RESUMO
BACKGROUND: Leishmania (L) are intracellular protozoan parasites that are able to survive and replicate within the harsh and potentially hostile phagolysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MPhi) striving to eliminate the pathogen and the parasite struggling for its own survival. To investigate this host-parasite conflict at the transcriptional level, in the context of monocyte-derived human MPhis (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used. RESULTS: After extracting mRNA from resting human MPhis, Leishmania-infected human MPhis and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 357,888 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, confidently discriminating host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MPhis' and 3,666 tags to L. major parasites transcripts. We focused on these, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MPhis genes, belonging to key immune response proteins (e.g., IFNgamma pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the parasite's intra-cellular developing stage. CONCLUSION: Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminating genes of human or parasitic origin in Leishmania-infected human MPhis. The findings presented in this work suggest that the Leishmania parasite modulates key transcripts in human MPhis that may be beneficial for its establishment and survival. Furthermore, these results provide an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes and indicate a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes will be useful in future rounds of data mining and gene annotation.
Assuntos
Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Parasita/genética , Leishmania major/genética , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Animais , Apoptose/genética , Sequência de Bases , Quimiocinas/genética , Espaço Extracelular/parasitologia , Regulação da Expressão Gênica , Biblioteca Gênica , Genes MHC Classe I , Genes MHC da Classe II , Humanos , Interferon gama/genética , Espaço Intracelular/parasitologia , Leishmania major/fisiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Proteínas S100/genéticaRESUMO
African trypanosomiases are parasitic diseases transmitted by tse-tse flies, considered as the main sanitary obstacle to animal production development in sub-Saharan Africa. However, if trypanosomiases have dramatic consequences on zebu (Bos indicus) populations, they have a weaker impact on the western African taurine (Bos taurus), which is known to be naturally tolerant to trypanosome infection. Mechanisms governing this trypanotolerant trait are still poorly understood, but today, recent postgenomic biotechnologies, such as the SAGE technique (serial analysis of gene expression) allow us to explore the full transcriptome. Twelve SAGE libraries were constructed from two trypanotolerant animals (N'Dama and Baoulé) and one susceptible species of cattle (the Sudanese zebu) during an experimental Trypanosoma congolense infection; 43,458 different tags were obtained at several particular points during the infection (before infection, at the maximum of parasitemia, the maximum of anemia, and at the end of the experiment after value normalization). Bioinformatics analyses highlighted some interesting gene variations with respect to the trypanotolerance status of the animal.
Assuntos
Doenças dos Bovinos/imunologia , RNA Mensageiro/genética , Tripanossomíase/imunologia , Tripanossomíase/veterinária , Animais , Bovinos , Doenças dos Bovinos/genética , Tripanossomíase/genéticaRESUMO
Analysis of several million expressed gene signatures (tags) revealed an increasing number of different sequences, largely exceeding that of annotated genes in mammalian genomes. Serial analysis of gene expression (SAGE) can reveal new Poly(A) RNAs transcribed from previously unrecognized chromosomal regions. However, conventional SAGE tags are too short to identify unambiguously unique sites in large genomes. Here, we design a novel strategy with tags anchored on two different restrictions sites of cDNAs. New transcripts are then tentatively defined by the two SAGE tags in tandem and by the spanning sequence read on the genome between these tagged sites. Having developed a new algorithm to locate these tag-delimited genomic sequences (TDGS), we first validated its capacity to recognize known genes and its ability to reveal new transcripts with two SAGE libraries built in parallel from a single RNA sample. Our algorithm proves fast enough to experiment this strategy at a large scale. We then collected and processed the complete sets of human SAGE tags to predict yet unknown transcripts. A cross-validation with tiling arrays data shows that 47% of these TDGS overlap transcriptional active regions. Our method provides a new and complementary approach for complex transcriptome annotation.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/análise , Sitios de Sequências Rotuladas , Sequência de Bases , Biologia Computacional , Biblioteca Gênica , Genômica/métodos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Alinhamento de Sequência , Transcrição GênicaRESUMO
