Identifying the molecular basis of the maternal to zygotic transition

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DESCRIPTION (provided by applicant): The development of an adult multi-cellular organism from a single fertilized egg requires the proliferation and differentiation of a large number of cells. In many species, the early post-fertilization divisions occur rapidly and synchronously without growth phases and cell cycle checkpoints. These early embryos are almost entirely transcriptionally inactive and therefore driven by maternally supplied RNAs. At the Mid-Blastula Transition (MBT), the embryo initiates large-scale transcription from the zygotic genome and cells gain growth phases and checkpoints. Previous work suggested that the MBT is initiated by the increased DNA-to-cytoplasmic ratio resulting from repeated rounds of DNA replication and cell division without cell growth. This led to the hypothesis that the progressive titration of an inhibitory factor present in the embryo allows the initiation of zygotic transcription. To understand the nature of this inhibitory activity we replicated early studies, which had been performed in intact embryos, in Xenopus laevis egg extracts and showed similar DNA concentration dependent transcriptional repression. Using this cell free system we have shown that adding DNA-coated beads to extracts lowers the threshold DNA concentration for transcriptional activation indicating that DNA beads titrate the inhibitory activity. Pre-incubatio of DNA-coated beads in egg extract reduces the beads' ability to induce transcription when the beads are added to a naive extract. Thus we are now able to biochemically assay and quantify the inhibitory activity. Using this assay we propose to biochemically purify the inhibitory factor() from Xenopus egg extract and to understand how the factor(s) contributes to transcriptional induction during development. )