ABSTRACT
MAIN CONCLUSIONS: HbNAC1 is a transcription factor in rubber plants whose expression is induced by dehydration, leading to latex biosynthesis. Laticifer is a special tissue in Hevea brasiliensis where natural rubber is biosynthesized and accumulated. In young stems of epicormic shoots, the differentiation of secondary laticifers can be induced by wounding, which can be prevented when the wounding site is wrapped. Using this system, differentially expressed genes were screened by suppression subtractive hybridization (SSH) and macroarray analyses. This led to the identification of several dehydration-related genes that could be involved in laticifer differentiation and/or latex biosynthesis, including a NAC transcription factor (termed as HbNAC1). Tissue sections confirmed that local tissue dehydration was a key signal for laticifer differentiation. HbNAC1 was localized at the nucleus and showed strong transcriptional activity in yeast, suggesting that HbNAC1 is a transcription factor. Furthermore, HbNAC1 was found to bind to the cis-element CACG in the promoter region of the gene encoding the small rubber particle protein (SRPP). Transgenic experiments also confirmed that HbNAC1 interacted with the SRPP promoter when co-expressed, and enhanced expression of the reporter gene ß-glucuronidase occurred in planta. In addition, overexpression of HbNAC1 in tobacco plants conferred drought tolerance. Together, the data suggest that HbNAC1 might be involved in dehydration-induced laticifer differentiation and latex biosynthesis.
Subject(s)
Cell Differentiation , Hevea/cytology , Latex/biosynthesis , Plant Proteins/metabolism , Adaptation, Physiological/genetics , Base Sequence , Dehydration , Droughts , Gene Expression Regulation, Plant , Genes, Plant , Hevea/genetics , Plant Bark/cytology , Plants, Genetically Modified , Promoter Regions, Genetic , Protein Binding , Reproducibility of Results , Saccharomyces cerevisiae/metabolism , Stress, Physiological , Subcellular Fractions/metabolism , Subtractive Hybridization Techniques , Nicotiana/genetics , Transcriptional Activation/geneticsABSTRACT
La expresión génica es el estudio de cómo el genotipo da lugar al fenotipo mediante la investigación de la cantidad de RNAm transcrito en un sistema biológico. Una gran cantidad de métodos fueron estandarizados para identificar variaciones en la expresión génica, incluyendo la hibridación sustractiva, differential display, análisis en serie de la expresión génica, la hibridación de microarrays, y la secuenciación por RNA-seq. La mayoría de las técnicas se han centrado en la investigación y diagnóstico del cáncer, produciendo una gran cantidad de datos, lo que permitió a entender la progresión del cáncer y las vías, descubrir y evaluar nuevas intervenciones de tratamiento, nuevas herramientas moleculares para el diagnóstico y el pronóstico, y analizar el tiempo de sobrevivencia en pacientes humanos y animales. De esta manera, las técnicas de expresión génica trajeron nuevas perspectivas importantes para el campo de la medicina veterinaria, y nuevas investigaciones centradas en oncología proporcionarán mucho más conocimiento acerca de las vías y la interacción de las células sanas y tumorales, mejorando las intervenciones diarias por los oncólogos y los clínicos.
Gene expression is the study of how the genotype gives rise to the phenotype by investigating the amount of transcribed mRNA in a biological system. A lot of methods have been standardized to identify the variation in gene expression, including subtractive hybridization, differential display, serial analysis of gene expression, microarray hybridization, and RNAseq sequencing. Most of techniques have been focused in cancer research and diagnosis, producing a huge amount of data, which allowed to understand the cancer progression and pathways, discover and evaluate new treatment interventions, new molecular tools for diagnosis and prognosis, and analyze the survival time in human and animal patients. In this way, gene expression techniques brought new important perspectives for the medical and veterinary fields, and further researches focusing oncology will provide much more knowledge concerning the pathways and interaction of healthy and tumor cells, improving the perspectives of the daily interventions by the oncologists and clinicians.
