RESUMO
A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.
Assuntos
Dictyostelium/química , Proteínas dos Microfilamentos/química , Fator 1 de Elongação de Peptídeos/química , Actinas/química , Actinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Clonagem Molecular , Citoesqueleto/química , Dictyostelium/genética , Biblioteca Gênica , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/imunologia , Mapeamento por Restrição , Alinhamento de SequênciaRESUMO
For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl(Pe) switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Processos de Determinação Sexual , Fatores de Transcrição/genética , Cromossomo X , Motivos de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Células Cultivadas , Sequência Conservada , Pegada de DNA , Análise Mutacional de DNA , Desoxirribonuclease I/metabolismo , Evolução Molecular , Feminino , Deleção de Genes , Sequências Hélice-Alça-Hélice , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição da Família Snail , Transcrição Gênica , Transgenes , beta-Galactosidase/metabolismoRESUMO
The choice of sexual identity in Drosophila is determined by a system that measures the X chromosome to autosome ratio (X/A). This system depends upon unequal expression of X-linked numerator genes in 1X and 2X nuclei. The numerators activate a special Sxl promoter, Sxl-Pe, in 2X/2A nuclei, but not 1X/2A nuclei. By multimerizing a conserved Sxl-Pe sequence block, we generated a gain-of-function promoter, Sxl-PeGOF, that is inappropriately active in 1X/2A nuclei. GOF activity requires the X-linked unpaired (upd) gene, which encodes a ligand for the Drosophila JAK/STAT signaling pathway. upd also functions as a numerator element in regulating wild-type Sxl-Pe reporters. We demonstrate that the JAK kinase, Hopscotch, and the STAT DNA-binding protein, Marelle, are also required for Sxl-Pe activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Proteínas Quinases/metabolismo , Processos de Determinação Sexual , Transativadores/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência Conservada , Drosophila melanogaster/genética , Feminino , Glicoproteínas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Janus Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Aminoácidos , Transdução de SinaisRESUMO
Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.
