嗜热四膜虫中Chromodomain蛋白Tcd3和Tcd4的表达调控与功能分析
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摘要
Chromodomain (chromatin organization modifier domain)是一个由30～50个氨基酸组成的保守的蛋白质结构域,含有Chromodomain的蛋白质广泛存在于多种生物中的染色质结合蛋白中,通过与组蛋白或RNA的作用参与了异染色质的形成及基因的表达调控。
四膜虫中程序性全基因组重排在有性生殖过程中发生,siRNA, MeH3K9, Pdd1p和Pdd3p参与这一过程,然而其详细机制仍不清楚。本研究从四膜虫基因组中筛选到两个含有Chromodomain的基因TCD3(Tetrahymena Chromo Domain 3)与TCD4(Tetrahymena Chromo Domain 4),对这两个基因的表达调控和细胞定位进行了研究,获得的主要结果如下：
1.TCD3和TCD4序列分析通过生物信息学的方法,从四膜虫大核基因数据库鉴定了TCD3 (TTHERM_00585180)和TCD4 (TTHERM_00585190),两个基因串联于同一条大核染色体上,间隔237bp,两个基因内均具有GT-AG的内含子。TCD3和TCD4基因拟编码的蛋白334个和344个氨基酸残基组成,分子量分别为40.26kDa和41.11kDa,等电点(pI)分别为4.590和5.435。全基因表达Microarray分析表明TCD3和TCD4在四膜虫营养生殖阶段和饥饿期不表达,在有性生殖过程中8h时表达量最高,并且两者的表达谱一致。
2.TCD3和TCD4的5'和3'-RACE TCD3和TCD4的ORF分别为1005bp和1035bp,大核染色体中基因全长分别为1241bp和1429bp,各含有两个内含子。5’RACE和3'RACE表明TCD3和TCD4的5’端非编码区(5'-UTR)长度分别为40bp和43bp,3'端非编码区(3’-UTR)长度分别为90bp和189bp。Chromodomain聚类分析表明Tcd3和Tcd4与pddlp蛋白的Chromodomain最为相似,SWISS—MODEL预测发现Tcd4的Chromodomain缺失α-螺旋结构。
3.Tcd3和Tcd4的免疫荧光定位构建含有HA标签的表达载体OE-PBX-TCD3和OE-PBX-TCD4,转化四膜虫CU428和B2086细胞株,通过巴龙霉素筛选,PCR鉴定,获得HA-Tcd3和HA-Tcd4的过表达细胞株。激光共聚焦显微镜对HA-Tcd3和HA-Tcd4的免疫荧光定位发现：HA-Tcd3定位于四膜虫有性生殖核发育的亲本大核和新发育的大核中；HA-Tcd4则定位于conjusome和新发育的大核中。
TCD3和TCD4相似的序列特征,保守的Chromodomain位置,一致的表达谱,表明两个基因中的一个很可能源于基因复制,进而加强该基因的功能。TCD3和TCD4在四膜虫接合生殖时期特异表达,都能够定位于新的发育的大核中,表明Tcd3和Tcd4极有可能参与了新的大核发育过程中程序性基因组重排。Tcd3和Tcd4在亲本大核和conjusome特异的定位,以及不同的模型结构,暗示基因TCD3和TCD4在进化过程中产生了新的功能。这些研究为进一步探讨chromodomain蛋白的功能提供了新的数据。
Chromodomain is a conservative protein domain consisting of 30-50 amino acids. Chromodomain-containing Proteins were identified in different organism. They participate in the formation of heterochromatin and expression regulation of genes through interaction with the histone or RNA.
