Protein Microarray On-Demand: A Novel Protein Microarray System

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (∼3–7×10 −13 M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This “all DNA” microarray can be converted to a protein microarray on-demand without need for any additional capture reagent.

Differential gene expression in Trypanosoma cruzi strains by microarrays DNA technology

Com o objetivo de analisar a expressão diferencial de genes em cepas de Trypanosoma cruzi pela técnica de microarranjos de DNA, construímos um protótipo de lâminas contendo 710 etiquetas de seqüências transcritas (ESTs) de epimastigotas de CL Brener e 20 genes de várias cepas do parasita. A seqüência nucleotídica estava disponível para 576 ESTs. Desta forma, seqüenciamos as 134 ESTs restantes e sua seqüência foi depositada no dbEST do GenBank. O agrupamento das sondas indicou que a lâmina continha 665 seqüências únicas. Inicialmente, avaliamos o microarranjo para a análise de genômica comparativa entre as cepas CL Brener (T. cruzi II) e Silvio (T. cruzi I). Um total de 4 hibridizações foram realizadas. Verificamos, com uma significância estatística (P≤ 0,01 no teste-t) que 31 seqüências do microarranjo (4,3%) apresentaram maior sinal de hibridização com o DNA de CL Brener, enquanto 37 sondas (5,0%) apresentaram situação inversa. Esse resultado sugere diferenças no número de cópias gênicas e/ou variação na similaridade das seqüências. Várias sondas com hibridização diferencial foram confirmadas em experimentos de Southern blot com DNA genômico dos dois isolados. Em seguida, o microarranjo foi avaliado para a análise da expressão diferencial de genes. Para isto, a lâmina foi hibridizada com cDNA de formas epimastigotas (fase midlog de crescimento) de CL Brener e Silvio. Um total de 8 experimentos de hibridização foi realizado com preparações de RNA obtidas de três cultivos independentes. Identificamos, com uma significância estatística (P≤ 0,01), 84 sondas (84/730; 11,5%) diferencialmente expressas: 35 sondas mais expressas em Silvio e 49 sondas mais expressas em CL Brener. Algumas destas sondas foram confirmadas por Northern blot. A comparação dos dados dos experimentos de hibridização do microarranjo com DNA e cDNA das duas cepas permitiu concluir que não há uma correlação direta entre a abundância de determinado ene e seu nível de expressão. Em alguns casos, observamos que seqüências com maior hibridização com o DNA de uma cepa são mais expressas na outra. Visando iniciar os estudos de expressão diferencial de genes em cepas isoladas de pacientes chagásicos, escolhemos as cepas Famema e Hem 179 (T. cruzi II), isoladas, respectivamente, de um paciente com a forma indeterminada da doença de Chagas e de um paciente com alterações cardíacas e digestivas. Observamos que aproximadamente 2,5% (18/730) das sondas apresentaram expressão diferencial nas formas epimastigotas das duas cepas e que 9,0% (65/730) das sondas mostraram razões de hibridização diferente nas formas infectantes (tripomastigotas metacíclicos). Algumas destas sondas foram confirmadas por Northern blot. Entre as sondas mais expressas em Hem 179, está a seqüência que codifica a subunidade 7 da NADH desidrogenase (ND7). O conjunto de dados permitiu concluir que microarranjos contendo predominantemente ESTs de CL Brener são uma ferramenta útil para estudos de genômica comparativa, análise de expressão gênica em isolados de T. cruzi, bem como para a descoberta de novos genes. Além disto, este estudo indicou uma regulação pós-transcricional intensa dos níveis de RNA em T. cruzi. Ampliamos os estudos para um maior número de cepas e construímos outro microarranjo contendo 710 ESTs de CL Brener (forma epimastigota), 45 ESTs da cepa Tulahuen (forma amastigota) e 32 genes de várias cepas do parasita. As 787 seqüências representam 714 seqüências únicas. O RNA total foi isolado de formas epimastigotas de cepas de três pacientes com a forma cardíaca (cepas 115, B13 e 147), e de três pacientes assintomáticos (cepas VL10, Famema e Berenice 62). Cinco da seis cepas são de regiões endêmicas de Minas Gerais (exceto Famema que é do estado de São Paulo). Após hibridização competitiva, a análise estatística dos dados indicou expressão diferencial de 14 sondas (14/787; 17,7%) entre os dois grupos de cepas. Destas, 9 sondas estão