RESUMO
Alternate forms of the general transcription machinery have been described in several tissues or cell types. However, the role of tissue-specific TBP-associated factors (TAF(II)s) and other tissue-specific transcription components in regulating differential gene expression during development was not clear. Here we show that the cannonball gene of Drosophila encodes a cell type-specific homolog of a more ubiquitously expressed component of the general transcription factor TFIID. cannonball is required in vivo for high level transcription of a set of stage- and tissue-specific target genes during male gametogenesis. Regulation of transcription by cannonball is absolutely required for spermatogenesis, as null mutations block meiotic cell cycle progression and result in a complete failure of spermatid differentiation. Our results demonstrate that cell type-specific TAF(II)s play an important role in developmental regulation of gene expression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Retroviridae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Alelos , Sequência de Aminoácidos , Animais , Northern Blotting , Ciclo Celular/genética , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Drosophila/genética , Proteínas de Drosophila , Genes Reporter , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Isoformas de Proteínas , Proteínas dos Retroviridae/química , Homologia de Sequência de Aminoácidos , Espermatogênese/genética , Espermatogênese/fisiologia , Transativadores/química , Fator de Transcrição TFIID , Fatores de Transcrição/fisiologia , Fatores de Transcrição TFII/genéticaRESUMO
We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.
Assuntos
Proteínas de Ligação a Calmodulina/fisiologia , Proteínas Fúngicas/genética , Genes Fúngicos , Fosfoproteínas Fosfatases/fisiologia , ATPases Translocadoras de Prótons/deficiência , Saccharomyces cerevisiae/genética , Vacúolos/enzimologia , Sequência de Bases , Calcineurina , Proteínas de Ligação a Calmodulina/antagonistas & inibidores , Ciclosporina/farmacologia , Proteínas Fúngicas/fisiologia , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/fisiologia , Tacrolimo/farmacologiaRESUMO
The yeast KAR1 gene is essential for mitotic growth and important for nuclear fusion. Mutations in KAR1 prevent duplication of the spindle pole body (SPB), and affect functions associated with both the nuclear and cytoplasmic microtubules. The localization of hybrid Kar1-lacZ proteins, described elsewhere (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press), suggest that the protein is associated with the SPB. In this paper, we report a deletion analysis demonstrating that the mitotic and karyogamy functions of KAR1 are separate and independent, residing in discrete functional domains. One region, here shown to be essential for mitosis, coincided with a part of the protein that is both necessary and sufficient to target Karl-lacZ hybrid proteins to the SPB (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Complementation testing demonstrated that deletions in this interval did not affect nuclear fusion. A second region, required only for karyogamy, was necessary for the localization of a Kar3-lacZ hybrid protein to the SPB. These data suggest a model for the roles of Kar1p and Kar3p, a kinesin-like protein, in nuclear fusion. Finally, a third region of KAR1 was found to be important for both mitosis and karyogamy. This domain included the hydrophobic carboxy terminus and is sufficient to target a lacZ-Kar1 hybrid protein to the nuclear envelope (Vallen E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Altogether, the essential mitotic regions of KAR1 comprised 20% of the coding sequence. We propose a model for Kar1p in which the protein is composed of several protein-binding domains tethered to the nuclear envelope via its hydrophobic tail.