Programmed genome rearrangement occurs during the conjugation stage in Tetrahymena. siRNA, MeH3K9, Pddlp and Pdd3p are involved in this process. However, the detailed programmed genome rearrangement mechanism is still unclear. In this study, we identified TCD3 and TCD4 from Tetrahymena. Transcriptional regulation and cellular localization of TCD3 and TCD4 were analysed, the results are as follows:
1. Bioinformatics analysis of TCD3 and TCD4 TCD3 (TTHERM_00585180) and TCD4 (TTHERM 00585190) are in the same chromosome. There is 237bp interval between them. They contain two introns, respectively. TCD3 and TCD4 encoded predicted polypeptide of 334 and 344 amino acids, respectively. The predicted molecur weight is 40.26kDa and 41.11kDa, isoelectric point is 4.590 and 5.435, respectively. Microarray analysis showed that TCD3 and TCD4 are unexpressed in growing stages and starvation stages, but, expressed during conjugation stage and upregulated at 8h. Furthermore, TCD3 and TCD4 showed same expression profile.
2. The 5 'and 3'-RACE of TCD3 and TCD4 The ORF of TCD3 and TCD4 contain 1005bp and 1035bp, respectively. Macronuclear chromosome counterpart is 1241bp and 1429bp.5'RACE of TCD3 and TCD4 showed 5' untranslated region is 40bp and 43bp, respectively.3'RACE of TCD3 and TCD4 showed 3'untranslated region is 90bp and 189bp, respectively. The cluster analysis of Chromodomain showed Tcd3 and Tcd4 is similar to pddlp.
3. The immunofluorescence localization of Tcd3 and Tcd4 HA-TCD3 and HA-TCD4 were constructed and transformed into CU428 and B2086 cells, respectively. Transformed cells were selected by paromomycin and identified by PCR. Immunofluorescence localization of HA-Tcd3 and HA-Tcd4 showed Tcd3 is located in the parental macronucleus and the new development macronucleus and Tcd4 is located in the conjusome and the new development macronucleus.
TCD3 and TCD4 showed similar sequence features, conserved chromodomain location, the same expression profile. It implies that two genes are likely derived from gene duplication to strengthen the function of the gene. TCD3 and TCD4 specifically expressed during conjugation stage and localize in the new development macronucleus. The results showed that Tcd3 and Tcd4 likely involved in the programmed genome rearrangement during the process of new macronucleus development. Specifically, Tcd3 and Tcd4 also localized in parent macronucleus and conjusome, respectively. It implies that two genes could evolve new functions. These studies provide new data for further exploring the function of the chromodomain protein.
四膜虫中程序性全基因组重排在有性生殖过程中发生,siRNA, MeH3K9, Pdd1p和Pdd3p参与这一过程,然而其详细机制仍不清楚。本研究从四膜虫基因组中筛选到两个含有Chromodomain的基因TCD3(Tetrahymena Chromo Domain 3)与TCD4(Tetrahymena Chromo Domain 4),对这两个基因的表达调控和细胞定位进行了研究,获得的主要结果如下：
1.TCD3和TCD4序列分析通过生物信息学的方法,从四膜虫大核基因数据库鉴定了TCD3 (TTHERM_00585180)和TCD4 (TTHERM_00585190),两个基因串联于同一条大核染色体上,间隔237bp,两个基因内均具有GT-AG的内含子。TCD3和TCD4基因拟编码的蛋白334个和344个氨基酸残基组成,分子量分别为40.26kDa和41.11kDa,等电点(pI)分别为4.590和5.435。全基因表达Microarray分析表明TCD3和TCD4在四膜虫营养生殖阶段和饥饿期不表达,在有性生殖过程中8h时表达量最高,并且两者的表达谱一致。
2.TCD3和TCD4的5'和3'-RACE TCD3和TCD4的ORF分别为1005bp和1035bp,大核染色体中基因全长分别为1241bp和1429bp,各含有两个内含子。5’RACE和3'RACE表明TCD3和TCD4的5’端非编码区(5'-UTR)长度分别为40bp和43bp,3'端非编码区(3’-UTR)长度分别为90bp和189bp。Chromodomain聚类分析表明Tcd3和Tcd4与pddlp蛋白的Chromodomain最为相似,SWISS—MODEL预测发现Tcd4的Chromodomain缺失α-螺旋结构。
