Identifying the molecular basis of the maternal to zygotic transition

描述(申请人提供)：从单个受精卵发育成成年多细胞生物体需要大量细胞的增殖和分化。在许多物种中，受精后的早期分裂迅速而同步地发生，没有生长阶段和细胞周期检查点。这些早期胚胎在转录上几乎完全不活跃，因此由母体提供的RNA驱动。在中期-囊胚期，胚胎启动合子基因组的大规模转录，细胞获得生长期和检查点。以前的工作表明，MBT是由重复几轮DNA复制和细胞分裂而不是细胞生长导致的DNA与细胞质比例的增加启动的。这导致了一种假设，即胚胎中存在的抑制因子的渐进滴定允许合子转录的启动。为了了解这种抑制活性的性质，我们在非洲爪哇卵提取液中复制了在完整胚胎中进行的早期研究，并显示了类似的DNA浓度依赖的转录抑制。利用这个无细胞体系，我们发现在提取物中加入包被DNA的小球可以降低转录激活的DNA浓度阈值，这表明DNA小球可以滴定抑制活性。在鸡蛋提取物中预先孵育DNA包裹的珠子，当珠子被添加到幼稚的提取物中时，会降低珠子诱导转录的能力。因此，我们现在能够对抑制活性进行生化检测和量化。利用这一方法，我们建议从非洲爪哇卵提取物中生化纯化抑制因子()，并了解该因子(S)在发育过程中如何在转录诱导中发挥作用。)

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. )