Disease relapse sometimes occurs after acute promyelocytic leukemia (APL) therapy with all-trans retinoic acid (ATRA). Among the diagnostic parameters predicting relapse, heterogeneity in the in vitro differentiation rate of blasts is an independent factor. To identify biologic networks involved in resistance, we conducted pharmacogenomic studies in APL blasts displaying distinct ATRA sensitivities. Although the expression profiles of genes invested in differentiation were similarly modulated in low- and high-sensitive blasts, low-sensitive cells showed higher levels of transcription of ATRA-target genes, transcriptional regulators, chromatin remodelers, and transcription factors. In opposition, only high-sensitive blasts expressed the CYP26A1 gene, encoding the p450 cytochrome which is known to be involved in retinoic acid catabolism. In NB4 cells, ATRA treatment activates a novel signaling pathway, whereby interleukin-8 stimulates the expression of the homeobox transcription factor HOXA10v2, an effective enhancer of CYP26A1 transcription. These data were corroborated in primary APL cells, as maturation levels correlated with CYP26A1 expression. Treatment with a retinoic acid metabolism blocking agent (RAMBA) results in high-nucleoplasmic concentrations of retinoid and growth of NB4-resistant subclones. Hence, for APL blasts associated with poor prognosis, the low CYP26A1 expression may explain high risk of resistance installation, by increased retinoid pressure. Pharmacogenomic profiles of genes involved in retinoid acid metabolism may help to optimize anticancer therapies, including retinoids.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Farmacogenética , Tretinoína/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Proteínas Homeobox A10 , Proteínas de Homeodomínio/genética , Humanos , Interleucina-8/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Modelos Biológicos , Ácido Retinoico 4 Hidroxilase , Transcrição Gênica/efeitos dos fármacos , Tretinoína/metabolismo , Células Tumorais CultivadasRESUMO
In central and sub-Saharan Africa, trypanosomosis is a tsetse fly-transmitted disease, which is considered as the most important impediment to livestock production in the region. However, several indigenous West African taurine breeds (Bos taurus) present remarkable tolerance to the infection. This genetic capability, named trypanotolerance, results from numerous biological mechanisms most probably under multigenic dependences, among which are control of the trypanosome infection by limitation of parasitemia and control of severe anemia due to the pathogenic effects. Today, some postgenomic biotechnologies, such as transcriptome analyses, allow characterization of the full expressed genes involved in the majority of animal diseases under genetic control. One of them is serial analysis of gene expression (SAGE) technology, which consists of the construction of mRNA transcript libraries for qualitative and quantitative analysis of the entire genes expressed or inactivated at a particular step of cellular activation. We developed four different mRNA transcript libraries from white blood cells on a N'Dama trypanotolerant animal during an experimental Trypanosoma congolense (T. congolense) infection: one before experimental infection (ND0), one at the parasitemia peak (NDm), one at the minimal packed cell volume (NDa), and the last one at the end of the experiment after normalization (NDf). Bioinformatic comparisons in bovine genomic databases allowed us to obtain more than 75,000 sequences, among which are several known genes, some others are already described as expressed sequence tags (ESTs), and the last are completely new, but probably functional in trypanotolerance. The knowledge of all identified named or unnamed genes involved in trypanotolerance characteristics will allow us to use them in a field marker-assisted selections strategy and in microarrays prediction sets for bovine trypanotolerance.
