Identifying the molecular basis of the maternal to zygotic transition

确定母体向合子转变的分子基础

• 批准号: 8508726
• 负责人: Jan M Skotheim
• 金额: $ 23.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508726
• 关键词: Adult; Binding; Biochemical; Biological Assay; Cell Count; Cell Cycle; Cell Cycle Checkpoint; Cell Nucleus; Cell division; Cell-Free System; Cells; Chromatin; Cytoplasm; DNA; DNA biosynthesis; Defect; Development; Embryo; Epigenetic Process; Fertilization; Fishes; Gene Expression; Genetic Transcription; Genome; Growth; Health; Human; Modeling; Molecular; Nature; Nuclear; Organism; Phase; Process; Rana; Sperm Count Procedure; Staging; System; Titrations; Transcriptional Activation; Transcriptional Regulation; Work; Xenopus; Xenopus laevis; base; blastocyst; cell growth; cell motility; egg; flexibility; fly; gene repression; human disease; in vivo; insight; public health relevance; synchronous cell division; zygote

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. )

描述（由申请人提供）：从单个受精卵发育成成体多细胞生物体需要大量细胞的增殖和分化。在许多物种中，早期受精后分裂快速而同步地发生，没有生长阶段和细胞周期检查点。这些早期胚胎几乎完全没有转录活性，因此由母体提供的RNA驱动。在囊胚中期转换（MBT），胚胎启动合子基因组的大规模转录，细胞获得生长阶段和检查点。先前的工作表明，MBT是由DNA与细胞质的比例增加引起的，这是由于重复的DNA复制和细胞分裂而没有细胞生长。这导致了这样的假设，即胚胎中存在的抑制因子的逐步滴定允许合子转录的启动。为了了解这种抑制活性的性质，我们复制了早期的研究，这是在完整的胚胎中进行的，在非洲爪蟾卵提取物中，并显示出类似的DNA浓度依赖性转录抑制。使用这种无细胞系统，我们已经表明，添加DNA包被的珠提取物降低了阈值DNA浓度的转录激活，表明DNA珠滴定的抑制活性。DNA包被的珠在卵提取物中的预孵育降低了当珠被添加到初始提取物中时珠诱导转录的能力。因此，我们现在能够生化测定和量化抑制活性。使用这种检测方法，我们建议从非洲爪蟾卵提取物中生物化学纯化抑制因子（），并了解该因子如何在发育过程中促进转录诱导。)