A expressão genética é o estudo de como o genótipo dá origem ao fenótipo a partir da investigação da quantidade de RNAm transcrito em um sistema biológico. Vários métodos já foram padronizados para identificar variações na expressão gênica, dentre eles a hibridização subtrativa, differential display, análise em série da expressão genética, hibridização de microarranjo, e sequenciamento por RNA-seq. A maioria das técnicas tem focado na pesquisa e diagnóstico do câncer, gerando enorme quantidade de dados, o que permitiu compreender a progressão do câncer e suas vias, descobrir e analisar novas intervenções terapêuticas, novas ferramentas moleculares para o diagnóstico e prognóstico, e analisar o tempo de sobrevivência em pacientes humanos e animais. Desta forma, as diferentes técnicas de expressão gênica trouxeram novas e importantes perspectivas para a área médica e veterinária, e novas pesquisas focadas em oncologia fornecerão muito mais conhecimento sobre as vias e interações entre células saudáveis e tumorais, melhorando as perspectivas das intervenções diárias pelos oncologistas e clínicos.
Subject(s)
Humans , Animals , Methods , Neoplasms/genetics , RNA, Messenger/analysis , Subtractive Hybridization Techniques/veterinaryABSTRACT
La expresión génica es el estudio de cómo el genotipo da lugar al fenotipo mediante la investigación de la cantidad de RNAm transcrito en un sistema biológico. Una gran cantidad de métodos fueron estandarizados para identificar variaciones en la expresión génica, incluyendo la hibridación sustractiva, differential display, análisis en serie de la expresión génica, la hibridación de microarrays, y la secuenciación por RNA-seq. La mayoría de las técnicas se han centrado en la investigación y diagnóstico del cáncer, produciendo una gran cantidad de datos, lo que permitió a entender la progresión del cáncer y las vías, descubrir y evaluar nuevas intervenciones de tratamiento, nuevas herramientas moleculares para el diagnóstico y el pronóstico, y analizar el tiempo de sobrevivencia en pacientes humanos y animales. De esta manera, las técnicas de expresión génica trajeron nuevas perspectivas importantes para el campo de la medicina veterinaria, y nuevas investigaciones centradas en oncología proporcionarán mucho más conocimiento acerca de las vías y la interacción de las células sanas y tumorales, mejorando las intervenciones diarias por los oncólogos y los clínicos.(AU)
Gene expression is the study of how the genotype gives rise to the phenotype by investigating the amount of transcribed mRNA in a biological system. A lot of methods have been standardized to identify the variation in gene expression, including subtractive hybridization, differential display, serial analysis of gene expression, microarray hybridization, and RNAseq sequencing. Most of techniques have been focused in cancer research and diagnosis, producing a huge amount of data, which allowed to understand the cancer progression and pathways, discover and evaluate new treatment interventions, new molecular tools for diagnosis and prognosis, and analyze the survival time in human and animal patients. In this way, gene expression techniques brought new important perspectives for the medical and veterinary fields, and further researches focusing oncology will provide much more knowledge concerning the pathways and interaction of healthy and tumor cells, improving the perspectives of the daily interventions by the oncologists and clinicians.(AU)
A expressão genética é o estudo de como o genótipo dá origem ao fenótipo a partir da investigação da quantidade de RNAm transcrito em um sistema biológico. Vários métodos já foram padronizados para identificar variações na expressão gênica, dentre eles a hibridização subtrativa, differential display, análise em série da expressão genética, hibridização de microarranjo, e sequenciamento por RNA-seq. A maioria das técnicas tem focado na pesquisa e diagnóstico do câncer, gerando enorme quantidade de dados, o que permitiu compreender a progressão do câncer e suas vias, descobrir e analisar novas intervenções terapêuticas, novas ferramentas moleculares para o diagnóstico e prognóstico, e analisar o tempo de sobrevivência em pacientes humanos e animais. Desta forma, as diferentes técnicas de expressão gênica trouxeram novas e importantes perspectivas para a área médica e veterinária, e novas pesquisas focadas em oncologia fornecerão muito mais conhecimento sobre as vias e interações entre células saudáveis e tumorais, melhorando as perspectivas das intervenções diárias pelos oncologistas e clínicos.(AU)
Subject(s)
Humans , Animals , Methods , RNA, Messenger/analysis , Neoplasms/genetics , Subtractive Hybridization Techniques/veterinaryABSTRACT
