嗜热四膜虫Tcd1蛋白功能结构域的突变分析

有性生殖过程特异表达的Tcd1在四膜虫大核基因组重排和修复中起到重要调节作用, Tcd1含有进化中保守的chromodomain(CD)结构域以及chromo shadow domain (CSD)结构域, 然而不同结构域的具体功能并不清楚。本研究首先鉴定了TCD1基因仅含有1个CD结构域的选择性剪切本TCD1β，免疫荧光定位表明,Tcd1β定位在胞质中。定点突变Tcd1中CD1内159位色氨酸为丙氨酸, Tcd1W159A点状定位在亲本大核,然后转移到新发育的大核上围绕核膜致密分布,发育的晚期逐步消失。进一步突变CD2中437位的色氨酸为丙氨酸后，Tcd1W159AW437A在早期亲本大核形成异常的环状分布, 而在新发育大核中形成点状分布。截短CSD结构域C端35个氨基酸后, Tcd1Δ35在亲本大核和新大核上的定位不受影响。 然而，截短CSD结构域C端的53个氨基酸后, Tcd1Δ53定位在细胞质中, 无核内定位。结果表明, Tcd1中的CD1和CD2结构域决定了Tcd1蛋白在核内的分布, CSD结构域决定了Tcd1入核转运, Tcd1的3个功能结构域共同决定了Tcd1在四膜虫中的功能定位。

Mutational Analysis of Functional Domain of Tcd1 from Tetrahymena thermophila

Specificity of Tcd1 expression is required for the genome wide DNA rearrangement and repair during macronuclear differentiation of Tetrahymena thermophila. Tcd1 contains evolutionally conserved chromo domain (CD) and chromo shadow domain (CSD), however, the functions were not clear. In this study, we identified a TCD1 alternative splice variant TCD1β, which contains only one CD domain. Immuno-fluorescence staining showed that Tcd1β localized in the cytoplasm. When conserved tryptophan 159 in the CD1 domain was mutated to alanine (Tcd1W159A), it formed dark stained puncta in the parental macronucleus (Mac), and then disappeared within the new Mac at late conjugation stage. If tryptophan 437 in the CD2 domain was further mutated into alanine (Tcd1W159AW437A), it formed abnormal ring structure in the parental Mac, and dot-like signals in the conjugated new Mac. Tcd1Δ35 (35 amino acids removed from the C-terminus) localized in the cytoplasm instead of in the parental Mac and new Mac. These results implied that in Tetrahymena thermophila, the CD domains of Tcd1 determined its localization and the CSD domain was required for nuclear import.