Sequencing and genome annotation of the party of Xylella fastidiosa

A citricultura paulista pode ser considerada uma das mais competitivas e importantes atividades agroindustriais do Brasil, gerando aproximadamente 400 mil empregos e adicionando anualmente U 1,4 bilhões ao país. Entretanto ainda apresenta uma produtividade inferior àquela encontrada na Flórida, principalmente devido a deficiências nutricionais e hídricas e a doenças que há diversos anos atingem às lavouras. Nos últimos dez anos a clorose variegada dos citros (CVC), conhecida popularmente como a doença do amarelinho, apresenta-se como o principal problema, sendo a bactéria gram-negativa Xylella fastidiosa seu agente causal. Em vista da importância do cultivo da laranja no país, o projeto \"Genoma Xylella fastidiosa\" foi proposto, visando o seqüenciamento total do genoma deste fitopatógeno bem como o treinamento de pessoal capacitado na utilização das modernas técnicas de biologia molecular. Segmentos de DNA provenientes de 7 cosmídeos e diversos clones provenientes de bibliotecas de \"shotgun\" genômico foram seqüenciados em nosso laboratório, totalizando 271.220 pb do genoma da bactéria. Em seguida foi feita a anotação das orfs preditas por programa GLIMMER, sendo que em nosso laboratório foram anotadas aproximadamente 290 ORFs. Seqüenciamentos diretamente do genoma e de clones das bibliotecas de RDA foram também realizados, complementando as metodologias de primer walking e construção de bibliotecas de fago utilizadas em outros laboratórios do projeto para o fechamento de \"gaps\". Todos os resultados obtidos em nosso laboratório, somados às contribuições de todos os grupos do projeto, serão base para a melhor compreensão dos mecanismos utilizados pela bactéria, podendo favorecer o desenvolvimento de novas estratégias para o combate desta praga.The São Paulo\'s citriculture can be considered one of the most competitive and important agroindustrial activity from Brazil. It provides aproximately 400,000 jobs and adds US 1,4000,000,000 for the country\'s economy. This activity, however, still shows less productivity than the one from Florida, mainly due to nutritional and hydric deficiencies and plagues that are already present for a long time. In the last ten years, the citrus variegated chlorosis (CVC), also known as little yellow disease, constitutes the main problem for the orange farmers. This disease is caused by gram-negative bacterium Xylella fastidiosa. Due to the importance of the orange cultive in the country, the project called Xylella fastidiosa\'s Genome was proposed. The main goals of this project are to sequence the entire genome of this phytopathogen and the trainning of specialized people in the use of modern techniques of molecular biology. DNA fragments cloned in 7 cosrnids and also form genomic shotgun libraries were sequenced in our laboratory. A total of 271,220 bp of bacteria genome were obtained. The next step was the annotation of the open reading frames (ORFs). This was made using the GLIMMER computer program which generates, in our laboratory, aproximately 290 ORFs. Direct sequencing of the genome and clones of RDA libraries were also done for obtaining nucleotide sequences form gaps. These methodologies complement the primer walking and phage library construction used by other laboratories included in the project. All results, obtained either by our laboratory or the other groups from the project, will be used for a better understanding of the mechanisms used by the bacteria, Altogether, they can be favor the development of new strategies in the plague combact

Sequencing and genome annotation of the party of Xylella fastidiosa

A citricultura paulista pode ser considerada uma das mais competitivas e importantes atividades agroindustriais do Brasil, gerando aproximadamente 400 mil empregos e adicionando anualmente U 1,4 bilhões ao país. Entretanto ainda apresenta uma produtividade inferior àquela encontrada na Flórida, principalmente devido a deficiências nutricionais e hídricas e a doenças que há diversos anos atingem às lavouras. Nos últimos dez anos a clorose variegada dos citros (CVC), conhecida popularmente como a doença do amarelinho, apresenta-se como o principal problema, sendo a bactéria gram-negativa Xylella fastidiosa seu agente causal. Em vista da importância do cultivo da laranja no país, o projeto \"Genoma Xylella fastidiosa\" foi proposto, visando o seqüenciamento total do genoma deste fitopatógeno bem como o treinamento de pessoal capacitado na utilização das modernas técnicas de biologia molecular. Segmentos de DNA provenientes de 7 cosmídeos e diversos clones provenientes de bibliotecas de \"shotgun\" genômico foram seqüenciados em nosso laboratório, totalizando 271.220 pb do genoma da bactéria. Em seguida foi feita a anotação das orfs preditas por programa GLIMMER, sendo que em nosso laboratório foram anotadas aproximadamente 290 ORFs. Seqüenciamentos diretamente do genoma e de clones das bibliotecas de RDA foram também realizados, complementando as metodologias de primer walking e construção de bibliotecas de fago utilizadas em outros laboratórios do projeto para o fechamento de \"gaps\". Todos os resultados obtidos em nosso laboratório, somados às contribuições de todos os grupos do projeto, serão base para a melhor compreensão dos mecanismos utilizados pela bactéria, podendo favorecer o desenvolvimento de novas estratégias para o combate desta praga.The São Paulo\'s citriculture can be considered one of the most competitive and important agroindustrial activity from Brazil. It provides aproximately 400,000 jobs and adds US 1,4000,000,000 for the country\'s economy. This activity, however, still shows less productivity than the one from Florida, mainly due to nutritional and hydric deficiencies and plagues that are already present for a long time. In the last ten years, the citrus variegated chlorosis (CVC), also known as little yellow disease, constitutes the main problem for the orange farmers. This disease is caused by gram-negative bacterium Xylella fastidiosa. Due to the importance of the orange cultive in the country, the project called Xylella fastidiosa\'s Genome was proposed. The main goals of this project are to sequence the entire genome of this phytopathogen and the trainning of specialized people in the use of modern techniques of molecular biology. DNA fragments cloned in 7 cosrnids and also form genomic shotgun libraries were sequenced in our laboratory. A total of 271,220 bp of bacteria genome were obtained. The next step was the annotation of the open reading frames (ORFs). This was made using the GLIMMER computer program which generates, in our laboratory, aproximately 290 ORFs. Direct sequencing of the genome and clones of RDA libraries were also done for obtaining nucleotide sequences form gaps. These methodologies complement the primer walking and phage library construction used by other laboratories included in the project. All results, obtained either by our laboratory or the other groups from the project, will be used for a better understanding of the mechanisms used by the bacteria, Altogether, they can be favor the development of new strategies in the plague combact

DNA Microarray-Based Genome Comparison of a Pathogenic and a Nonpathogenic Strain of Xylella fastidiosa Delineates Genes Important for Bacterial Virulence

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease

Shotgun sequencing of the human transcriptome with ORF expressed sequence tags

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription–PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html)