Davis W, De Sousa P A, Schultz R M
Department of Biology, University of Pennsylvania, Philadelphia, 19104-6018, USA.
Dev Biol. 1996 Mar 15;174(2):190-201. doi: 10.1006/dbio.1996.0065.
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.
合子基因激活（ZGA）在小鼠胚胎的2细胞阶段肯定会发生。通过二维凝胶电泳分析蛋白质合成，发现一类基因的表达在2细胞胚胎中短暂增加。尽管生物材料的匮乏阻碍了对这些基因的系统鉴定，但mRNA差异显示方法解决了这个问题。使用这种方法，我们发现翻译起始因子eIF-4C的mRNA丰度短暂增加，这种增加被α-鹅膏蕈碱抑制，并且与eIF-4C蛋白质合成的相对速率的短暂增加相关。我们使用eIF-4C特异性引物通过基于逆转录PCR的检测方法证实了eIF-4C mRNA丰度的短暂增加。第一轮DNA复制似乎对eIF-4C的表达至关重要，因为在1细胞胚胎的S期之前添加阿非迪霉素会抑制eIF-4C表达增加的幅度。阿非迪霉素处理还会抑制一种公认的ZGA标志物——转录所需复合物（TRC）的合成，TRC在2细胞阶段也会短暂表达。在含有阿非迪霉素的培养基中培养晚期1细胞/早期2细胞胚胎表明，第二轮DNA复制对于eIF-4C表达的增加不是必需的，但DNA复制对于eIF-4C表达和TRC合成的减少是必需的。然而，eIF-4C表达的减少不需要胞质分裂或有丝分裂，因为当2细胞胚胎分别在细胞松弛素D或诺考达唑存在的情况下培养时就会发生这种减少。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细胞胚胎在曲古抑菌素（一种组蛋白脱乙酰酶的特异性不可逆抑制剂）中培养时，这两种减少都不会发生。这些实验结果表明，第一轮DNA复制对ZGA是允许的，而第二轮是抑制性的。