Assuntos
Doenças dos Bovinos/genética , Doenças dos Bovinos/imunologia , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/veterinária , Trypanosoma congolense , Tripanossomíase Africana/veterinária , Animais , Bovinos , Biologia Computacional , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Predisposição Genética para Doença , RNA Mensageiro/metabolismo , Transcrição Gênica , Tripanossomíase Africana/genética , Tripanossomíase Africana/imunologia , Moscas Tsé-Tsé/parasitologiaRESUMO
BACKGROUND: The kidney development involves a wide variety of developmental processes requiring a lot of genes expressed in a sequential manner. The aim of the present study is to identify new genes involved in these processes. METHODS: To obtain a view of the mouse embryonic kidney transcriptome we used the SADE method, which allows large-scale quantitative gene expression measurements. RESULTS: 7,689 tags were sequenced from our library. Among the 4,507 unique transcripts yielded, 64% correspond to known genes, 22% ESTs, 12% unidentified genes. 472 genes were differentially expressed as compared to published adult kidney library. Among these, we identified several candidate genes and focused on a particular one: thymosin beta4 (Tbeta4), an actin-sequestering protein more highly expressed in fetal kidney. First we studied the in vivo expression patterns of Tbeta4 transcript during kidney development. Tbeta4 increases throughout the kidney development and remains high during active nephrogenesis. Moreover, the spatial distribution of Tbeta4 mRNA was analysed and reveals that during active nephrogenesis (i.e., 18 dpc) Tbeta4 is localised in differentiating glomeruli. In adult kidney, Tbeta4 remains expressed in podocytes and collecting ducts. CONCLUSION: Our results provide the first demonstration of Tbeta4 production in vivo by embryonic kidney and further show that Tbeta4 is implicated in kidney organogenesis.
Assuntos
Envelhecimento/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Rim/embriologia , Rim/metabolismo , Proteoma/genética , Proteoma/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Post genomic biotechnologies, such as transcriptome analysis, are now efficient enough to characterize the full complement of genes involved in the expression of specific biological functions. One of them is the Serial Analysis of Gene Expression (SAGE) technique. SAGE involves the construction of transcript libraries for a quantitative analysis of the entire set of genes expressed or inactivated at particular stages of cellular activation. Bioinformatic comparisons in hosts and pathogens genomic databases allow the identification of several up- and down-regulated genes, ESTs and unknown transcripts directly involved in the host-pathogen immunological interaction mechanisms. Based on the first results obtained during an experimental Trypanosoma congolense infection in trypanotolerant cattle, the efficiency and limits of such a technique, from the data acquisition level to the data analysis level, is discussed in this analysis.
Assuntos
Doenças dos Bovinos/genética , Perfilação da Expressão Gênica/veterinária , Trypanosoma congolense , Tripanossomíase Africana/veterinária , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/imunologia , Biologia Computacional , DNA/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/estatística & dados numéricos , Genômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Tripanossomíase Africana/genética , Tripanossomíase Africana/imunologiaRESUMO
BACKGROUND AND OBJECTIVES: We studied the gene expression profile of human purified reticulocytes to provide a transcriptional basis for the study of erythroid biology, differentiation and hematologic disorders. DESIGN AND METHODS: We screened highly purified blood reticulocytes from ten healthy adult volunteers. We chose a modified protocol of serial analysis of gene expression (SAGE), the serial analysis of downsized extracts (SADE). RESULTS: Data analysis revealed that 64% of gene signatures (tags) matched with known genes; mainly hemoglobin. In addition to the abundant globin mRNA, SAGE analysis identified previously described genes and new transcripts. In reticulocytes, which are poor in mRNA, we also identified 9% of EST and 27% of tags that did not match with any known genes. Mining our data, 70% of the unknown tags and 39% of tags identifying EST were found to be specific to the reticulocyte. We demonstrated the presence of a mRNA that matched with the reverse sequence of the hemoglobin b (HBB) transcript. INTERPRETATION AND CONCLUSIONS: This is the first description of an antisense transcript of the human HBB gene suggesting regulation by way of sense-antisense pairing. The well-characterized genes found in the SAGE library were genes specific to the blood cell lineage, housekeeping genes and, interestingly, genes not previously described in the reticulocyte. Furthermore the study provides markers of the erythroid lineage regulated during the differentiation process as observed in in vitro experiments.
