Tanaka Tetsuya S, Nishiumi Fumiko, Komiya Tohru, Ikenishi Kohji
Department of Animal Sciences, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, IL, USA.
Int J Dev Biol. 2010;54(8-9):1347-53. doi: 10.1387/ijdb.092862tt.
We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.
我们在其他地方报道过，非洲爪蟾65号雌性的后代正常分裂，但它们的发育在原肠胚形成开始时就停止了，就像美西螈卵缺陷（o）突变体一样，无论与不同的野生型雄性交配情况如何，并且该雌性的卵中缺乏野生型卵中存在的一种酸性38 kDa蛋白质。在当前的研究中，我们首先确定了该蛋白质中一条分离良好的胰蛋白酶肽的部分氨基酸序列（VANLE），该序列在非洲爪蟾的延伸因子1δ（Ef1δ）中被发现，最后通过肽质量谱鉴定该蛋白质为Ef1δ同工型之一，即Ef1δ2。对野生型卵母细胞和胚胎中的Ef1δ2及其紧密同源物Ef1δ1进行的RT-PCR分析表明，这两种转录本都是母源的，并且在整个检测阶段Ef1δ1的含量都比Ef1δ2丰富。重要的是，原肠胚形成后每个胚胎中Ef1δ2转录本的量逐渐减少，这与我们早期研究中报道的每个胚胎中38 kDa蛋白质的逐渐减少一致。由于翻译的药理学抑制会导致野生型胚胎的原肠胚形成停滞，因此可以合理地得出结论，突变胚胎由于缺乏对翻译必不可少的Ef1δ2而在发育中停滞。因此，本研究提供了关于两栖动物原肠胚形成缺陷突变原因的首个分子信息。