Bananas and plantains are considered an important crop around the world. Banana production is affected by several constraints, of which Black Sigatoka Disease, caused by the fungus Mycosphaerella fijiensis, is considered one of the most important diseases in banana plantations. The banana accession 'Calcutta-4' has a natural resistance to Black Sigatoka; however, the fruit is not valuable for commercialization. Gene identification and expression studies in 'Calcutta-4' might reveal possible gene candidates for resistant to the disease and elucidate mechanisms for resistance. A subtracted cDNA library was generated from leaves after 6, 9 and 12 days inoculated with M. fijiensis conidia on greenhouse banana plants of the accession 'Calcutta-4'. Bioinformatic analysis revealed 99 good quality sequences. Blast2go analysis revealed that 31% of the sequences could not be categorized and, according to the Biological Process Category, 32 and 28 ESTs are related to general metabolic and cellular processes, respectively; while 10 ESTs response to stimulus. Seven sequences were redundant and one was similar to genes that may be involved in pathogen resistance including the putative disease resistance protein RGA1. Genes encoding zinc finger domains were identified and may play an important role in pathogen resistance by inducing the expression of downstream genes. Expression analysis of four selected genes was performed using RT-qPCR during the early stage of the disease development at 6, 9, 12 and 15 days post inoculation showing a peak of up regulation at 9 or 12 days post inoculation. Three of the four genes showed an up-regulation of expression in 'Calcutta-4' when compared to 'Williams' after inoculation with M. fijiensis, suggesting a fine regulation of specific gene candidates that may lead to a resistance response. The genes identified in early responses in a plant-pathogen interaction may be relevant for the resistance response of 'Calcutta-4' to Black Sigatoka. Genes with different functions may play a role in plant response to the disease. The present study suggests a fine up regulation of these genes that might be needed to perform an incompatible interaction. Further gene functional studies need to be performed to validate their use as candidate resistance genes in susceptible banana cultivars.
Subject(s)
Ascomycota/immunology , Disease Resistance/genetics , Gene Expression Regulation, Plant , Musa/genetics , Musa/immunology , Ascomycota/pathogenicity , Expressed Sequence Tags , Gene Expression Profiling , Gene Library , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Plant Diseases/genetics , Plant Diseases/immunology , Spores, Fungal/immunology , Subtractive Hybridization TechniquesABSTRACT
Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of ß-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.
Subject(s)
DNA, Protozoan/genetics , Leishmania major/genetics , Leishmaniasis, Cutaneous/parasitology , Animals , Cricetinae , DNA, Complementary/genetics , Galactosyltransferases/metabolism , Glycosphingolipids/metabolism , Humans , Leishmania major/pathogenicity , Macrophages/parasitology , Mesocricetus , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neglected Diseases/parasitology , Protozoan Proteins/metabolism , Sequence Homology, Nucleic Acid , Subtractive Hybridization Techniques , Virulence/geneticsABSTRACT
Spontaneous leaf color variation in bamboo provides the opportunity to study the mechanisms of leaf color formation and the breeding of ornamental bamboos. Despite the fact that many genes are known to be involved in leaf color variation in model plants, molecular mechanisms governing natural leaf color variation in bamboo have remained obscure. This study aimed to identify the genes responsible for the occurrence of such phenomena in bamboo using the suppression subtractive hybridization (SSH) method between green and albino leaves in Pseudosasa japonica f. A total of 1062 and 1004 differentially expressed transcripts were obtained from the forward and reverse SSH libraries, respectively. Subsequently, 59 differentially expressed unigenes with potential roles in leaf color formation, predicted via computational analysis of their functional relevance, were selected for further analysis using qPCR. Ten genes, involved in photosynthesis, plastid development, and cation signal transduction, showed 2-fold changes in expression levels between green and albino leaves. Further expression pattern analyses of these genes at three developmental stages revealed much lower expression abundance of Lhca1-encoded chlorophyll a/b binding protein in the albino leaves than in the green leaves. Our results suggest that, together with the concatenated negative pressure for subsequent photosynthetic processes, the albino phenotype is at least partly attributable to chloroplast inner membrane damage or to the impairment of photosynthetic pigment accumulation, which results from low Lhca1 expression.