Assuntos
RNA Antissenso/sangue , RNA Mensageiro/genética , Reticulócitos/química , Adulto , Proteínas Sanguíneas/genética , Células Cultivadas/metabolismo , Sistemas Computacionais , Células Precursoras Eritroides/química , Etiquetas de Sequências Expressas , Feminino , Sangue Fetal/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Globinas/genética , Humanos , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase , RNA Antissenso/genética , RNA Antissenso/isolamento & purificação , RNA Mensageiro/sangue , RNA Mensageiro/isolamento & purificaçãoRESUMO
New postgenomic biotechnologies, such as transcriptome analyses, are now able to characterize the full complement of genes involved in the expression of specific biological functions. One of these is the Serial Analysis of Gene Expression (SAGE) technique, which consists of the construction of transcripts libraries for a quantitative analysis of the entire gene(s) expressed or inactivated at a particular step of cellular activation. Bioinformatic comparisons in the bovine genomic databases allow the identification of several up- and downregulated genes, expressed sequence tags, and unknown functional genes directly involved in the genetic control of the studied biological mechanism. We present and discuss the preliminary results in comparing the expressed genes in two total mRNA transcripts libraries obtained during an experimental Trypanosoma congolense infection in one trypanotolerant N'Dama animal cow. Knowing all the functional genes involved in the trypanotolerance control will permit validation of some results obtained with the quantitative trait locus approach, to set up specific microarrays sets for further metabolic and pharmacological studies, and to design field marker-assisted selection by introgression programs.
Assuntos
Doenças dos Bovinos/imunologia , Biologia Computacional , Perfilação da Expressão Gênica , Trypanosoma congolense/genética , Trypanosoma congolense/patogenicidade , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/veterinária , Medicina Veterinária/tendências , Animais , Bovinos , Doenças dos Bovinos/genética , Feminino , Biblioteca Gênica , Imunidade Inata , RNA Mensageiro/genética , Transcrição Gênica , Tripanossomíase Africana/genéticaRESUMO
As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3'UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense-antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples.
Assuntos
Perfilação da Expressão Gênica/métodos , RNA Antissenso/genética , Biologia Computacional , DNA Complementar/genética , Etiquetas de Sequências Expressas , Humanos , RNA Antissenso/biossíntese , Células U937RESUMO
BACKGROUND AND OBJECTIVES: Enhancement of oxygen delivery to tissues is associated with improved sporting performance. One way of enhancing oxygen delivery is to take recombinant human erythropoietin (rHuEpo), which is an unethical and potentially dangerous practice. However, detection of the use of rHuEpo remains difficult in situations such as: i) several days after the end of treatment ii) when a treatment with low doses is conducted iii) if the rHuEpo effect is increased by other substances. In an attempt to detect rHuEpo abuse, we selected erythroid gene markers from a SAGE library and analyzed the effects of rHuEpo administration on expression of the HBB, FTL and OAZ genes. DESIGN AND METHODS: Ten athletes were assigned to the rHuEpo or placebo group. The rHuEpo group received subcutaneous injections of rHuEpo (50 UI/kg three times a week, 4 weeks; 20 UI/kg three times a week, 2 weeks). HBB, FTL and OAZ gene profiles were monitored by real time-polymerase chain reaction (PCR) quantification during and for 3 weeks after drug administration. RESULTS: The global analysis of these targeted genes detected in whole blood samples showed a characteristic profile of subjects misusing rHuEpo with a increase above the threshold levels. The individual analysis of OAZ mRNA seemed indicative of rHuEpo treatment. INTERPRETATION AND CONCLUSIONS: The performance-enhancing effect of rHuEpo treatment is greater than the duration of hematologic changes associated with rHuEpo misuse. Although direct electrophoretic methods to detect rHuEpo have been developed, recombinant isoforms of rHuEpo are not detectable some days after the last subcutaneous injection. To overcome these limitations indirect OFF models have been developed. Our data suggest that, in the near future, it will be possible to consolidate results achievable with the OFF models by analyzing selected erythroid gene markers as a supplement to indirect methods.