induzidas em todas as cepas isoladas de pacientes cardíacos e 4 sondas estão reprimidas em todas as cepas de cardíacos quando comparadas com as cepas de assintomáticos. Dentre as sondas mais expressa em cepas de cardíacos estão: as ESTs que codificam a subunidade 7 da NADH desidrogenase (ND7), a aspartato aminotransferase, enzima importante no processo de regeneração de metionina, e a triparedoxina peroxidase de T. cruzi, envolvida na resistência a peróxidos exógenos. Também foram reveladas 8 ESTs que não apresentam similaridade em bancos de dados. Parte destas sondas foi confirmada por Northern blot com RNA total de todas as cepas. Em alguns casos, identificamos diferenças do tamanho de transcritos entre as cepas. Em resumo, a análise da expressão diferencial de genes em cepas de pacientes com a forma cardíaca e indeterminada da doença de Chagas permitiu identificar alguns genes que podem estar relacionados à patogênese e/ou serem usados como alvo para o prognóstico da evolução doença. Posteriormente, realizamos um experimentos-piloto para investigar a resposta transcricional de fibroblastos humanos em cultura à infecção com duas cepas de T. cruzi: VL10 (isolada de indivíduo assintomático) e 147 (isolada de indivíduo com cardiopatia severa). Após 24 horas de infecção, os RNAs das monocamadas foram extraídos. No mesmo período, RNA foi extraído de fibroblastos humanos não infectados e usado como referência. Experimentos de hibridização competitiva foram realizados em lâminas de microarranjo contendo oligonucleotídios derivados de 24.000 genes humanos, construídas o Laboratório de Tecnologia Molecular do Instituto Nacional do Câncer do NIH, em Frederick, EUA chefiado pelo Dr. David Munroe. Identificamos 28 sondas diferencialmente transcritas nas células infectadas com a cepa 147 (paciente cardíaco). Destas, 27 sondas (27/28; 96,4%) estão com expressão reduzida nas células infectadas. Por outro lado, apenas uma sonda, correspondente ao gene da \"heme oxigenase (decycling) 1\", apresentou indução nas células infectadas com 147. Na infecção de células com a cepa VL10, encontramos 17 sondas diferencialmente transcritas. Destas, 16 sondas (16/17; 94,1 %) estão com expressão aumentada nas células infectadas (dentre elas, a heme oxigenase), enquanto que apenas uma sonda (proteína 5 ligante do fator de crescimento do tipo insulina) mostra situação inversa. Concluímos que as diferenças de resposta da mesma célula hospedeira à infecção pelas duas cepas são marcantes, o que determina um estudo temporal ao longo da infecção para a confirmação das observações acima. Comprovamos por PCR em tempo real o aumento da expressão da heme oxigenase na infecção pelas duas cepas e repressão da expressão da proteína 5 ligante do fator de crescimento do tipo insulina apenas em células infectadas por VL10.To investigate differential gene expression in Trypanosoma cruzi strains by microarray DNA technology, we have constructed a prototype slide that contains 710 expression sequence tags (ESTs) of CL Brener epimastigotes and 20 genes of various parasite strains. The nucleotide sequence was available for 576 ESTs. Therefore, we have sequenced 134 additional ESTs and the sequences have been deposited in dbEST of GenBank. Clustering of these probes indicated that slide contains 665 unique sequences. Initially, we have evaluated the microarray for comparative genomic analysis between CL Brener (T. cruzi II) and Silvio (T. cruzi I) strains. Four independent experiments were performed. We verified that 31 probes (4.3%) showed statistical significant (P≤, 0.01 in t-test) higher hybridization with CL Brener DNA, whereas 37 probes (5.0%) showed the inverse situation. This suggests differences in the abundance of the genes in the genomes of the strains and/or variation in sequence similarity. Some of these probes were confirmed by Southern blot with genomic DNA of both strains. Next, we evaluated the microarray for the analysis of differential gene expression. For this purpose, the slide with hybridized with cDNA obtained from epimastigote forms in mid-log growth phase of CL Brener and Silvio strains. A total of eight hybridization experiments were perfonned with RNA preparations obtained from three independent parasite harvests. We identified 84 probes (841730; 11.5%) that showed statistically