out by a network of 34 biology laboratories and one bioinformatics centre. The network is called the Organization for Nucleotide Sequencing and Analysis (ONSA)42, and is entirely located in the state of São Paulo, Brazil. Sequencing and assembly The sequence was generated using a combination of ordered cosmid and shotgun strategies43. A cosmid library was constructed, providing roughly 15-fold genome cover- age, containing 1,056 clones with average insert size of 40 kilobases (kb). High-density colony filters of the library were made, and a physical map of the genome was constructed using a strategy of hybridization without replacement44. A total of 113 cosmid clones was selected for sequencing on the basis of the hybridization map and end-sequence analysis. The cosmid sequences were assembled into 15 contigs covering 90% of the genome. Additionally, shotgun libraries with different insert sizes (0.8–2.0 kb and 2.0–4.5 kb) were constructed from nebulized or restricted genomic DNA cloned into plasmids, and sequenced to achieve a 3.74-fold coverage of high-quality sequence (29,140 reads). Most of the sequencing was performed with BigDye terminators on ABI Prism 377 DNA sequencers. Cosmid and shotgun sequences were assembled into six contigs. We identified sequence gaps by linking information from forward and reverse reads, and closed either by primer walking or insert subcloning. The remaining physical gaps were closed by combinatorial PCR and by lambda clones selected from a lDash library by end-sequencing. The collinearity between the genome and the obtained sequence was confirmed by digestion of genomic DNA with AscI, NotI, SfiI, SmiI and SrfI, followed by comparison of the digestion pattern with the electronic digestion of the generated sequence. In addition, sequences from both ends of most cosmid clones and 236 l clones were used to confirm the orientation and integrity of the contigs. The sequence was assembled using phred+phrap+consed45. All consensus bases have quality with Phred value of at least 20. There are no unexplained high quality discrepancies, each consensus base is confirmed by at least one read from each strand, and the overall error estimate is less than 1 in every 10,000 bases. ORF prediction and annotation ORFs were determined using glimmer 2.0 (ref. 46) and the glimmer post-processor RBSfinder (S. L. Salzberg, personal communication). A few ORFs were found by hand guided by BLAST41 results. Annotation was carried out in a cooperative way, mostly by comparison with sequences in public databases, using BLAST41 and tRNAscan-SE (ref. 47) and was based on the functional categories for E. coli48. Only one tmRNA was located (K. Williams, personal communication). To help annotate transport proteins, we built a custom BLAST41 database using sequences from http://www-biology.ucsd.edu/,msaier/ transport/toc.html and compared our ORFs with these sequences. Phylogenetic trees for conserved COGs12 were built using ClustalX49 for multiple alignment and Phylip50. Paralogous gene families (Table 2) were determined using BLASTX with the E-value cut-off equal to e-5 and such that at least 60% of the query sequence and at least 30% of the subject sequence were aligned. Received 24 March; accepted 24 May 2000. 1. Rosseti, V. et al. Présence de bactéries dans le xylème d’orangers atteints de chlorose variégée, une nouvelle maladie des agrumes au Brésil. C. R. Acad. Sci. 