RESUMEN
Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.
Asunto(s)
Sondas de ADN/síntesis química , Colorantes Fluorescentes/síntesis química , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ADN Complementario/química , Células HeLa/fisiología , Humanos , Deficiencias de Hierro , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Coloración y Etiquetado/métodosRESUMEN
Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, we now identify the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.
Asunto(s)
Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Proteínas Hierro-Azufre/genética , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada , Cartilla de ADN/genética , ADN Complementario/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteína 1 Reguladora de Hierro , Proteína 2 Reguladora de Hierro , Proteínas Reguladoras del Hierro , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.
Asunto(s)
Ciclo del Ácido Cítrico/genética , Ciclo del Ácido Cítrico/fisiología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Aconitato Hidratasa/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Bovinos , Secuencia Conservada , ADN Complementario/genética , Humanos , Proteína 1 Reguladora de Hierro , Proteínas Reguladoras del Hierro , Mitocondrias/enzimología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , PorcinosRESUMEN
The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the iron-regulatory factor. Iron-regulatory factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to iron-responsive elements and were immunoprecipitated by iron-regulatory-factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in iron-responsive-element-binding of iron-regulatory factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant iron-regulatory factor is a bifunctional protein; after purification, it binds to iron-responsive elements and represses translation in vitro. Following iron treatment, iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-factor seems to be required for the conversion of the iron-responsive-element binding to the aconitase form of the protein.
Asunto(s)
Aconitato Hidratasa/metabolismo , Ferritinas/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Cromatografía de Afinidad , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Células HeLa , Humanos , Proteínas Reguladoras del Hierro , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virus Vaccinia/genéticaRESUMEN
The biosynthetic rates for both the transferrin receptor (TfR) and ferritin are regulated by iron. An iron-responsive element (IRE) in the 5' untranslated portion of the ferritin messenger RNA (mRNA) mediates iron-dependent control of its translation. In this report the 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels. Deletion studies identified a 678-nucleotide fragment of the TfR complementary DNA that is critical for this iron regulation. Five potential stem-loops that resemble the ferritin IRE are contained within the region critical for TfR regulation. Each of two of the five TfR elements was independently inserted into the 5' untranslated region of an indicator gene transcript. In this location they conferred iron regulation of translation. Thus, an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.