significant changes (P≤, 0.01 in t-test) in expression: 35 probes up regulated in Silvio and 49 probes up regulated in CL Brener. Some of these probes were confirmed by Northern blot. When we compared the data of the hybridization of the microarray with genomic DNA and cDNA of CL Brener and Silvio, we verified no correlation between the putative abundance of a particular gene and its expression level. In a few cases, we noticed that sequences with higher hybridization with genomic DNA of one strain were more expressed in the other. In an initial attempt of investigate differential gene expression in human T. cruzi isolates, we selected the strains Famema and Hem 179 (both T. cruz II), obtained, respectively, from an asymptomatic individual and from a patient with severe cardiopathy and digestive disorders. Differential expression of 2.5% (181730) and 9.0% (651730) of the probes was observed, respectively, in epimastigotes and metacyclic trypomastigotes of the two strains. Some of the probes were confirmed by Northern blot. Among the probes more expressed in Hem 179 is the gene encoding subunit 7 of theNADH dehydrogenase (ND7). We concluded that microarrays, containing predominantly ESTs of CL Brener, are a powerful tool for comparative genomics and gene expression analyses in T. cruzi as well as for discovery of new genes. In addition, this study has provided further evidence for a high level of post-transcriptional regulation of RNA abundance in T. cruzi. We have extended the analysis to a larger number of strains and constructed a new set of microarray slides, which contain 710 ESTs of CL Brener epimastigotes, 45 ESTs Tulahuen strain amastigotes and 32 characterized genes of various strains. These probes represent 714 unique sequences. Total RNA was extracted from mid-log phase epimastigotes of three strains isolated from patients with cardiac disorders (115, B13 and 147) and three strains from asymptomatic patients (VL10, Famema and Berenice 62). Five of these strains are from endemic areas of Minas Gerais (except Famema that is from the state of São Paulo). After competitive hybridization with the microarray, statistical analysis of the data indicated differential expression of 14 probes (14/787, 17.7%) between the two groups of strains. Among these sequences, 9 probes are up regulated in all the strains from cardiac patients and 4 probes are down regulated in all these strains as compared with all the strains isolated from asymptomatic individuals. Among the probes up regulated in the strains from cardiac patients there are ESTs encoding subunit 7 of the NADH dehydrogenase (ND7); aspartate aminotransferase, key enzyme in the methionine regeneration process; and T. cruzi tryparedoxin peroxidase, an important enzyme for resistance to exogenous peroxides. Eight ESTs with no similarity matches in databases were also disclosed. Some of these probes were confirmed by Northern blot assays with RNA of all the strains. In some cases, we noticed differences of the molecular size of the transcripts among the strains. In summary, the analysis of differential gene expression in strains isolated from human chagasic patients with the cardiac and indeterminate forms allowed the identification of some potential genes that may be related to pathogenesis and/or may be used as targets for prognosis of the evolution of Chagas disease. Subsequently, we performed a pilot experiment to investigate the transcriptional response of human fibroblasts in culture to the infection by two T. cruzi strains: VL10 (isolated from an asymptomatic patient) and 147 (isolated from a patient with severe cardiomyopathy). After 24 hours of infection, total RNA was extracted from the infected monolayers. In the same period, RNA was extracted from not infected human fibroblasts, and used as a control. Competitive hybridization experiments were performed in microarray slides containing oligonucleotides representing 24,000 human genes, constructed in the Laboratory of Molecular Technology of the National Institute of Cancer of N1H, in Frederick, EUA headed by Dr. David Munroe. We identified 28 probes differentially transcribed in cells infected with 147 strain. Of these, 27 probes (27/28; 96.4%) were down regulated in the infected celIs, and only one probe, corresponding to the heme oxygenase (decycling) 1 