Subject(s)
Bambusa/genetics , Gene Expression Regulation, Plant , Genes, Plant , Pigmentation/genetics , Plant Leaves/genetics , Bambusa/anatomy & histology , Bambusa/growth & development , Chlorophyll/genetics , Chlorophyll/metabolism , Chlorophyll A , Chlorophyll Binding Proteins/genetics , Chlorophyll Binding Proteins/metabolism , Chloroplasts/genetics , Color , Photosynthesis , Plant Leaves/anatomy & histology , Plant Leaves/growth & development , Signal Transduction , Subtractive Hybridization TechniquesABSTRACT
Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC.
Subject(s)
Isothiocyanates , Drug Resistance, Fungal , Alternaria/genetics , Alternaria/metabolism , Fungicides, Industrial , Computational Biology , Subtractive Hybridization Techniques , Hybridization, GeneticABSTRACT
The development of a genetic transformation system is needed to address the problem of the low efficiency associated with soybean regeneration. To contribute to the enhancement of the soybean regenerative capacity, we explored the developmental mechanisms of soybean regeneration at the molecular level using a suppression subtractive hybridization cDNA library constructed from cotyledonary nodes of soybean cultivar DN50. A total of 918 positive clones were identified and screened, with most inserted fragments ranging from 100 to 750 bp. Of these, 411 differentially expressed functional expressed sequence tags were identified and annotated based on their similarity to orthologs and paralogs detected in GenBank using the nucleotide and translated nucleotide Basic Local Alignment Search Tools. Functional analysis revealed that the associated genes were involved in signal transduction, synthesis, and metabolism of macromolecules, glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, cell defense, cell wall differentiation, and a variety of hormone and cytokinin-mediated signaling pathways. The information uncovered in our study should serve as a foundation for the establishment of an efficient and stable genetic transformation system for soybean regeneration.
Subject(s)
Glycine max/genetics , Regeneration/genetics , Subtractive Hybridization Techniques/methods , Cell Differentiation/genetics , Expressed Sequence Tags , Gene Expression Regulation, Plant , Gene Library , Glycine max/growth & developmentABSTRACT
The swimming crab, Portunus trituberculatus, is an important marine animal and is widely cultured in China. In the present study, suppression subtractive hybridization was applied to identify the differentially expressed genes in the ovaries of mature and immature P. trituberculatus. One hundred and seventy six expressed sequence tag (ESTs) were identified, of which 100 were down-regulated, and 76 up-regulated. BLAST analysis identified 51 unigenes, of which 27 were down-regulated, and 24 up-regulated. Quantitative real-time reverse transcriptase polymerase chain reaction results indicated that the SSH technique is valuable in screening genes related to ovarian development. Genes identified in this study encoded proteins corresponding to a wide range of functions and included immune response protein, transcription initiation factor, metabolic proteins, chromosome, histone h3, ovarian development-related protein, and vitellogenin. In addition, 64 metabolic pathways were annotated in differentially expressed ESTs by using the Kyoto Encyclopedia of Genes and Genomes pathway. Four annotated pathways (oxidative phosphorylation, carbon metabolism, fatty acid degradation, and protein digestion and absorption) appeared to be involved in ovarian development. In ontology analysis, 5.83% of the cellular process genes in reverse subtraction cDNA library are involved in reproduction, and 5.88% involved in developmental process. In up-regulated genes, myosin II-expressed polehole-like protein; histone h3; ovigerous-hair stripping substance; peritrophin 48; and ovarian development-related protein appeared to be involved in ovarian development. Identification of differentially expressed genes in the mature and immature ovary of the swimming crab provides new insights for further studies on the mechanism underlying ovarian development in this species.
