Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide.
Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide.
复制标题
表达人 U1 小核核糖核蛋白 C 多肽的互补 DNA 的分离和表征。
作者：
K. Yamamoto;H. Miura;Y. Moroi;S. Yoshinoya;M. Goto;K. Nishioka;T. Miyamoto
用含有高滴度抗核糖核蛋白(RNP)抗体的患者血清筛选后，从噬菌体表达载体lambda gt11中的人成纤维细胞c DNA文库中克隆到一段互补DNA，命名为PS2。通过将PS2亚克隆到pEX-2表达载体中，获得了适量的cro-β-半乳糖苷酶融合蛋白(PS2EX)。用Sepharose4B与pS2EX偶联，从患者血清中纯化出抗pS2EX抗体。用抗pS2EX抗体对HeLa细胞进行免疫荧光染色，间期细胞核呈典型的斑点状。在免疫印迹分析中，抗pS2EX抗体识别了22 kDa的蛋白。细胞裂解物的免疫沉淀和随后的RNA分析表明，抗pS2EX抗体仅沉淀U1RNP。各种抗RNP血清对pS2EX的反应性与免疫印迹中C多肽的阳性反应有很好的相关性。这些结果表明，PS2是U1SnRNP的C多肽的cDNA。在用人RNA和放射性标记的PS2进行的Northern杂交中，观察到一条约800碱基的单一条带。PS2的核苷酸序列与已知蛋白质没有明显的同源性。
A cloned complementary DNA, termed pS2, was isolated from a human fibroblast cDNA library in the bacteriophage expression vector lambda gt11 after screening with a patient's serum containing a high titer of anti-ribonucleoprotein (RNP) antibodies. A reasonable amount of cro-beta-galactosidase fusion protein (pS2EX) was obtained through subcloning of the pS2 insert into a plasmid expression vector pEX-2. Antibody against pS2EX (anti-pS2EX) was purified from this patient's serum by Sepharose 4B conjugated with pS2EX. Immunofluorescent staining of HeLa cells with anti-pS2EX antibody exhibited a typical speckled pattern in the interphase nuclei. In the immunoblot analysis, the anti-pS2EX antibody recognized the 22 kDa protein. Using immunoprecipitation of cell lysate and subsequent RNA analysis, anti-pS2EX antibody was shown to precipitate U1 RNP only. The reactivities of various anti-RNP sera to pS2EX correlated well with the positive reaction to C polypeptide in the immunoblot. These findings indicate that pS2 is a cDNA for C polypeptide of U1 snRNP. In the Northern blot using human RNA and radiolabeled pS2, a single band about 800 base was observed. The nucleotide sequence of pS2 showed no significant homologies to known proteins.