RESUMO
The early cell cycles of Drosophila embryogenesis involve rapid oscillations between S phase and mitosis. These unique S-M cycles are driven by maternal stockpiles of components necessary for DNA replication and mitosis. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) are required to control the cell cycle specifically at the onset of Drosophila development by inhibiting DNA replication and promoting mitosis. PNG is a protein kinase that is in a complex with PLU. We employed a sensitized png mutant phenotype to screen for genes that when reduced in dosage would dominantly suppress or enhance png. We screened deficiencies covering over 50% of the autosomes and identified both enhancers and suppressors. Mutations in eIF-5A and PP1 87B dominantly suppress png. Cyclin B was shown to be a key PNG target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. The plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein.
Assuntos
Ciclina B/metabolismo , Proteínas de Drosophila , Drosophila/enzimologia , Drosophila/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Ligação a RNA , Supressão Genética , Animais , Ciclo Celular , Núcleo Celular/metabolismo , Cruzamentos Genéticos , Genótipo , Temperatura Alta , Immunoblotting , Mutação , Fatores de Iniciação de Peptídeos/genética , Fenótipo , Biossíntese de Proteínas , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Following completion of meiosis, DNA replication must be repressed until fertilization. In Drosophila, this replication block requires the products of the pan gu (png), plutonium (plu) and giant nuclei (gnu) genes. These genes also ensure that S phase oscillates with mitosis in the early division cycles of the embryo. We have identified the png gene and shown that it encodes a Ser/Thr protein kinase expressed only in ovaries and early embryos, and that the predicted extent of kinase activity in png mutants inversely correlates with the severity of the mutant phenotypes. The PLU and PNG proteins form a complex that has PNG-dependent kinase activity, and this activity is necessary for normal levels of mitotic cyclins. Our results reveal a novel protein kinase complex that controls S phase at the onset of development apparently by stabilizing mitotic cyclins.
Assuntos
Proteínas de Drosophila , Drosophila/citologia , Drosophila/enzimologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fase S/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Ciclinas/metabolismo , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Feminino , Genes de Insetos , Masculino , Dados de Sequência Molecular , Mutação , Filogenia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Mapeamento por RestriçãoRESUMO
The Drosophila brahma (brm) gene encodes an activator of homeotic genes related to the yeast chromatin remodeling factor SWI2/SNF2. Here, we report the phenotype of null and dominant-negative brm mutations. Using mosaic analysis, we found that the complete loss of brm function decreases cell viability and causes defects in the peripheral nervous system of the adult. A dominant-negative brm mutation was generated by replacing a conserved lysine in the ATP-binding site of the BRM protein with an arginine. This mutation eliminates brm function in vivo but does not affect assembly of the 2-MD BRM complex. Expression of the dominant-negative BRM protein caused peripheral nervous system defects, homeotic transformations, and decreased viability. Consistent with these findings, the BRM protein is expressed at relatively high levels in nuclei throughout the developing organism. Site-directed mutagenesis was used to investigate the functions of conserved regions of the BRM protein. Domain II is essential for brm function and is required for the assembly or stability of the BRM complex. In spite of its conservation in numerous eukaryotic regulatory proteins, the deletion of the bromodomain of the BRM protein has no discernible phenotype.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Drosophila/genética , Proteínas de Insetos/genética , Proteínas Nucleares , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteínas de Ligação a DNA/química , Drosophila/embriologia , Proteínas de Drosophila , Proteínas de Insetos/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transativadores/química , Fatores de Transcrição/químicaRESUMO
Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.
Assuntos
Ciclo Celular/genética , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila/embriologia , Embrião não Mamífero/fisiologia , Proteínas de Insetos/fisiologia , Fatores de Transcrição/fisiologia , Animais , Núcleo Celular/metabolismo , Drosophila/genética , Olho/embriologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Insetos/genética , Masculino , Meiose , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenômenos Fisiológicos Oculares , Óvulo/metabolismo , Proteína Fosfatase 1 , Proteínas/genética , Proteínas/metabolismo , Fatores de Transcrição/genéticaRESUMO
The Drosophila brahma (brm) gene encodes an activator of homeotic genes that is highly related to the yeast transcriptional activator SWI2 (SNF2), a potential helicase. To determine whether brm is a functional homolog of SWI2 or merely a member of a family of SWI2-related genes, we searched for additional Drosophila genes related to SWI2 and examined their function in yeast cells. In addition to brm, we identified one other Drosophila relative of SWI2: the closely related ISWI gene. The 1,027-residue ISWI protein contains the DNA-dependent ATPase domain characteristic of the SWI2 protein family but lacks the three other domains common to brm and SWI2. In contrast, the ISWI protein is highly related (70% identical) to the human hSNF2L protein over its entire length, suggesting that they may be functional homologs. The DNA-dependent ATPase domains of brm and SWI2, but not ISWI, are functionally interchangeable; a chimeric SWI2-brm protein partially rescued the slow growth of swi2- cells and supported transcriptional activation mediated by the glucocorticoid receptor in vivo in yeast cells. These findings indicate that brm is the closest Drosophila relative of SWI2 and suggest that brm and SWI2 play similar roles in transcriptional activation.