Subject(s)
Crustacea/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental , Ovary/embryology , Animals , Computational Biology/methods , Expressed Sequence Tags , Female , Gene Library , Metabolic Networks and Pathways , Ovary/metabolism , Subtractive Hybridization TechniquesABSTRACT
Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.
Subject(s)
Entamoeba histolytica/genetics , Entamoeba histolytica/pathogenicity , Gene Expression Profiling , Gene Expression Regulation , Subtractive Hybridization Techniques/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Cytopathogenic Effect, Viral/genetics , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Humans , Liver/parasitology , Male , Trophozoites/physiology , Virulence/geneticsABSTRACT
Calamus palustris Griff. is an economically important dioecious rattan species in Southeast Asia. However, dioecy and onset of flowering at 3-4 years old render uncertainties in desired female:male seedling ratios to establish a productive seed orchard for this rattan species. We constructed a subtractive library for male floral tissue to understand the genetic mechanism for gender determination in C. palustris. The subtractive library produced 1536 clones with 1419 clones of high quality. Reverse Northern screening showed 313 clones with differential expression, and sequence analyses clustered them into 205 unigenes, including 32 contigs and 173 singletons. The subtractive library was further validated with reverse transcription-quantitative polymerase chain reaction analysis. Homology identification classified the unigenes into 12 putative functional proteins with 83% unigenes showing significant match to proteins in databases. Functional annotations of these unigenes revealed genes involved in male flower development, including MADS-box genes, pollen-related genes, phytohormones for flower development, and male flower organ development. Our results showed that the male floral genes may play a vital role in sex determination in C. palustris. The identified genes can be exploited to understand the molecular basis of sex determination in C. palustris.
Subject(s)
Calamus/genetics , Flowers/genetics , Genes, Plant , Subtractive Hybridization Techniques , Computational Biology , DNA, Complementary/genetics , Gene Expression Regulation, Plant , Gene LibraryABSTRACT
The influence of warm day and cool night conditions on induction of spikes in Phalaenopsis orchids has been studied with respect to photosynthetic efficiency, metabolic cycles and physiology. However, molecular events involved in spike emergence induced by warm day and cool night conditions are not clearly understood. We examined gene expression induced by warm day and cool night conditions in the Phalaenopsis hybrid Fortune Saltzman through suppression subtractive hybridization, which allowed identification of flowering-related genes in warm day and cool night conditions in spikes and leaves at vegetative phase grown under warm daily temperatures. In total, 450 presumably regulated expressed sequence tags (ESTs) were identified and classified into functional categories, including metabolism, development, transcription factor, signal transduction, transportation, cell defense, and stress. Furthermore, database comparisons revealed a notable number of Phalaenopsis hybrid Fortune Saltzman ESTs that matched genes with unknown function. The expression profiles of 24 genes (from different functional categories) have been confirmed by quantitative real-time PCR in induced spikes and juvenile apical leaves. The results of the real-time PCR showed that, compared to the vegetative apical leaves, the transcripts of genes encoding flowering locus T, AP1, AP2, KNOX1, knotted1-like homeobox protein, R2R3-like MYB, adenosine kinase 2, S-adenosylmethionine synthetase, dihydroflavonol 4-reductase, and naringenin 3-dioxygenase accumulated significantly higher levels, and genes encoding FCA, retrotransposon protein Ty3 and C3HC4-type RING finger protein accumulated remarkably lower levels in spikes of early developmental stages. These results suggested that the genes of two expression changing trends may play positive and negative roles in the early floral transition of Phalaenopsis orchids. In conclusion, spikes induced by warm day and cool night conditions were complex in Phalaenopsis orchids; nevertheless, several molecular flowering pathway-related genes were found. The acquired data form the basis for a molecular understanding of spike induction by warm day and cool night conditions in Phalaenopsis orchids.
