RESUMEN
Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over.
Asunto(s)
Intercambio Genético , Recombinación Homóloga/genética , Meiosis/genética , Dominios RING Finger/genética , Animales , Roturas del ADN de Doble Cadena , Drosophila melanogaster/genética , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Dominios Proteicos/genética , Interferencia de ARN , Técnicas del Sistema de Dos Híbridos , Cromosoma X/genéticaRESUMEN
Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). In most organisms only a fraction of DSBs become crossovers. Here we report a novel meiotic gene, vilya, which encodes a protein with homology to Zip3-like proteins shown to determine DSB fate in other organisms. Vilya is required for meiotic DSB formation, perhaps as a consequence of its interaction with the DSB accessory protein Mei-P22, and localizes to those DSB sites that will mature into crossovers. In early pachytene Vilya localizes along the central region of the synaptonemal complex and to discrete foci. The accumulation of Vilya at foci is dependent on DSB formation. Immuno-electron microscopy demonstrates that Vilya is a component of recombination nodules, which mark the sites of crossover formation. Thus Vilya links the mechanism of DSB formation to either the selection of those DSBs that will become crossovers or to the actual process of crossing over.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Intercambio Genético , Roturas del ADN de Doble Cadena , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Meiosis , Animales , Proteínas Cromosómicas no Histona/metabolismo , Microscopía Inmunoelectrónica , Unión ProteicaRESUMEN
In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, we aim to identify its components and to determine how each of these components contributes to SC function. Here, we report the identification of a novel SC component in Drosophila melanogaster female oocytes, which we have named Corolla. Using structured illumination microscopy, we demonstrate that Corolla is a component of the central region of the SC. Consistent with its localization, we show by yeast two-hybrid analysis that Corolla strongly interacts with Cona, a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complejo Sinaptonémico/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Datos de Secuencia Molecular , Oocitos/metabolismo , Unión ProteicaRESUMEN
Homologous recombination in meiosis is initiated by the programmed induction of double strand breaks (DSBs). Although the Drosophila Spo11 ortholog Mei-W68 is required for the induction of DSBs during meiotic prophase, only one other protein (Mei-P22) has been shown to be required for Mei-W68 to exert this function. We show here that the chromatin-associated protein Trade Embargo (Trem), a C2H2 zinc finger protein, is required to localize Mei-P22 to discrete foci on meiotic chromosomes, and thus to promote the formation of DSBs, making Trem the earliest known function in the process of DSB formation in Drosophila oocytes. We speculate that Trem may act by either directing the binding of Mei-P22 to preferred sites of DSB formation or by altering chromatin structure in a manner that allows Mei-P22 to form foci.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiosis , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Reparación del ADN , Proteínas de Drosophila/química , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Alineación de SecuenciaRESUMEN
The synaptonemal complex (SC) is an intricate structure that forms between homologous chromosomes early during the meiotic prophase, where it mediates homolog pairing interactions and promotes the formation of genetic exchanges. In Drosophila melanogaster, C(3)G protein forms the transverse filaments (TFs) of the SC. The N termini of C(3)G homodimers localize to the Central Element (CE) of the SC, while the C-termini of C(3)G connect the TFs to the chromosomes via associations with the axial elements/lateral elements (AEs/LEs) of the SC. Here, we show that the Drosophila protein Corona (CONA) co-localizes with C(3)G in a mutually dependent fashion and is required for the polymerization of C(3)G into mature thread-like structures, in the context both of paired homologous chromosomes and of C(3)G polycomplexes that lack AEs/LEs. Although AEs assemble in cona oocytes, they exhibit defects that are characteristic of c(3)G mutant oocytes, including failure of AE alignment and synapsis. These results demonstrate that CONA, which does not contain a coiled coil domain, is required for the stable 'zippering' of TFs to form the central region of the Drosophila SC. We speculate that CONA's role in SC formation may be similar to that of the mammalian CE proteins SYCE2 and TEX12. However, the observation that AE alignment and pairing occurs in Tex12 and Syce2 mutant meiocytes but not in cona oocytes suggests that the SC plays a more critical role in the stable association of homologs in Drosophila than it does in mammalian cells.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Oocitos/química , Oocitos/metabolismo , Complejo Sinaptonémico/química , Animales , Cromosomas/metabolismo , Proteínas de Drosophila/análisis , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiosis , Transporte de Proteínas , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismoRESUMEN
Using an FLP/FRT-based method to create germline clones, we screened Drosophila chromosome arms 2L and 3R for new female meiotic mutants. The screen was designed to recover mutants with severe effects on meiotic exchange and/or segregation. This screen yielded 11 new mutants, including six alleles of previously known meiotic genes (c(2)M and ald/mps1). The remaining five mutants appear to define at least four new genes whose ablation results in severe meiotic defects. Three of the novel meiotic mutants were identified at the molecular level. Two of these, mcm5(A7) and trem(F9), define roles in meiotic recombination, while a third, cona(A12), is important for synaptonemal complex assembly. Surprisingly, five of the nine mutants for which the lesion has been identified at the molecular level are not the result of mutations characteristic of EMS mutagenesis, but rather due to the insertion of the transposable element Doc. This study demonstrates the utility of germline clone-based screens for the discovery of strong meiotic mutants, including mutations in essential genes, and the use of molecular genetic techniques to map the loci.