gene, showed up regulation. For the fibroblast infected with VL 10 strain, we found 17 probes differentially transcribed. Of these, 16 probes (16/17; 94.1%) were up regulated (among these probes there is the heme oxigenase sequence), and only one probe (insulin-like growth factor binding protein 5) showed the inverse situation. We concluded that there are drastic differences in the response of the same cell line to the infection by two parasite stmins. This determines that a temporal analysis of gene expression during the infection process should be performed to corroborate the above observations. The increase of heme oxigenase transcripts in fibroblasts infected by the two strains was comrrmed by real time-PCR This methodology also confirmed down-regulation of the insulin-like growth factor binding protein 5 rnRNA in fibroblasts infected with VL 10 strain

Differential gene expression in Trypanosoma cruzi strains by microarrays DNA technology

Com o objetivo de analisar a expressão diferencial de genes em cepas de Trypanosoma cruzi pela técnica de microarranjos de DNA, construímos um protótipo de lâminas contendo 710 etiquetas de seqüências transcritas (ESTs) de epimastigotas de CL Brener e 20 genes de várias cepas do parasita. A seqüência nucleotídica estava disponível para 576 ESTs. Desta forma, seqüenciamos as 134 ESTs restantes e sua seqüência foi depositada no dbEST do GenBank. O agrupamento das sondas indicou que a lâmina continha 665 seqüências únicas. Inicialmente, avaliamos o microarranjo para a análise de genômica comparativa entre as cepas CL Brener (T. cruzi II) e Silvio (T. cruzi I). Um total de 4 hibridizações foram realizadas. Verificamos, com uma significância estatística (P≤ 0,01 no teste-t) que 31 seqüências do microarranjo (4,3%) apresentaram maior sinal de hibridização com o DNA de CL Brener, enquanto 37 sondas (5,0%) apresentaram situação inversa. Esse resultado sugere diferenças no número de cópias gênicas e/ou variação na similaridade das seqüências. Várias sondas com hibridização diferencial foram confirmadas em experimentos de Southern blot com DNA genômico dos dois isolados. Em seguida, o microarranjo foi avaliado para a análise da expressão diferencial de genes. Para isto, a lâmina foi hibridizada com cDNA de formas epimastigotas (fase midlog de crescimento) de CL Brener e Silvio. Um total de 8 experimentos de hibridização foi realizado com preparações de RNA obtidas de três cultivos independentes. Identificamos, com uma significância estatística (P≤ 0,01), 84 sondas (84/730; 11,5%) diferencialmente expressas: 35 sondas mais expressas em Silvio e 49 sondas mais expressas em CL Brener. Algumas destas sondas foram confirmadas por Northern blot. A comparação dos dados dos experimentos de hibridização do microarranjo com DNA e cDNA das duas cepas permitiu concluir que não há uma correlação direta entre a abundância de determinado ene e seu nível de expressão. Em alguns casos, observamos que seqüências com maior hibridização com o DNA de uma cepa são mais expressas na outra. Visando iniciar os estudos de expressão diferencial de genes em cepas isoladas de pacientes chagásicos, escolhemos as cepas Famema e Hem 179 (T. cruzi II), isoladas, respectivamente, de um paciente com a forma indeterminada da doença de Chagas e de um paciente com alterações cardíacas e digestivas. Observamos que aproximadamente 2,5% (18/730) das sondas apresentaram expressão diferencial nas formas epimastigotas das duas cepas e que 9,0% (65/730) das sondas mostraram razões de hibridização diferente nas formas infectantes (tripomastigotas metacíclicos). Algumas destas sondas foram confirmadas por Northern blot. Entre as sondas mais expressas em Hem 179, está a seqüência que codifica a subunidade 7 da NADH desidrogenase (ND7). O conjunto de dados permitiu concluir que microarranjos contendo predominantemente ESTs de CL Brener são uma ferramenta útil para estudos de genômica comparativa, análise de expressão gênica em isolados de T. cruzi, bem como para a descoberta de novos genes. Além disto, este estudo indicou uma regulação pós-transcricional intensa dos níveis de RNA em T. cruzi. Ampliamos os estudos para um maior número de cepas e construímos outro microarranjo contendo 710 ESTs de CL Brener (forma epimastigota), 45 ESTs da cepa Tulahuen (forma amastigota) e 32 genes