RESUMO
We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/fisiologia , Fuso Acromático/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação , Cromatina/genética , Cromatina/fisiologia , Proteínas Cromossômicas não Histona , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/genética , Quinase 3 da Glicogênio Sintase , Cinetocoros , Mutagênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologiaRESUMO
A preparation of decapitated Drosophila melanogaster has been used for direct application of drugs to the nerve cord. Serotonin, dopamine, and octopamine stimulate locomotion and grooming, showing distinguishable effects that often are potentiated by addition of the vertebrate monoamine oxidase-inhibitor hydrazaline. Many of the hydrazaline-induced effects are sexually dimorphic, with males showing greater responses than females. Behaviors similar to those induced by dopamine can be induced by application of the vertebrate dopamine D2-like receptor agonist quinpirole, whose effects are also sexually dimorphic. In contrast, vertebrate D2-like and D1-like dopamine antagonists result in akinesic states, and D1-like agonists selectively stimulate grooming. These data indicate that Drosophila nerve cord amine receptors are coupled to reflexive behaviors similar to those stimulated by brain dopamine receptors in vertebrates.
