Yamamoto S, Kondo Y, Hanada H, Nakamura M
Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Japan.
J Exp Zool. 1996 Aug 15;275(6):431-43. doi: 10.1002/(SICI)1097-010X(19960815)275:6<431::AID-JEZ5>3.0.CO;2-P.
In the present paper we report the purification of calreticulin (CLT) from livers of the frog, Rana rugosa, the cloning and sequencing of its cDNA, and the CLT gene expression. CLT with M(r) = 52 kDa, estimated by SDS-PAGE, was purified from frog livers. Using rat CLT cDNA as a probe, a 2.4-kilobase frog cDNA clone was isolated from a frog liver cDNA library. The cDNA encoded 419 amino acids including an 18-residue NH2-terminal signal sequence that was 76% homologous to the rat CLT sequence and was 84% homologous to the partial sequence of Xenopus laevis CLT (Treves et al. [1992] Biochem. J. 287:579-581). Phylogenetic relationships estimated from the amino acid sequence of CLTs showed no pronounced variation between the two frog species, R. rugosa and X. laevis. Northern blot analysis indicated that the CLT mRNA level was very high in the liver of tadpoles, but extremely low in adult frogs. Expression levels were also very high in the premature ovary, while moderate expression was observed in the testis and brain of adult frogs. However, there was little histological change in the liver of tadpoles during development. Furthermore, CLT was recognized by Western blot analysis of total proteins in the liver of adult frogs. Immunostaining showed that CLT was distributed in the cytoplasm of liver cells. These results suggest that the expression of the CLT gene is tissue-dependent in the frog, R. rugosa, and that CLT probably functions biochemically in liver cells even when its gene expression is low.
在本论文中，我们报道了从皱皮蛙肝脏中纯化钙网蛋白（CLT）、其cDNA的克隆与测序以及CLT基因表达情况。通过SDS-PAGE估算，从蛙肝脏中纯化出了分子量（M(r)）为52 kDa的CLT。以大鼠CLT cDNA为探针，从蛙肝脏cDNA文库中分离出一个2.4千碱基的蛙cDNA克隆。该cDNA编码419个氨基酸，包括一个18个残基的NH2末端信号序列，其与大鼠CLT序列的同源性为76%，与非洲爪蟾CLT的部分序列同源性为84%（Treves等人[1992]《生物化学杂志》287:579 - 581）。根据CLT氨基酸序列估算的系统发育关系表明，皱皮蛙和非洲爪蟾这两种蛙之间没有明显差异。Northern印迹分析表明，CLT mRNA水平在蝌蚪肝脏中非常高，但在成年蛙中极低。在未成熟卵巢中的表达水平也非常高，而在成年蛙的睾丸和大脑中观察到中等程度的表达。然而，蝌蚪发育过程中肝脏几乎没有组织学变化。此外，通过对成年蛙肝脏总蛋白的Western印迹分析可识别出CLT。免疫染色显示CLT分布在肝细胞的细胞质中。这些结果表明，在皱皮蛙中CLT基因的表达是组织依赖性的，并且即使其基因表达水平较低，CLT可能在肝细胞中仍发挥生化功能。
