TRANSLATIONAL CONTROL OF GENE EXPRESSION BY MRNA

在海胆卵的卵子发生和成熟过程中， mRNA的储存积累起来，以供早期使用。 胚胎发生 储存的母体mRNA的利用发生在 在受精前的低水平。 受精后，戏剧性的 mRNA的可用性和募集发生变化， 在早期，许多蛋白质的合成速率迅速增加， 乳沟 变化已记录在两个总比率， 母体mRNA的利用和在选择特定的 不同发育阶段的mRNA种类。 我们以前的 研究已经允许无细胞翻译的发展， 从卵子和胚胎中提取的系统， 在完整系统中观察到的差异。 本研究提出利用这些无细胞系统， 结合其他方法，以识别细胞 参与利用母体mRNA的组分。 使用 在无细胞系统中，eIF-4F活性的抑制剂已经被 在未受精卵中发现， 受精后2-3小时内的活性。 身份和 将表征eIF-4F抑制活性的性质。 这种活动抑制启动的机制 序列也将被研究。 的方式 抑制剂在受精后失活的可能性将被探索。 到 作为对这些研究的补充， 发育调控的mRNA相互作用蛋白将是 采用蛋白质印迹和体外结合 技术，在体外合成加帽的32 P标记的mRNA。

During oogenesis and maturation of the sea urchin egg, large stores of mRNA are accumulated for subsequent use during early embryogenesis. Utilization of stored maternal mRNA occurs at a very low rate prior to fertilization. After fertilization, dramatic changes in mRNA availability and recruitment occur, allowing rapid increases in the synthetic rate of many proteins during early cleavage. Changes have been documented in both overall rates of maternal mRNA utilization and in the selection of particular mRNA species at different developmental stages. Our previous studies have allowed the development of cell-free translation systems from eggs and embryos that reproduce the overall differences observed in the intact system. This investigation proposes to utilize these cell-free systems, in conjunction with other approaches, to identify the cellular components involved in the utilization of maternal mRNA. Using the cell-free systems an inhibitor of eIF-4F activity has been discovered in the unfertilized egg, that decreases gradually in activity over the first 2-3 hr after fertilization. The identity and nature of the eIF-4F inhibitory activity will be characterized. The mechanism by which this activity inhibits the initiation sequence will also be investigated. The manner in which the inhibitor is inactivated after fertilization will be explored. To complement these studies, a systematic search for developmentally regulated mRNA-interactive proteins will be undertaken, using a protein blotting and in vitro binding technique, with capped 32p-labelled mRNA's synthesized in vitro.