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Molecular Cloning and Characterization of Novel Porcine Ftsj1 and Ftsj2 Genes which are Homologous to E. coli Ribosomal RNA Methyltransferase
heat shock protein
大腸桿菌RrmJ (ribosomal RNA large subunit methyltransferase J)屬於一個熱休克蛋白，其蛋白功能主要作用在23S rRNA之A-loop U2552 核苷酸位置，進行2’-O-ribose甲基轉化作用。缺乏A-loop U2552位置之2’-O-ribose甲基化會造成轉譯作用的效率降低，以及大腸桿菌的生長缺陷。大腸桿菌RrmJ同時也是一個高保留性的蛋白質，從原核生物到哺乳動物中均具有此同源蛋白的存在；其中在人類基因體裡已被確認出含有Ftsj1、Ftsj2以及Ftsj3三個類似RrmJ的同源基因，然而在其他哺乳動物中並未有任何相關之研究報告。本研究即利用RT-PCR、degenerated PCR及3’-RACE方式新選殖出位於豬隻中之Ftsj1與Ftsj2 mRNA，分別含有1867與1589個核苷酸，並可轉譯出含有329與245個胺基酸之蛋白質。在利用演化樹分析、蛋白質序列比對以及蛋白質三級結構的預測結果，顯示豬隻FTSJ1與FTSJ2蛋白質與大腸桿菌RrmJ具有很高度的相似性，並且亦推測兩者於其在酵母菌中之同源蛋白Trm7p與Mrm2p具有相同的功能，分別作用於tRNA的anticodon loop與粒線體rRNA的A-loop位置。而利用半定量RT-PCR進行豬隻中13個組織的Ftsj1與Ftsj2 mRNA表現量結果，顯示所有的組織中均有Ftsj1與Ftsj2 mRNA的表現，並且呈現不同程度的表現量。進一步利用real-time RT-PCR分別對25oC、30oC與35oC環境溫控畜舍中處理一週之豬隻進行Ftsj1與Ftsj2 mRNA表現量之測定，其結果顯示在少數組織中Ftsj1與Ftsj2 mRNA的表現量會隨著溫度的提升而有上升的趨勢；而在大多數的組織中其表現則是呈現不變或受到抑制的情形，顯示在少數組織中Ftsj1與Ftsj2基因仍保留著熱休克蛋白的特性，然而在大部分的組織中則已喪失了這項功能。另外在Ftsj2基因甲基化的實驗顯示，其intron1在各組織中均呈現高度甲基化，因而在Ftsj2基因表現之抑制作用中，並沒有明顯的受到該檢測區段之甲基化機制所調控；相反的，Ftsj2在35oC下mRNA表現量上升的組織中則具有甲基化程度下降的趨勢。而在更廣泛的基因調控區域中，如基因之啟動子，甲基化程度受到環境效應之變化，更值得繼續進行更深入之研究。|
Abstract An E. coli heat shock protein called RrmJ (ribosomal RNA large subunit methyltransferase J) is responsible for the 2'-O-ribose methylation of A-loop U2552 in the 23S rRNA. Absence of this methlyation causes to reduce either the efficiency of protein synthesis or the growth rate of E. coli. The RrmJ is also a highly conserved protein from eubacteria to mammalia. In human, three closed homologs of RrmJ have been identified and designated as FTSJ1, FTSJ2 and FTSJ3 proteins. In this study, we have cloned two novel porcine Ftsj1 and Ftsj2 full length cDNAs by RT-PCR, degenerated PCR and 3'-RACE, which encoded two polypeptides with 329 and 245 amino acids, respectively. Under bioinformatic analysis, the significantly similarity of porcine FTSJ1 and FTSJ2 to E. coli RrmJ has been demonstrated that both proteins may perform a similar characteristic to RrmJ. Semi-quantitative RT-PCR results showed that porcine Ftsj1 and Ftsj2 mRNA were expressed in all examined thirteen tissues with different levels. Growing piglets were kept at 25oC, 30oC and 35oC thermal-controlled environment for one week and tissue mRNAs were extracted for detection of the Ftsj1 and Ftsj2 genes expression. Using real-time RT-PCR technique, the results of Ftsj1 and Ftsj2 mRNA expression in the thermal-controlled piglet tissues indicate that porcine Ftsj1 and Ftsj2 mRNA expressions were up regulation in only few tissues, and the roles of FTSJ1 and FTSJ2 in mammalia might be different from E. coli under heat shock stress. DNA methylation status was also examined to understand the correlation between Ftsj2 gene methylation and Ftsj2 mRNA downregulation under heat shock stress. The results showed that all the selected tissues are highly methylated in Ftsj2 intron1, and did not regulated the mechanism of Ftsj2 mRNA expression inhibition; conversely, the methylation status was decreased in the tissues where Ftsj2 mRNA were expression increasely. But if Ftsj2 gene still contains other CpG islands, likely promoter region, which involve the regulation of Ftsj2 expression will be study further.
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