嗜热四膜虫染色体浓缩调节因子(RCC1)的定位及功能

Localization and Function of Regulator of Chromosome Condensation 1 from Tetrahymena thermophila

真核细胞中染色体浓缩调节因子(regulator of chromosome condensation 1,RCC1)是RanGTPase唯一的鸟嘌呤核苷酸交换因子.染色质结合的RCC1和RanGTPase相互作用,催化细胞核内RanGDP向RanGTP的转化,进而调控了核质间的定向运送、有丝分裂期纺锤体的组装以及核膜的形成.本实验从原生生物嗜热四膜虫大核基因组中鉴定了1个新的RCC1(TTHERM_00530380)基因.该基因全长2 541 bp,包含2个内含子序列,开放阅读框为2 181 bp,编码726个氨基酸.实时荧光定量PCR表明,RCC1在四膜虫营养生长、饥饿以及有性生殖时期都有表达,且在有性生殖转录水平达到最高.免疫荧光定位分析表明,HA-RCC1在营养生长和饥饿时期,定位于大核和小核中;在有性生殖时期,定位于亲本大核、减数分裂的小核、新生成的大核和凋亡的大核中.过表达RCC1导致大核的无丝分裂异常,细胞增殖变慢,最终产生无大核的后代细胞.敲减RCC1导致了多小核的产生.结果表明,RCC1参与调控了四膜虫细胞核的分裂,RCC1的正常表达对核分裂以及细胞增殖起到重要的调控作用.

In eukaryotic cell, regulator of chromosome condensation 1 （RCC1） is unique guanine nucleotide exchange factor for the nuclear GTP binding protein RanGTPase. RCC1 binds to chromatin and interacts with RanGTPase to promote the loss of bound GDP and the uptake of fresh GTP. The interaction of RCC1 with Ran regulates nuclear transport and spindle assembly during mitosis, as well as nuclear envelope formation. In this study, we identified a homolog of RCC1 （TTHERM_O0530380） gene from Tetrahymena thermophila. The RCC1 sequence analysis revealed that the gene length was 2541 bp and interrupted by two introns. The open reading frame was 2181 bp and theoretically encoded a protein of 726 amino acids. Real-time PCR showed that RCC1 was expressed in the growing and starvation stage. Furthermore, RCC1 showed a higher expression level during conjugation stage. Immuno-fluorescence staining showed that HA-RCC1 was localized in the macronucleus and micronucleus during growing and starvation stage; HA-RCC1 was localized in the parental macronucleus, meiotic micronuclei, new macronuclei and apoptotic macronucleus during conjugation stage. Over-expression of RCC1 leaded toabnormal macronuclear amitosis, affected cell proliferation and produced amacronuclear progeny. Knockdown of RCC1 leaded to a multi - micronuclear cell. These results showed that RCC1 was involved in nuclear division and its normal expression plays an important role for nuclear division and cellular proliferation in T. thermophila.