Subject(s)
Flowers/genetics , Gene Expression Regulation, Plant , Orchidaceae/genetics , Photoperiod , Subtractive Hybridization Techniques , Temperature , Expressed Sequence Tags , Flowers/metabolism , Gene Expression Profiling , Gene Library , Metabolic Networks and Pathways , Orchidaceae/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
In Brazil, common bean (Phaseolus vulgaris L.) productivity is severely affected by drought stress due to low technology cultivation systems. Our purpose was to identify differentially expressed genes in roots of a genotype tolerant to water deficit (BAT 477) when submitted to an interruption of irrigation during its development. A SSH library was constructed taking as "driver" the genotype Carioca 80SH (susceptible to drought). After clustering and data mining, 1572 valid reads were obtained, resulting in 1120 ESTs (expressed sequence tags). We found sequences for transcription factors, carbohydrates metabolism, proline-rich proteins, aquaporins, chaperones and ubiquitins, all of them organized according to their biological processes. Our suppressive subtractive hybridization (SSH) library was validated through RT-qPCR experiment by assessing the expression patterns of 10 selected genes in both genotypes under stressed and control conditions. Finally, the expression patterns of 31 ESTs, putatively related to drought responses, were analyzed in a time-course experiment. Our results confirmed that such genes are more expressed in the tolerant genotype during stress; however, they are not exclusive, since different levels of these transcripts were also detected in the susceptible genotype. In addition, we observed a fluctuation in gene regulation over time for both the genotypes, which seem to adopt and adapt different strategies in order to develop tolerance against this stress.
Subject(s)
Adaptation, Physiological/genetics , Droughts , Gene Expression Profiling , Gene Expression Regulation, Plant , Phaseolus/genetics , Phaseolus/physiology , Plant Roots/genetics , Cluster Analysis , Expressed Sequence Tags , Genes, Plant , Genotype , Humidity , Plant Roots/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Software , Soil , Subtractive Hybridization Techniques , Time Factors , Transcription Factors/metabolism , Transcription, Genetic , WaterABSTRACT
Uma das áreas mais promissoras na pesquisa sobre o câncer de mama é a identificação de marcadores moleculares que possam ser utilizados na detecção precoce da doença, na predição do prognóstico do paciente ou no desenvolvimento de abordagens terapêuticas alternativas, reduzindo, assim, as taxas de mortalidade e morbidade associadas à doença. Alterações epigenéticas têm sido associadas à tumorigênese, e a metilação do DNA é um dos mecanismos epigenéticos mais estudados. A metilação do DNA está associada ao silenciamento gênico e alterações no padrão de metilação têm sido utilizadas com freqüência na caracterização de genes relacionados ao câncer, assim como marcador tumoral para o diagnóstico e prognóstico da doença. Neste trabalho, nós identificamos genes silenciados por metilação do DNA em linhagens celulares tumorais de mama, como uma forma de aumentar o número de marcadores moleculares disponíveis para o câncer de mama. Para tanto, 3 linhagens celulares tumorais de mama (MCF-7, MDA-MB-231, MDA-MB-435) foram tratadas com o agente desmetilante 5-aza-2'-deoxicitidina, capaz de induzir a expressão de genes silenciados por metilação do DNA. RNA total extraído de culturas celulares submetidas ou não ao tratamento com o agente desmetilante foi utilizado para a construção de bibliotecas subtrativas de cDNA utilizando a metodologia de RaSH (Rapid Subtraction Hybridization). Duas bibliotecas diferentes foram construídas para cada linhagem celular: uma utilizando como tester cDNA sintetizado a partir de RNA extraído da linhagem celular tratada com o agente desmetilante, e contendo genes induzidos pelo tratamento; e a outra, utilizando como tester cDNA da linhagem celular não submetida ao tratamento, e que foi utilizada como controle da subtração. Um total de 2367 seqüências foram geradas a partir de 3 bibliotecas subtraídas utilizando o cDNA das linhagens MCF-7, MDA-MB-435 e MDA-MB-231 tratadas como tester. A comparação entre cada biblioteca subtrativa e seu experimento controle