de várias cepas do parasita. As 787 seqüências representam 714 seqüências únicas. O RNA total foi isolado de formas epimastigotas de cepas de três pacientes com a forma cardíaca (cepas 115, B13 e 147), e de três pacientes assintomáticos (cepas VL10, Famema e Berenice 62). Cinco da seis cepas são de regiões endêmicas de Minas Gerais (exceto Famema que é do estado de São Paulo). Após hibridização competitiva, a análise estatística dos dados indicou expressão diferencial de 14 sondas (14/787; 17,7%) entre os dois grupos de cepas. Destas, 9 sondas estão induzidas em todas as cepas isoladas de pacientes cardíacos e 4 sondas estão reprimidas em todas as cepas de cardíacos quando comparadas com as cepas de assintomáticos. Dentre as sondas mais expressa em cepas de cardíacos estão: as ESTs que codificam a subunidade 7 da NADH desidrogenase (ND7), a aspartato aminotransferase, enzima importante no processo de regeneração de metionina, e a triparedoxina peroxidase de T. cruzi, envolvida na resistência a peróxidos exógenos. Também foram reveladas 8 ESTs que não apresentam similaridade em bancos de dados. Parte destas sondas foi confirmada por Northern blot com RNA total de todas as cepas. Em alguns casos, identificamos diferenças do tamanho de transcritos entre as cepas. Em resumo, a análise da expressão diferencial de genes em cepas de pacientes com a forma cardíaca e indeterminada da doença de Chagas permitiu identificar alguns genes que podem estar relacionados à patogênese e/ou serem usados como alvo para o prognóstico da evolução doença. Posteriormente, realizamos um experimentos-piloto para investigar a resposta transcricional de fibroblastos humanos em cultura à infecção com duas cepas de T. cruzi: VL10 (isolada de indivíduo assintomático) e 147 (isolada de indivíduo com cardiopatia severa). Após 24 horas de infecção, os RNAs das monocamadas foram extraídos. No mesmo período, RNA foi extraído de fibroblastos humanos não infectados e usado como referência. Experimentos de hibridização competitiva foram realizados em lâminas de microarranjo contendo oligonucleotídios derivados de 24.000 genes humanos, construídas o Laboratório de Tecnologia Molecular do Instituto Nacional do Câncer do NIH, em Frederick, EUA chefiado pelo Dr. David Munroe. Identificamos 28 sondas diferencialmente transcritas nas células infectadas com a cepa 147 (paciente cardíaco). Destas, 27 sondas (27/28; 96,4%) estão com expressão reduzida nas células infectadas. Por outro lado, apenas uma sonda, correspondente ao gene da \"heme oxigenase (decycling) 1\", apresentou indução nas células infectadas com 147. Na infecção de células com a cepa VL10, encontramos 17 sondas diferencialmente transcritas. Destas, 16 sondas (16/17; 94,1 %) estão com expressão aumentada nas células infectadas (dentre elas, a heme oxigenase), enquanto que apenas uma sonda (proteína 5 ligante do fator de crescimento do tipo insulina) mostra situação inversa. Concluímos que as diferenças de resposta da mesma célula hospedeira à infecção pelas duas cepas são marcantes, o que determina um estudo temporal ao longo da infecção para a confirmação das observações acima. Comprovamos por PCR em tempo real o aumento da expressão da heme oxigenase na infecção pelas duas cepas e repressão da expressão da proteína 5 ligante do fator de crescimento do tipo insulina apenas em células infectadas por VL10.To investigate differential gene expression in Trypanosoma cruzi strains by microarray DNA technology, we have constructed a prototype slide that contains 710 expression sequence tags (ESTs) of CL Brener epimastigotes and 20 genes of various parasite strains. The nucleotide sequence was available for 576 ESTs. Therefore, we have sequenced 134 additional ESTs and the sequences have been deposited in dbEST of GenBank. Clustering of these probes indicated that slide contains 665 unique sequences. Initially, we have evaluated the microarray for comparative genomic analysis between CL Brener (T. cruzi II) and Silvio (T. cruzi I) strains. Four independent experiments were performed. We verified that 31 probes (4.3%) showed statistical significant (P≤, 0.01 in t-test) higher hybridization with CL Brener DNA, whereas 37 probes (5.0%) showed the inverse situation. This suggests differences in the abundance of the genes in the genomes of the strains