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Perez, Scientific Director of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), for his strategic vision in creating and nurturing this project as well as C. A. de Pian and Juçara Parra for their administrative coordination. We thank our Steering Committee: S. Oliver, A. Goffeau, J. Sgouros, A. C. M. Paiva and J. L. Azevedo for their critical accompaniment of the work. We also thank R. Fulton and P. Minx for their timely contribution and advice. Project funding was from FAPESP, the RHAE programme of the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) and Fundecitrus. For the full list of individuals who contributed to the completion of this project see (http://www.lbi. ic.unicamp.br/xf). Correspondence and requests for materials should be addressed to J.C.S. (e-mail: setubal@ic.unicamp.br). The sequence has been deposited in GenBank with accession numbers AE003849 (chromosome), AE003850 (pXF1.3) and AE003851 (pXF51). articles 156 NATURE | VOL 406 | 13 JULY 2000 | www.nature.com Authors: A. J. G. Simpson1, F.C. Reinach2, P. Arruda3, F. A. Abreu4, M. Acencio5, R. Alvarenga2, L. M. C. Alves6, J. E. Araya7, G. S. Baia2, C. S. Baptista8, M. H. Barros8, E. D. Bonaccorsi2, S. Bordin9, J. M. Bové10, M. R. S. Briones7, M. R. P. Bueno11, A. A. Camargo1, L. E. A. Camargo12, D. M. Carraro12, H. Carrer12, N. B. Colauto13, C. Colombo14, F. F. Costa9, M. C. R. Costa15, C. M. Costa-Neto16, L. L. Coutinho12, M. Cristofani17, E. Dias-Neto1, C. Docena2, H. El-Dorry2, A. P. Facincani6, A. J. S. Ferreira2, V. C. A. Ferreira18, J. A. Ferro6, J. S. Fraga4, S. C. França19, M. C. Franco20, M. Frohme21, L. R. Furlan22, M. Garnier10, G. H. Goldman23, M. H. S. Goldman24, S. L. Gomes2, A. Gruber4, P. L. Ho25, J. D. Hoheisel21, M. L. Junqueira26, E. L. Kemper3, J. P. Kitajima27, J. E. Krieger26, E. E. Kuramae28, F. Laigret10, M. R. Lambais12, L. C. C. Leite25, E. G. M. Lemos6, M. V. F. Lemos29, S. A. Lopes19, C. R. Lopes13, J. A. Machado30†, M. A. Machado17, A. M. B. N. Madeira4, H. M. F. Madeira12†, C. L. Marino13, M. V. Marques8, E. A. L. Martins25, E. M. F. Martins18, A. Y. Matsukuma2, C. F. M. Menck8, E. C. Miracca5, C. Y. Miyaki11, C. B. Monteiro-Vitorello12, D. H. Moon20, M. A. Nagai5, A. L. T. O. Nascimento25, L. E. S. Netto11, A. Nhani Jr6, F. G. Nobrega8†, L. R. Nunes31, M. A. Oliveira32, M. C. de Oliveira33, R. C. de Oliveira31, D. A. Palmieri13, A. Paris13, B. R. Peixoto2, G. A. G. Pereira32, H. A. Pereira Jr6, J. B. Pesquero16, R. B. Quaggio2, P. G. Roberto19, V. Rodrigues34, A. J. de M. Rosa34, V. E. de Rosa Jr28, R. G. de Sá34, R. V. Santelli2, H. E. Sawasaki14, A. C. R. da Silva2, A. M. da Silva2, F. R. da Silva3,27, W. A. Silva Jr15, J. F. da Silveira7, M. L. Z. Silvestri2, W. J. Siqueira14, A. A. de Souza17, A. P. de Souza3, M. F. Terenzi23, D. Truffi12, S. M. Tsai20, M. H. Tsuhako18, H. Vallada35, M. A. Van Sluys33, S. Verjovski-Almeida2, A. L. Vettore3, M. A. Zago15, M. Zatz11, J. Meidanis27 & J. C. Setubal27. Addresses: 1, Instituto Ludwig dePesquisa sobre oCâncer, Rua Prof. Antonio Prudente,109 – 4o andar, 01509-010, São Paulo - SP, Brazil; 2, Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo - SP, Brazil; 3, Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Caixa Postal 6010,13083-970, Campinas – SP, Brazil; 4, Laboratório de BiologiaMolecular, Departamento de Patologia, Faculdade de MedicinaVeterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, 05508-000, São Paulo – SP, Brazil; 5, Disciplina de Oncologia, Departamento de Radiologia, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Arnaldo, 455, 01296-903, São Paulo – SP, Brazil; 6, Departamento de Tecnologia, Faculdadede Ciências Agrárias e Veterinárias de Jaboticabal, UniversidadeEstadual Paulista, Via © 2000 Macmillan Magazines Ltd articles NATURE | VOL 406 | 13 JULY 2000 | www.nature.com 157 de Acesso Prof. Paulo D. Castellane s/n, Km 5,14884-900, Jaboticabal – SP, Brazil; 7, Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina,UniversidadeFederal de São Paulo, Rua Botucatu, 862, 04023-062, São Paulo – SP, Brazil; 8, Departamento deMicrobiologia, Instituto deCiênciasBiomédicas,UniversidadedeSãoPaulo,Av. Prof. LineuPrestes,1374, 05508-900, São Paulo – SP, Brazil; 9, Hemocentro, Faculdade de Ciências Médicas, Universidade Estadual de Campinas,13083-97, Campinas – SP, Brazil; 10, Institut National de la Recherche Agronomique et Université Victor Ségalen Bordeaux 2, 71 Avenue Edouard Bourleaux, Laboratoire de Biologie Cellulaire et Moléculaire, 33883 