Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embryo: Identification by mRNA differential display and the role of DNA replication in zygotic gene activation
Transient expression of translation initiation factor eIF-4C during the 2-cell stage of the preimplantation mouse embryo: Identification by mRNA differential display and the role of DNA replication in zygotic gene activation
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DOI：
10.1006/dbio.1996.0065
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发表时间：
1996-03-15
影响因子：
2.7
通讯作者：
Schultz, RM
中科院分区：
文献类型：
--
作者：
Davis, W;DeSousa, PA;Schultz, RM
合子基因激活（ZGA）肯定发生在小鼠胚胎的 2 细胞阶段。通过二维凝胶电泳对蛋白质合成的分析揭示了一类在双细胞胚胎中表达瞬时增加的基因。尽管生物材料的缺乏阻碍了对这些基因的系统鉴定，但 mRNA 差异显示方法却解决了这个问题。使用这种方法，我们发现被 α-鹅膏蕈碱抑制的翻译起始因子 eIF-4C 的 mRNA 丰度瞬时增加，并且与 eIF-4C 蛋白质合成相对速率的瞬时增加相关。我们通过使用 eIF-4C 特异性引物进行基于逆转录 PCR 的测定，证实了 eIF-4C mRNA 丰度的短暂增加。第一轮 DNA 复制似乎对于 eIF-4C 表达至关重要，因为在 1 细胞胚胎 S 期之前添加 aphidicolin 会抑制 eIF-4C 表达增加的幅度。 Aphidicolin 处理还会抑制 ZGA 公认标记物（转录需要复合物 (TRC)）的合成，该标记物也在 2 细胞阶段短暂表达。在含有 aphidicolin 的培养基中孵育晚期 1 细胞/早期 2 细胞胚胎表明，第二轮 DNA 复制不是 eIF-4C 表达增加所必需的，但 eIF-4C 表达和 TRC 合成减少需要 DNA 复制。然而，eIF-4C 表达的减少不需要胞质分裂或有丝分裂，因为它是在分别存在细胞松弛素 D 或诺考达唑的情况下培养 2 细胞胚胎时发生的。染色质结构的变化可能与 eIF-4C 和 TRC 表达的减少有关，因为当 2 细胞胚胎在 trapoxin（组蛋白脱乙酰酶的特异性且不可逆的抑制剂）中培养时，这两种情况都不会发生减少。这些实验的结果表明，第一轮 DNA 复制相对于 ZGA 是允许的，而第二轮是抑制性的。 (C) 1996 学术出版社
Zygotic gene activation (ZGA) definitely occurs by the 2-cell stage in the mouse embryo. Analysis of protein synthesis by two-dimensional gel electrophoresis reveals a class of genes whose expression transiently increases in the 2-cell embryo. Although the paucity of biological material has prevented a systematic identification of these genes, the mRNA differential display method circumvents this problem. Using this approach we find a transient increase in the mRNA abundance of the translation initiation factor eIF-4C that is inhibited by alpha-amanitin and correlated with a transient increase in the relative rate of protein synthesis for eIF-4C. We confirm the transient increase in eIF-4C mRNA abundance by a reverse transcription-PCR-based assay using eIF-4C-specific primers. The first round of DNA replication seems critical for eIF-4C expression, since addition of aphidicolin prior to S phase in the 1-cell embryo inhibits the magnitude of the increase in eIF-4C expression. Aphidicolin treatment also inhibits the synthesis of an accepted marker for ZGA, the transcription-requiring complex (TRC), which is also transiently expressed during the 2-cell stage. Incubating late 1-cell/early 2-cell embryos in medium containing aphidicolin reveals that the second round of DNA replication is not required for the increase in eIF-4C expression but DNA replication is required for the decrease in both eIF-4C expression and TRC synthesis. The decrease in eIF-4C expression, however, does not require cytokinesis or mitosis, since it occurs when 2-cell embryos are cultured in the presence of cytochalasin D or nocodazole, respectively. Changes in chromatin structure may be involved in the decrease in both eIF-4C and TRC expression, since neither decrease occurs when 2-cell embryos are cultured in trapoxin, which is a specific and irreversible inhibitor of histone deacetylase. Results of these experiments suggest that the first round of DNA replication is permissive with respect to ZGA and that the second round is repressive. (C) 1996 Academic Press, Inc.