RESUMO
An inducible RNA interference (RNAi) construct composed of inverted repeating alcA promoters flanking the developmental regulatory gene brlAbeta was tested in Aspergillus nidulans. On inducing medium, the RNAi strains failed to sporulate and lacked brlAalpha and brlAbeta expression. RNAi was specific for brlAbeta, but not brlAalpha, silencing, indicating brlAalpha regulation by brlAbeta.
Assuntos
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Interferência de RNA , Aspergillus nidulans/fisiologia , Proteínas Fúngicas/metabolismo , Regiões Promotoras Genéticas , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologiaRESUMO
The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix-loop-helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5' and 3' flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.
Assuntos
Proteínas de Ligação a DNA/genética , Peixes/genética , Genes Reguladores , Sequências Hélice-Alça-Hélice , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Proteínas de Peixe-Zebra , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Mapeamento Cromossômico , Feminino , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Rearranjo Gênico , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Long-range comparative sequence analysis provides a powerful strategy for identifying conserved regulatory elements. The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis, and it displays a highly conserved expression pattern. We present here a detailed sequence comparison of 193 kb of the human SCL locus to 234 kb of the mouse SCL locus. Four new genes have been identified together with an ancient mitochondrial insertion in the human locus. The SCL gene is flanked upstream by the SIL gene and downstream by the MAP17 gene in both species, but the gene order is not collinear downstream from MAP17. To facilitate rapid identification of candidate regulatory elements, we have developed a new sequence analysis tool (SynPlot) that automates the graphical display of large-scale sequence alignments. Unlike existing programs, SynPlot can display the locus features of more than one sequence, thereby indicating the position of homology peaks relative to the structure of all sequences in the alignment. In addition, high-resolution analysis of the chromatin structure of the mouse SCL gene permitted the accurate positioning of localized zones accessible to restriction endonucleases. Zones known to be associated with functional regulatory regions were found to correspond precisely with peaks of human/mouse homology, thus demonstrating that long-range human/mouse sequence comparisons allow accurate prediction of the extent of accessible DNA associated with active regulatory regions.
Assuntos
Sequência Conservada/genética , Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Animais , Composição de Bases , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/genética , Genes Neoplásicos , Marcadores Genéticos , Variação Genética , Humanos , Hidrólise , Leucemia-Linfoma de Células T do Adulto/genética , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Células-Tronco/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Vertebrados/genética , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Galinhas , Sequências Hélice-Alça-Hélice , Humanos , Camundongos , Dados de Sequência Molecular , Rombencéfalo/embriologia , Homologia de Sequência de Aminoácidos , Proteína 1 de Leucemia Linfocítica Aguda de Células T , XenopusAssuntos
Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice , Hematopoese/fisiologia , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transcrição GênicaRESUMO
We describe here Tdr2, a new class of Tc1-like transposons in zebrafish. Tdr2 was identified from the genomic sequence of a zebrafish PAC (P1 artificial chromosome) clone, and fragments of Tdr2 were found in several zebrafish EST (expressed sequence tag) sequences. Predicted translation of the Tdr2 transposase gene showed that it was most closely related to Caenorhabditis elegans Tc3A, suggesting an ancient origin of the Tdr2 transposon. Tdr2 spans 1. 1kb and is flanked by inverted repeats of approx. 100bp. The 5' repeat is itself composed of an inverted repeat, raising the possibility of the formation of a cruciform DNA structure. Tdr2 transposons may facilitate the development of novel transposon-based tools for the genetic analysis of zebrafish.
Assuntos
Elementos de DNA Transponíveis/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/química , DNA/genética , Dados de Sequência Molecular , Filogenia , Sequências Repetitivas de Ácido Nucleico/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transposases/genéticaRESUMO
The SCL gene encodes a basic helix-loop-helix transcription factor with a pivotal role in the development of endothelium and of all hematopoietic lineages. SCL is also expressed in the central nervous system, although its expression pattern has not been examined in detail and its function in neural development is unknown. In this article we present the first analysis of SCL transcriptional regulation in vivo. We have identified three spatially distinct regulatory modules, each of which was both necessary and sufficient to direct reporter gene expression in vivo to three different regions within the normal SCL expression domain, namely, developing endothelium, midbrain, and hindbrain/spinal cord. In addition we have demonstrated that GATA factor binding sites are essential for neural expression of the SCL constructs. The midbrain element was particularly powerful and axonal lacZ expression revealed the details of axonal projections, thus implicating SCL in the development of occulomotor, pupillary, or retinotectal pathways. The neural expression pattern of the SCL gene was highly conserved in mouse, chicken, and zebrafish embryos and the 5' region of the chicken SCL locus exhibited a striking degree of functional conservation in transgenic mice. These data suggest that SCL performs critical functions in neural development. The regulatory elements identified here provide important tools for analyzing these functions.
Assuntos
Encéfalo/embriologia , Proteínas de Ligação a DNA/fisiologia , Endotélio/embriologia , Proteínas Proto-Oncogênicas , Medula Espinal/embriologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Proteínas de Peixe-Zebra , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Encéfalo/metabolismo , Embrião de Galinha , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero , Endotélio/metabolismo , Genes Reporter , Hibridização In Situ , Óperon Lac/genética , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Medula Espinal/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Distribuição Tecidual , Peixe-Zebra/embriologiaRESUMO
The SCL/TAL-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor essential for the development of all hemopoietic lineages and also acts as a T-cell oncogene. Four related genes have been described in mammals (LYL-1, TAL-2, NSCL1, and NSCL2), all of which exhibit a high degree of sequence similarity to SCL/TAL-1 in the bHLH domain and two of which (LYL-1 and TAL-2) have also been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia. In this study we describe the identification and characterization of a pufferfish gene termed SLP-1, which represents a new member of this gene family. The genomic structure and sequence of SLP-1 suggests that it forms a subfamily with SCL/TAL-1 and LYL-1 and is most closely related to SCL/TAL-1. However, unlike SCL/TAL-1, SLP-1 is widely expressed. Sequence analysis of a whole cosmid containing SLP-1 shows that SLP-1 is flanked upstream by a zinc finger gene and a fork-head-domain gene and downstream by a heme-oxygenase and a RING finger gene.