possibilitou a exclusão de um total de 50 falso-positivos. A comparação das seqüências oriundas dessas bibliotecas com seqüências depositadas em bancos de dados públicos, permitiu a identificação de 533 genes induzidos pelo tratamento e possivelmente regulados por metilação. Para a identificação de genes induzidos na linhagem MDA-MB-231, foi realizado um experimento de Northern Blot Reverso, e 4 genes que apresentaram indução superior a 3 vezes foram escolhidos para validação por PCR em Tempo Real. Ao todo, 41 candidatos foram selecionados para validação experimental por PCR em Tempo Real utilizando o cDNA sintetizado a partir do mesmo RNA utilizado para a construção das bibliotecas de RaSH. Dos 37 genes para os quais foi possível padronizar a reação de PCR em Tempo Real, 5 genes mostraram aumento da expressão de pelo menos 2 vezes após o tratamento (Sequestosome 1, SQSTM1; Superoxide dismutase 2, SOD2; Connective Tissue Growth Factor, CTGF; Small Ubiquitin-like Modifier 2, SUMO2 e Fibronectin 1, FN1). Uma análise preliminar do padrão de expressão destes genes em 18 amostras de tumores de mama revelou a redução da expressão do gene SUMO2 em 10 amostras tumorais de mama quando comparadas a um pool de amostras de mama normal.
One of the most promising areas in breast cancer research is the identification of molecular markers that can be used in the early detection of the disease, for prediction patient prognosis and for the development of alternative therapeutic approaches, reducing, in this mortality and morbidity associated with the disease. Epigenetic alterations have been frequently associated with tumorigenesis, and DNA methylation is one of the most studied epigenetic mechanisms. DNA methylation is associated with gene silencing and changes in the methylation pattern have been used frequently in the characterization of cancer-related genes, as well as a tumor marker for the diagnosis and prognosis of the disease. In this work, we identified genes silenced by DNA methylation in breast tumor cell lines, as a way to enlarge the number of molecular markers available for breast cancer. For this purpose, 3 breast tumor cell lines (MCF-7, MDA-MB-435, MDA-MB-231) were treated with the demethylating agent 5-aza-2'-deoxycytidine (5aza-dC), which can induce the the expression of genes silenced by DNA methylation. Total RNA extracted from cell cultures submitted or not to treatment with the demethylating agent was used for the construction of subtractive cDNA libraries using the RaSH (Rapid Subtraction Hybridization) methodology. Two different libraries were constructed for each cell line: one using as a tester cDNA synthesized from RNA extracted from the cell line treated with the demethylating agent, and containing genes induced by the treatment; and the other using as a tester cDNA from the cell line not submitted to treatment, and that was used as control for the subtracion. A total of 2367 sequences were generated from 3 cDNA libraries subtracted using cDNA of the treated MCF-7, MDA-MB-435 and MDA-MB-231 cell lines as a tester. The comparison between each subtractive library and its control allowed the exclusion of a total of 50 false-positives candidate genes. Comparison of the sequences generated from these libraries with sequences submitted to public databases allowed the identification of 533 genes induced by treatment and possibly regulated by DNA methylation. For the identification of induced genes in the MDA-MB-231 cell line, a reverse Northern blot exeriment was performed, and 4 genes with a more than three-fold induction were selected for validation by Real-Time PCR. In all, 41 candidates were selected for experimental validation by Real-Time PCR using the cDNA synthesized from the same RNA source used for the construction of the RaSH libraries. From the 37 genes for which it was possible to carry on Real-Time PCR analysis, 5 genes showed more than 2-fold intuction upon treatment (Sequestosome 1, SQSTM1; Superoxide dismutase 2, SOD2; Connective Tissue Growth Factor, CTGF; Small Ubiquitin-like Modifier 2, SUMO2 and Fibronectin 1, FN1). A preliminary analysis of the expression pattern of these genes in 18 breast tumor samples revealed reduction expression of the SUMO2 gene, in 10 breast tumor samples when compared to a pool of normal breast samples.