Assuntos
Dopagem Esportivo , Eritropoese/genética , Eritropoetina/sangue , Reticulócitos/fisiologia , Esportes , Adolescente , Adulto , Biomarcadores/sangue , Regulação da Expressão Gênica , Humanos , Placebos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Proteínas RecombinantesRESUMO
Because of its short life span in blood, its low RNA content and its highly condensed nucleus, the granulocyte was initially considered as a terminally differentiated cell unable to express novel genes. However, mature granulocytes still contain a variety of mRNAs and may respond to external stimuli by rapid and complex changes in gene expression. The present work was undertaken to provide a wider view of the gene expression profile in unstimulated circulating PMNs. We used serial analysis of gene expression (SAGE) adapted to downsized extracts (SADE) to cope with their small mRNA content. As cell samples of the highest degree of purity were crucial for this project, we adapted a magnetic cell sorting method to reach the required high levels of purity (99.55 +/- 0.19%) together with low activation rates (1.37 +/- 0.28). We analyzed 20,787 tags identifying 8547 different transcripts, of which 47.8% were unknown, 34.6% were transcripts of known genes, and 13.8% matched with expressed sequence tags (EST). Highly expressed genes were those involved in cell mobility, diapedesis, cell signaling and destruction of micro-organisms. In addition, this method led to the identification of genes which had not previously been reported in granulocytes. These results could provide new molecular markers and a reliable reference for the investigation of pathologies involving alteration of the granulocyte gene expression profile.
Assuntos
Perfilação da Expressão Gênica , Neutrófilos/metabolismo , Biomarcadores , Citometria de Fluxo , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3- or NH4NO3 as N source.
Assuntos
Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Sequência de Bases , DNA de Plantas/genética , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Expressão Gênica , Genoma de Planta , Canais Iônicos/genética , Transporte de Íons/genética , Nitrogênio/metabolismo , Raízes de Plantas/genética , RNA Mensageiro/genética , RNA de Plantas/genéticaRESUMO
In Africa, trypanosomosis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine Bos taurus) breeds, such as the Longhorn (N'Dama) cattle are well known to control trypanosome infections. This genetic ability named "trypanotolerance" results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New post genomic biotechnologies such as transcriptome analyses are efficient in characterising the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from Peripheral Blood Mononuclear Cells of an N'Dama cow, 2 total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down- regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow to set up specific microarray sets for further metabolic and pharmacological studies and to design field marker-assisted selection by introgression programmes.
Assuntos
Perfilação da Expressão Gênica , Imunidade Inata/imunologia , Trypanosoma congolense/genética , Tripanossomíase Africana/veterinária , Tripanossomíase Bovina/genética , África Subsaariana , Animais , Bovinos , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Biblioteca Gênica , Tripanossomíase Africana/genética , Tripanossomíase Africana/imunologia , Tripanossomíase Bovina/imunologiaRESUMO
Highly sensitive and specific diagnostic tools are essential for monitoring the health status of farmed species. After the development of genomic probe diagnostic systems in the 1990s, followed by PCR-based systems, a miniarray method has been developed allowing one-step multiple detection. The miniarray method was developed to enable the accessibility of powerful array technology. To use this system, hybridisation and washing process were modified, resulting into a significant increase in the test's rapidity and sensitivity. With miniarray technology, hybridisation time is reduced to 20 min, whereas other methods require a longer hybridisation time. Hybridisation of the PCR product on a nylon membrane and revelation of the hybrids by an antibody increase considerably the ability of pathogen's detection. A first application is developed for the diagnosis of two specific viruses which are, by their geographical range and their impact on the production, very important in shrimp pathology, namely, the White Spot Syndrome Virus (WSSV) and the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).