and/or variation in sequence similarity. Some of these probes were confirmed by Southern blot with genomic DNA of both strains. Next, we evaluated the microarray for the analysis of differential gene expression. For this purpose, the slide with hybridized with cDNA obtained from epimastigote forms in mid-log growth phase of CL Brener and Silvio strains. A total of eight hybridization experiments were perfonned with RNA preparations obtained from three independent parasite harvests. We identified 84 probes (841730; 11.5%) that showed statistically significant changes (P≤, 0.01 in t-test) in expression: 35 probes up regulated in Silvio and 49 probes up regulated in CL Brener. Some of these probes were confirmed by Northern blot. When we compared the data of the hybridization of the microarray with genomic DNA and cDNA of CL Brener and Silvio, we verified no correlation between the putative abundance of a particular gene and its expression level. In a few cases, we noticed that sequences with higher hybridization with genomic DNA of one strain were more expressed in the other. In an initial attempt of investigate differential gene expression in human T. cruzi isolates, we selected the strains Famema and Hem 179 (both T. cruz II), obtained, respectively, from an asymptomatic individual and from a patient with severe cardiopathy and digestive disorders. Differential expression of 2.5% (181730) and 9.0% (651730) of the probes was observed, respectively, in epimastigotes and metacyclic trypomastigotes of the two strains. Some of the probes were confirmed by Northern blot. Among the probes more expressed in Hem 179 is the gene encoding subunit 7 of theNADH dehydrogenase (ND7). We concluded that microarrays, containing predominantly ESTs of CL Brener, are a powerful tool for comparative genomics and gene expression analyses in T. cruzi as well as for discovery of new genes. In addition, this study has provided further evidence for a high level of post-transcriptional regulation of RNA abundance in T. cruzi. We have extended the analysis to a larger number of strains and constructed a new set of microarray slides, which contain 710 ESTs of CL Brener epimastigotes, 45 ESTs Tulahuen strain amastigotes and 32 characterized genes of various strains. These probes represent 714 unique sequences. Total RNA was extracted from mid-log phase epimastigotes of three strains isolated from patients with cardiac disorders (115, B13 and 147) and three strains from asymptomatic patients (VL10, Famema and Berenice 62). Five of these strains are from endemic areas of Minas Gerais (except Famema that is from the state of São Paulo). After competitive hybridization with the microarray, statistical analysis of the data indicated differential expression of 14 probes (14/787, 17.7%) between the two groups of strains. Among these sequences, 9 probes are up regulated in all the strains from cardiac patients and 4 probes are down regulated in all these strains as compared with all the strains isolated from asymptomatic individuals. Among the probes up regulated in the strains from cardiac patients there are ESTs encoding subunit 7 of the NADH dehydrogenase (ND7); aspartate aminotransferase, key enzyme in the methionine regeneration process; and T. cruzi tryparedoxin peroxidase, an important enzyme for resistance to exogenous peroxides. Eight ESTs with no similarity matches in databases were also disclosed. Some of these probes were confirmed by Northern blot assays with RNA of all the strains. In some cases, we noticed differences of the molecular size of the transcripts among the strains. In summary, the analysis of differential gene expression in strains isolated from human chagasic patients with the cardiac and indeterminate forms allowed the identification of some potential genes that may be related to pathogenesis and/or may be used as targets for prognosis of the evolution of Chagas disease. Subsequently, we performed a pilot experiment to investigate the transcriptional response of human fibroblasts in culture to the infection by two T. cruzi strains: VL10 (isolated from an asymptomatic patient) and 147 (isolated from a patient with severe cardiomyopathy). After 24 hours of infection, total RNA was extracted from the infected monolayers. In the same period, RNA was extracted from not infected