3.Tcd3和Tcd4的免疫荧光定位构建含有HA标签的表达载体OE-PBX-TCD3和OE-PBX-TCD4,转化四膜虫CU428和B2086细胞株,通过巴龙霉素筛选,PCR鉴定,获得HA-Tcd3和HA-Tcd4的过表达细胞株。激光共聚焦显微镜对HA-Tcd3和HA-Tcd4的免疫荧光定位发现：HA-Tcd3定位于四膜虫有性生殖核发育的亲本大核和新发育的大核中；HA-Tcd4则定位于conjusome和新发育的大核中。
TCD3和TCD4相似的序列特征,保守的Chromodomain位置,一致的表达谱,表明两个基因中的一个很可能源于基因复制,进而加强该基因的功能。TCD3和TCD4在四膜虫接合生殖时期特异表达,都能够定位于新的发育的大核中,表明Tcd3和Tcd4极有可能参与了新的大核发育过程中程序性基因组重排。Tcd3和Tcd4在亲本大核和conjusome特异的定位,以及不同的模型结构,暗示基因TCD3和TCD4在进化过程中产生了新的功能。这些研究为进一步探讨chromodomain蛋白的功能提供了新的数据。
Chromodomain is a conservative protein domain consisting of 30-50 amino acids. Chromodomain-containing Proteins were identified in different organism. They participate in the formation of heterochromatin and expression regulation of genes through interaction with the histone or RNA.
Programmed genome rearrangement occurs during the conjugation stage in Tetrahymena. siRNA, MeH3K9, Pddlp and Pdd3p are involved in this process. However, the detailed programmed genome rearrangement mechanism is still unclear. In this study, we identified TCD3 and TCD4 from Tetrahymena. Transcriptional regulation and cellular localization of TCD3 and TCD4 were analysed, the results are as follows:
1. Bioinformatics analysis of TCD3 and TCD4 TCD3 (TTHERM_00585180) and TCD4 (TTHERM 00585190) are in the same chromosome. There is 237bp interval between them. They contain two introns, respectively. TCD3 and TCD4 encoded predicted polypeptide of 334 and 344 amino acids, respectively. The predicted molecur weight is 40.26kDa and 41.11kDa, isoelectric point is 4.590 and 5.435, respectively. Microarray analysis showed that TCD3 and TCD4 are unexpressed in growing stages and starvation stages, but, expressed during conjugation stage and upregulated at 8h. Furthermore, TCD3 and TCD4 showed same expression profile.
2. The 5 'and 3'-RACE of TCD3 and TCD4 The ORF of TCD3 and TCD4 contain 1005bp and 1035bp, respectively. Macronuclear chromosome counterpart is 1241bp and 1429bp.5'RACE of TCD3 and TCD4 showed 5' untranslated region is 40bp and 43bp, respectively.3'RACE of TCD3 and TCD4 showed 3'untranslated region is 90bp and 189bp, respectively. The cluster analysis of Chromodomain showed Tcd3 and Tcd4 is similar to pddlp.
3. The immunofluorescence localization of Tcd3 and Tcd4 HA-TCD3 and HA-TCD4 were constructed and transformed into CU428 and B2086 cells, respectively. Transformed cells were selected by paromomycin and identified by PCR. Immunofluorescence localization of HA-Tcd3 and HA-Tcd4 showed Tcd3 is located in the parental macronucleus and the new development macronucleus and Tcd4 is located in the conjusome and the new development macronucleus.
TCD3 and TCD4 showed similar sequence features, conserved chromodomain location, the same expression profile. It implies that two genes are likely derived from gene duplication to strengthen the function of the gene. TCD3 and TCD4 specifically expressed during conjugation stage and localize in the new development macronucleus. The results showed that Tcd3 and Tcd4 likely involved in the programmed genome rearrangement during the process of new macronucleus development. Specifically, Tcd3 and Tcd4 also localized in parent macronucleus and conjusome, respectively. It implies that two genes could evolve new functions. These studies provide new data for further exploring the function of the chromodomain protein.
引文
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