RESUMEN
The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. Here, we discover that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, we detect that both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, we show that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm.
Asunto(s)
Células Germinativas/metabolismo , Animales , Citoplasma/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Genes Reporteros , Hidrogel de Polietilenoglicol-Dimetacrilato , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Biológicos , Oocitos/metabolismo , Proteínas de Unión al ARN/metabolismo , Xenopus , Pez CebraRESUMEN
Protein microarrays have emerged as an indispensable research tool for providing information about protein functions and interactions through high-throughput screening. Traditional methods for immobilizing biomolecules onto solid surfaces have been based on covalent and noncovalent binding, entrapment in semipermeable membranes, microencapsulation, sol gel, and hydrogel methods. Each of these techniques has its own strengths but fails to combine the most important tenets of a functional protein microarray such as covalent attachment, native protein conformation, homogeneity of the protein monolayer, control over active site orientation, and retention of protein activity. Here we present a selective and site-directed covalent immobilization technique for proteins via a benzoxazine ring formation through a Diels-Alder reaction in water and a genetically encoded 3-amino-L-tyrosine (3-NH(2)Tyr) amino acid. Fully functional protein microarrays, with monolayer arrangements and complete control over their orientations, were generated using this strategy.