Suzuki K, Sato K, Katsu K, Hayashita H, Kristensen D B, Yoshizato K
Department of Biological Science, Graduate School of Science, Hiroshima University, Higashihiroshima, Japan.
Differentiation. 2001 Aug;68(1):44-54. doi: 10.1046/j.1432-0436.2001.068001044.x.
The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.
利用新克隆的牛蛙角蛋白cDNA作为探针，研究了牛蛙（Rana catesbeiana）蝌蚪变态过程中幼虫表皮向成体表皮的转化。使用针对非洲爪蟾幼虫角蛋白（XLK）的高度特异性抗血清克隆了牛蛙幼虫角蛋白（RLK）cDNA（rlk）。牛蛙蝌蚪的尾部皮肤蛋白通过二维凝胶电泳分离，并用抗XLK抗血清进行蛋白质印迹分析。通过这种方法检测到的牛蛙抗原被测序并鉴定为II型角蛋白。我们利用根据RLK的这些肽序列设计的引物，通过PCR从蝌蚪皮肤中克隆了rlk。rlk核苷酸序列预测的RLK是一种549个氨基酸长的II型角蛋白。牛蛙蝌蚪身体皮肤和尾部皮肤之间的消减克隆产生了牛蛙成体角蛋白（RAK）的cDNA（rak）。RAK是一种433个氨基酸长的I型角蛋白。我们还从牛蛙蝌蚪表皮中克隆了牛蛙角蛋白8（RK8）cDNA（rk8）。RK8有502个氨基酸长，与细胞角蛋白8同源。Northern印迹分析和原位杂交实验表明，rlk在称为丝状细胞的幼虫特异性表皮细胞中通过变态前期活跃表达，并在变态高潮期和成年皮肤中完全失活。RAK mRNA在蝌蚪表皮的基底细胞和成年表皮的生发细胞中表达。rlk和rak的表达分别受到甲状腺激素（TH）的下调和上调。相比之下，在自发和TH诱导的变态过程中，RK8的表达没有变化。RK8 mRNA仅在幼虫表皮的顶端细胞中表达。这些角蛋白基因表达模式表明，TH对角蛋白基因表达的调控因幼虫表皮细胞类型而异。本研究证明了这些基因在研究胚胎后表皮发育和分化分子机制方面的有用性。
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