human fibroblasts, and used as a control. Competitive hybridization experiments were performed in microarray slides containing oligonucleotides representing 24,000 human genes, constructed in the Laboratory of Molecular Technology of the National Institute of Cancer of N1H, in Frederick, EUA headed by Dr. David Munroe. We identified 28 probes differentially transcribed in cells infected with 147 strain. Of these, 27 probes (27/28; 96.4%) were down regulated in the infected celIs, and only one probe, corresponding to the heme oxygenase (decycling) 1 gene, showed up regulation. For the fibroblast infected with VL 10 strain, we found 17 probes differentially transcribed. Of these, 16 probes (16/17; 94.1%) were up regulated (among these probes there is the heme oxigenase sequence), and only one probe (insulin-like growth factor binding protein 5) showed the inverse situation. We concluded that there are drastic differences in the response of the same cell line to the infection by two parasite stmins. This determines that a temporal analysis of gene expression during the infection process should be performed to corroborate the above observations. The increase of heme oxigenase transcripts in fibroblasts infected by the two strains was comrrmed by real time-PCR This methodology also confirmed down-regulation of the insulin-like growth factor binding protein 5 rnRNA in fibroblasts infected with VL 10 strain

Genomic organization and transcription analysis of the 195-bp satellite DNA in Trypanosoma cruzi

The 195-bp satellite DNA is the most abundant Trypanosoma cruzi repetitive sequence. Here we show by RNA blotting and RT-PCR that 195 SAT is intensely transcribed. We observed a positive correlation between the level of satellite RNA and the abundance of the satellite copies in the genome of T cruzi strains and that the satellite expression is not developmentally regulated. By analyzing CL Brener individual reads, we estimated that 195 SAT corresponds to approximately 5% of the CL Brener genome. 195 SAT elements were found in only 37 annotated contigs, indicating that a large number of satellite copies were not incorporated into the assembled data. The assembled satellite units are distributed in non-syntenic regions with Trypanosoma brucei and Leishmania major genomes, enriched with surface proteins, retroelements, RHS and hypothetical proteins. Satellite repeats were not observed in annotated subtelomeric regions. We report that 12 satellite sequences are truncated by the retroelement VIPER. (C) 2008 Elsevier B.V. All rights reserved

Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila

Myceliophthora thermophila is a thermophilic fungus whose genome encodes a wide range of carbohydrate-active enzymes (CAZymes) involved in plant biomass degradation. Such enzymes have potential applications in turning different kinds of lignocellulosic feedstock into sugar precursors for biofuels and chemicals. The present study examined and compared the transcriptomes and exoproteomes of M. thermophila during cultivation on different types of complex biomass to gain insight into how its secreted enzymatic machinery varies with different sources of lignocellulose. In the transcriptome analysis three monocot (barley, oat, triticale) and three dicot (alfalfa, canola, flax) plants were used whereas in the proteome analysis additional substrates, i.e. wood and corn stover pulps, were included. A core set of 59 genes encoding CAZymes was up-regulated in response to both monocot and dicot straws, including nine polysaccharide monooxygenases and GH10, but not GH11, xylanases. Genes encoding additional xylanolytic enzymes were up-regulated during growth on monocot straws, while genes encoding additional pectinolytic enzymes were up-regulated in response to dicot biomass. Exoproteome analysis was generally consistent with the conclusions drawn from transcriptome analysis, but additional CAZymes that accumulated to high levels were identified. Despite the wide variety of biomass sources tested some CAZy family members were not expressed under any condition. The results of this study provide a comprehensive view from both transcriptome and exoproteome levels, of how M. thermophila responds to a wide range of biomass sources using its genomic resources.Peer reviewed: YesNRC publication: N