Vilenave d’Ornon Cedex, France; 11, Departamento de Biologia, Instituto de Biociências,UniversidadedeSãoPaulo,RuadoMatão, 277, 05508-900,SãoPaulo – SP,Brazil; 12, EscolaSuperiordeAgricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias,11,13418-900, Piracicaba – SP, Brazil; 13, Departamento de Genética, Instituto de Biociências, Universidade Estadual Paulista, Distrito de Rubião Junior,18618-000, Botucatu – SP, Brazil; 14, Centro de Genética, Biologia Molecular e Fitoquı́mica, Instituto Agronômico de Campinas, Av. Barão de Itapura,1481, Caixa Postal 28,13001-970, Campinas – SP, Brazil; 15, Departamento de Clı́nica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900,14049-900, Ribeirão Preto – SP, Brazil; 16, Departamento de Biofı́sica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu, 862, 04023-062, São Paulo – SP, Brazil; 17, Centro de Citricultura Sylvio Moreira, Instituto Agronômico de Campinas, Caixa Postal 04,13490-970, Cordeirópolis – SP, Brazil; 18, Laboratório de Bioquı́mica Fitopatológica e Laboratório de Imunologia, Instituto Biológico, Av. Cons. Rodrigues Alves,1252, 04014-002, São Paulo – SP, Brazil; 19, Departamento de Biotecnologia dePlantasMedicinais, UniversidadedeRibeirão Preto,Av.Costábile Romano, 2201,14096-380, Ribeirão Preto – SP,Brazil; 20,Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, Caixa Postal 96,13400-970, Piracicaba – SP, Brazil; 21, Funktionelle Genomanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, D-69120, Heidelberg, Germany; 22, Departamento de Melhoramento e Nutrição Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista, Fazenda Lageado, Caixa Postal 560,18600-000, Botucatu - SP, Brazil; 23, Departamento de Ciências Farmacêuticas , Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n,14040-903, Ribeirão Preto – SP, Brazil; 24, Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900,14040-901,Ribeirão Preto – SP,Brazil; 25,Centro deBiotecnologia, InstitutoButantan,Av. Vital Brasil,1500, 05503-900, São Paulo – SP, Brazil; 26, Laboratório de Genética e Cardiologia Molecular/LIM 13, Instituto do Coração (InCor), Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 44, 05403-000, São Paulo – SP, Brazil; 27, Instituto de Computação, Universidade Estadual de Campinas, Caixa Postal 6176,13083-970, Campinas – SP, Brazil; 28, Departamento de Produção Vegetal, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Fazenda Lageado, Caixa Postal 237,18603-970, Botucatu – SP, Brazil; 29, Departamento de Biologia Aplicada à Agropecuária, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, UniversidadeEstadual Paulista, Via deAcessoProf. PauloDonato Castellane,14884-900, Jaboticabal – SP, Brazil; 30, Hospital doCâncer – A.C. Camargo, R. Antonio Prudente, 211, 01509-010, São Paulo – SP, Brazil; 31, Núcleo Integrado de Biotecnologia, Universidade de Mogi dasCruzes,Av.Dr.CândidoXavierdeAlmeidaSouza, 200, 08780-911,Mogi dasCruzes – SP,Brazil; 32,Departamento deGenética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Caixa Postal 6010,13083-970, Campinas– SP, Brazil; 33, Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, 05508-900, São Paulo - SP, Brazil; 34, Departamento de Parasitologia, Microbiologia e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900,14049-900, Ribeirão Preto – SP, Brazil; 35, Departamento de Psiquiatria, Instituto de Psiquiatria, Faculdade de Medicina, Universidade de São Paulo, Rua Dr. Ovı́deo Pires de Campos s/n, Sala 4051 (3o. andar), 05403-010, São Paulo – SP, Brazil. † Present addresses: Novartis Seeds LTDA, Av. Prof. Vicente Rao, 90, 04706-900, São Paulo – SP, Brazil (J. A. Machado); Centro de Ciências Agrárias e Ambientais, Pontifı́cia Universidade Católica do Paraná, BR-376, Km 14, Caixa Postal 129, 83010-500, São José dos Pinhais – PR, Brazil (H. M. F. Madeira); Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraı́ba, Av. Shishimi Hifumi, 2911, 12244-000, São José dos Campos – SP, Brazil (F. G. Nobrega). © 2000 Macmillan Magazines Ltd