Assuntos
Vírus de DNA/isolamento & purificação , Hemolinfa/virologia , Penaeidae/virologia , Animais , DNA Viral/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
The human leukemia cell line U937 is a well-established model for studying monocytic cell differentiation. We used a modified protocol (SADE) of serial analysis of gene expression (SAGE) and developed a SADE linker-anchored PCR assay to investigate the pattern of expression of known genes and to identify new transcripts in proliferating cells and during cell growth arrest and differentiation. We implemented new informatic tools to compare expression profiles before and after exposure of cells to differentiation inducers. From the analysis of 47,388 tags, we identified 13,806 distinct transcripts, 265 of which showed significant variations (P<0.01). Among 1219 well-identified genes, major changes concerned transcription and translation components, cytoskeleton, and macrophage-specific genes. Nearly half of the tags, some of them expressed at high levels, matched partially characterized genes or ESTs, or revealed yet-unknown transcripts, providing a wealth of new candidate genes that may reveal novel aspects of terminal monocytic differentiation.
Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Humanos , Transcrição Gênica , Células U937RESUMO
The availability of the sequences for whole genomes is changing our understanding of cell biology. Functional genomics refers to the comprehensive analysis, at the protein level (proteome) and at the mRNA level (transcriptome) of all events associated with the expression of whole sets of genes. New methods have been developed for transcriptome analysis. Serial Analysis of Gene Expression (SAGE) is based on the massive sequential analysis of short cDNA sequence tags. Each tag is derived from a defined position within a transcript. Its size (14 bp) is sufficient to identify the corresponding gene and the number of times each tag is observed provides an accurate measurement of its expression level. Since tag populations can be widely amplified without altering their relative proportions, SAGE may be performed with minute amounts of biological extract. Dealing with the mass of data generated by SAGE necessitates computer analysis. A software is required to automatically detect and count tags from sequence files. Criterias allowing to assess the quality of experimental data can be included at this stage. To identify the corresponding genes, a database is created registering all virtual tags susceptible to be observed, based on the present status of the genome knowledge. By using currently available database functions, it is easy to match experimental and virtual tags, thus generating a new database registering identified tags, together with their expression levels. As an open system, SAGE is able to reveal new, yet unknown, transcripts. Their identification will become increasingly easier with the progress of genome annotation. However, their direct characterization can be attempted, since tag information may be sufficient to design primers allowing to extend unknown sequences. A major advantage of SAGE is that, by measuring expression levels without reference to an arbitrary standard, data are definitively acquired and cumulative. All publicly available data can thus be stored in a unique database, facilitating whole-genome analysis of differential expression between cell types, normal and diseased samples, or samples with and without drug treatment. SAGE data are readily amenable to statistical comparisons, allowing to determine the level of confidence of the observed variations. A major limitation of SAGE is that, because each analysis is obligatory performed on the whole set of expressed genes, it can hardly be performed on multiple samples, for example in kinetics studies or to compare the effects of large numbers of drugs. To overcome this limitation, high-throughput detection of a subset of mRNAs is more rapidly performed by parallel hybridization of mRNAs on arrays of nucleic acids immobilized on solid supports. From this point of view, a SAGE platform is a powerful instrument for selecting the most informative subset of genes, assembling them to design microarrays dedicated to a specific problem and calibrating measurement by comparison with a standard cell model for which SAGE data are available. This approach is an attractive alternative to strategies based exclusively on pangenomic arrays. A very large amount of SAGE data are already available and the problem is now to extract their biological meaning. Knowledge on metabolic pathways is already organized so that its successful integration in a SAGE platform can be undertaken. For other cell components and pathways, the problem lies on the lack of controlled vocabulary to describe gene activities, starting form a clear definition of the concept of biological function itself. Progress in gene and cell ontology is expected to facilitate computer-based extraction of biological knowledge from existing and forthcoming SAGE data.