RESUMO
Cell-type specific genes were recognized by interrogating microarrays carrying Dictyostelium gene fragments with probes prepared from fractions enriched in prestalk and prespore cells. Cell-type specific accumulation of mRNA from 17 newly identified genes was confirmed by Northern analyses. DNA microarrays carrying 690 targets were used to determine expression profiles during development. The profiles were fit to a biologically based kinetic equation to extract the times of transcription onset and cessation. Although the majority of the genes that were cell-type enriched at the slug stage were first expressed as the prespore and prestalk cells sorted out in aggregates, some were found to be expressed earlier before the cells had even aggregated. These early genes may have been initially expressed in all cells and then preferentially turned over in one or the other cell type. Alternatively, cell type divergence may start soon after the initiation of development.
Assuntos
Dictyostelium/citologia , Dictyostelium/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários/genética , Animais , Northern Blotting , Diferenciação Celular/genética , Dictyostelium/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Esporos/citologia , Esporos/genética , Esporos/crescimento & desenvolvimento , Fatores de Tempo , Transcrição Gênica/genéticaRESUMO
MOTIVATION: The DNA microarray technology can generate a large amount of data describing the time-course of gene expression. These data, when properly interpreted, can yield a great deal of information concerning differential gene expression during development. Much current effort in bioinformatics has been devoted to the analysis of gene expression data, usually via some 'clustering analysis' on the raw data in some abstract high dimensional space. Here, we describe a method where we first 'process' the raw time-course data using a simple biologically based kinetic model of gene expression. This allows us to reduce the vast data to a few vital attributes characterizing each expression profile, e.g. the times of the onset and cessation of the expression of the developmentally regulated genes. These vital attributes can then be trivially clustered by visual inspection to reveal biologically significant effects. RESULTS: We have applied this approach to microarray expression data from samples isolated every 2 h throughout the 24 h developmental program of Dictyostelium discoideum. mRNA accumulation patterns for 50 developmental genes were found to fit the kinetic model with a p-value of 0.05 or better. Transcription of these genes appears to be initiated in bursts at well-defined periods during development, in a manner suggestive of a dependent sequence. This approach can be applied to analyses of other temporal gene expression patterns, including those of the cell cycle.
Assuntos
Dictyostelium/crescimento & desenvolvimento , Dictyostelium/genética , Perfilação da Expressão Gênica/estatística & dados numéricos , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Algoritmos , Animais , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários , Transcrição GênicaRESUMO
We present a novel approach to the clustering of gene expression patterns based on the mutual connectivity of the patterns. Unlike certain widely used methods (e.g., self-organizing maps and K-means) which essentially force gene expression data into a fixed number of predetermined clustering structures, our approach aims to reveal the natural tendency of the data to cluster, in analogy to the physical phenomenon of percolation. The approach is probabilistic in nature, and as such accommodates the possibility that one gene participates in multiple clusters. The result is cast in terms of the connectivity of each gene to a certain number of (significant) clusters. A computationally efficient algorithm is developed to implement our approach. Performance of the method is illustrated by clustering both constructed data and gene expression data obtained from Dictyostelium development.
Assuntos
Dictyostelium/genética , Perfilação da Expressão Gênica/estatística & dados numéricos , Algoritmos , Animais , Análise por Conglomerados , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Cellulose is a major component of the extracellular matrices formed during development of the social amoeba, Dictyostelium discoideum. We isolated insertional mutants that failed to accumulate cellulose and had no cellulose synthase activity at any stage of development. Development proceeded normally in the null mutants up to the beginning of stalk formation, at which point the culminating structures collapsed onto themselves, then proceeded to attempt culmination again. No spores or stalk cells were ever made in the mutants, with all cells eventually lysing. The predicted product of the disrupted gene (dcsA) showed significant similarity to the catalytic subunit of cellulose synthases found in bacteria. Enzyme activity and normal development were recovered in strains transformed with a construct expressing the intact dcsA gene. Growing amoebae carrying the construct accumulated the protein product of dcsA, but did not make cellulose until they had developed for at least 10 hr. These studies show directly that the product of dcsA is necessary, but not sufficient, for synthesis of cellulose.
Assuntos
Dictyostelium/enzimologia , Glucosiltransferases/genética , Proteínas de Protozoários , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Celulose/biossíntese , Dictyostelium/genética , Expressão Gênica , Dados de Sequência Molecular , Mutagênese , FenótipoRESUMO
A variety of extracellular signals lead to the accumulation of cAMP which can act as a second message within cells by activating protein kinase A (PKA). Expression of many of the essential developmental genes in Dictyostelium discoideum are known to depend on PKA activity. Cells in which the receptor-coupled adenylyl cyclase gene, acaA, is genetically inactivated grow well but are unable to develop. Surprisingly, acaA(-) mutant cells can be rescued by developing them in mixtures with wild-type cells, suggesting that another adenylyl cyclase is present in developing cells that can provide the internal cAMP necessary to activate PKA. However, the only other known adenylyl cyclase gene in Dictyostelium, acgA, is only expressed during germination of spores and plays no role in the formation of fruiting bodies. By screening morphological mutants generated by Restriction Enzyme Mediated Integration (REMI) we discovered a novel adenylyl cyclase gene, acrA, that is expressed at low levels in growing cells and at more than 25-fold higher levels during development. Growth and development up to the slug stage are unaffected in acrA(-) mutant strains but the cells make almost no viable spores and produce unnaturally long stalks. Adenylyl cyclase activity increases during aggregation, plateaus during the slug stage and then increases considerably during terminal differentiation. The increase in activity following aggregation fails to occur in acrA(-) cells. As long as ACA is fully active, ACR is not required until culmination but then plays a critical role in sporulation and construction of the stalk.
Assuntos
Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Dictyostelium/crescimento & desenvolvimento , Chaperonas Moleculares , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Clonagem Molecular , Dictyostelium/classificação , Dictyostelium/genética , Regulação da Expressão Gênica no Desenvolvimento , Histidina Quinase , Dados de Sequência Molecular , Mutagênese , Mutação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We have identified and disrupted the gene coding for alpha-glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two alpha 1,3-linked glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. Mutagenesis by restriction enzyme-mediated integration (REMI) generated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks alpha-glucosidase II activity and makes incompletely processed N-linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccharide profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene is highly homologous to alpha-glucosidase II genes in the human and the pig, C. elegans, and yeast. Although various cell lines have been reported to be defective in alpha-glucosidase II activity, disruption of the Dictyostelium gene gives the first example of a clear developmental phenotype associated with loss of this enzyme.