Regulation Of Expression And Functional Analysis Of Tcd3 And Tcd4 In Tetrahymena Thermophila

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 (TTHERM₀0585180) 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.