RESUMO
Invasive amoebiasis caused by Entamoeba histolytica is a major global health problem. Virulence is a rare outcome of infection, occurring in fewer than 1 in 10 infections. Not all strains of the parasite are equally virulent, and understanding the mechanisms and causes of virulence is an important goal of Entamoeba research. The sequencing of the genome of E. histolytica and the related avirulent species Entamoeba dispar has allowed whole-genome-scale analyses of genetic divergence and differential gene expression to be undertaken. These studies have helped elucidate mechanisms of virulence and identified genes differentially expressed in virulent and avirulent parasites. Here, we review the current status of the E. histolytica and E. dispar genomes and the findings of a number of genome-scale studies comparing parasites of different virulence.
Assuntos
Entamoeba histolytica/patogenicidade , Entamoeba/patogenicidade , Entamebíase/parasitologia , Genoma de Protozoário/genética , Interações Hospedeiro-Parasita/genética , Proteínas de Protozoários/metabolismo , Animais , Entamoeba/classificação , Entamoeba/genética , Entamoeba/fisiologia , Entamoeba histolytica/genética , Entamoeba histolytica/fisiologia , Humanos , Proteínas de Protozoários/genética , Especificidade da Espécie , Virulência/genéticaRESUMO
MOTIVATION: The focus of this paper is on two new normalization methods for cDNA microarrays. After the image analysis has been performed on a microarray and before differentially expressed genes can be detected, some form of normalization must be applied to the microarrays. Normalization removes biases towards one or other of the fluorescent dyes used to label each mRNA sample allowing for proper evaluation of differential gene expression. RESULTS: The two normalization methods that we present here build on previously described non-linear normalization techniques. We extend these techniques by firstly introducing a normalization method that deals with smooth spatial trends in intensity across microarrays, an important issue that must be dealt with. Secondly we deal with normalization of a new type of cDNA microarray experiment that is coming into prevalence, the small scale specialty or 'boutique' array, where large proportions of the genes on the microarrays are expected to be highly differentially expressed. AVAILABILITY: The normalization methods described in this paper are available via http://www.pi.csiro.au/gena/ in a software suite called tRMA: tools for R Microarray Analysis upon request of the authors. Images and data used in this paper are also available via the same link.
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Modelos Genéticos , Modelos Estatísticos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Espectrometria de Fluorescência/métodos , Espectrometria de Fluorescência/normas , Calibragem/normas , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normasRESUMO
Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;-amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl alpha-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Carboxipeptidases/química , Carboxipeptidases/genética , Genes de Plantas/genética , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Carboxipeptidases/metabolismo , Clonagem Molecular , Citocininas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Meristema/genética , Meristema/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Plantas Geneticamente Modificadas , RNA de Plantas/genética , RNA de Plantas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Powdery mildew diseases are economically important diseases, caused by obligate biotrophic fungi of the Erysiphales. To understand the complex inheritance of resistance to the powdery mildew disease in the model plant Arabidopsis thaliana, quantitative trait loci analysis was performed using a set of recombinant inbred lines derived from a cross between the resistant accession Kashmir-1 and the susceptible accession Columbia glabrous1. We identified and mapped three independent powdery mildew quantitative disease resistance loci, which act additively to confer disease resistance. The locus with the strongest effect on resistance was mapped to a 500-kbp interval on chromosome III.
Assuntos
Arabidopsis/genética , Ascomicetos/patogenicidade , Característica Quantitativa Herdável , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Sequência de Bases , Primers do DNA , FenótipoRESUMO
Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA. Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I [SLI]) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA. Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA. Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot. Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different. The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI. Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI. By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules. These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.
Assuntos
DNA Helicases , Replicação do DNA , Proteínas de Ligação a DNA , Conformação de Ácido Nucleico , Plasmídeos/biossíntese , RNA Antissenso/genética , RNA Bacteriano/genética , Transativadores , Proteínas de Bactérias/biossíntese , Sequência de Bases , Simulação por Computador , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Biossíntese de Proteínas , RNA Antissenso/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Relação Estrutura-AtividadeRESUMO
The translation of RepA, the replication initiation protein of the IncB plasmid pMU720, requires that its mRNA (RNAII) folds to form a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. The formation of this pseudoknot is dependent in turn on the translation and correct termination of a leader peptide, RepB. A small countertranscript RNA, RNAI, controls the replication of pMU720 by interacting with RNAII to negatively regulate the expression of repA both directly, by sequestering the proximal bases required for pseudoknot formation, and indirectly, by inhibiting the translation of repB. Inhibition of the translation of repB by RNAI was found to depend on the close proximity of the RNAI-RNAII complex to the translational initiation region of repB, indicating that the primary mechanism of RNAI control involves steric hindrance. Disruption of RNAI control of repB had only a small effect on the copy number of the IncB plasmid, indicating that inhibition of the expression of repA by RNAI is achieved predominantly by inhibition of pseudoknot formation rather than by inhibition of repB translation.
Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , Biossíntese de Proteínas , RNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Análise Mutacional de DNA , Replicação do DNA , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica , Plasmídeos/biossíntese , Sinais Direcionadores de Proteínas/biossíntese , RNA/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Replicon/genética , Deleção de SequênciaRESUMO
The translational initiation region of the mRNA for the replication initiation protein (RepA) of pMU720 is predicted to be sequestered in an inhibitory secondary structure designated stem-loop III. Activation of repA translation requires both the disruption of stem-loop III by ribosomes involved in the translation and termination of the leader peptide RepB and the formation of a pseudoknot, a tertiary RNA structure. Disruption of stem-loop III by site-directed mutagenesis was found to be insufficient to allow high repA expression in the absence of pseudoknot formation, indicating that the pseudoknot acts as an enhancer of repA translation. Furthermore, extending the length of the leader peptide RepB and changing the distance between the pseudoknot and repA Shine-Dalgarno sequence were found to have major effects on the translation of repA.
Assuntos
Proteínas de Bactérias/genética , DNA Helicases , Replicação do DNA , DNA Super-Helicoidal/ultraestrutura , Proteínas de Ligação a DNA , Plasmídeos , Proteínas , Transativadores , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Mensageiro/genéticaRESUMO
The nature of translational coupling between repB and repA, the overlapping rep genes of the IncB plasmid pMU720, was examined. Mutations in the start codon of the promoter proximal gene, repB, reduced the efficiency of translation of both rep genes. Moreover, there was no independent initiation of repA translation in the absence of repB translation. The position of the repB stop codon was crucial for the efficient expression of repA, with the wild-type positioning being optimal. Translational coupling was found to be totally dependent on the formation of a pseudoknot structure. A model which invokes formation of a pseudoknot to facilitate initiation of repA is proposed.
