Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India.
Gene. 2012 Jan 10;491(2):165-72. doi: 10.1016/j.gene.2011.09.036. Epub 2011 Oct 12.
In the present study, we cloned and characterized the buffalo (Bubalus bubalis) OCT4 ortholog expressed in embryonic stem cell (ESC) like cells and its promoter region. The 5'- and 3'-RACE experiments were conducted to analyze the transcription initiation site and regulatory regions. The comparative analysis of buffalo OCT4 promoter with other mammalian orthologs revealed high conservation. Among the regulatory regions highest similarity was observed between buffalo, bovine and sheep. Interestingly, buffalo OCT4 promoter exhibited a 78 bp deletion between two proximal enhancers (PE-1A and PE-1B) when compared to other mammalian orthologs. 5'-RACE revealed four different transcription start sites for OCT4 gene. As far as we know there is no previous report regarding multiple transcription initiation sites for OCT4 gene in any species. In addition, we identified expression of four pseudogenes in buffalo ESC-like cells. Among the multiple transcripts characterized, we found four cDNA clones (1083 bp) derived from ESC-like cells sharing 96.9-99.3% sequence homology with the parent gene and having the capacity of encoding 139, 206, 206 and 324 amino acid long truncated proteins. Multiple pseudogenes have been proposed for OCT4 which might contribute to the false detection of this gene during expression studies. However, only few of them were reported to be transcribed and none were reported to be translated in stem cells. Western blot analysis of OCT4 protein using ESC-like cells revealed multiple bands, indicating that some of the hypothetical pseudogenes are being translated. These novel pseudogenes or their protein products may have some important regulatory functions.
在本研究中，我们克隆并鉴定了水牛（Bubalus bubalis）OCT4 同源基因在胚胎干细胞（ESC）样细胞中的表达及其启动子区域。通过 5'和 3'RACE 实验分析转录起始位点和调控区。与其他哺乳动物的同源基因进行比较分析发现，水牛 OCT4 启动子具有高度保守性。在调控区中，水牛、牛和绵羊之间的相似度最高。有趣的是，与其他哺乳动物的同源基因相比，水牛 OCT4 启动子在两个近端增强子（PE-1A 和 PE-1B）之间存在 78bp 的缺失。5'RACE 揭示 OCT4 基因有四个不同的转录起始位点。据我们所知，在任何物种中，OCT4 基因都没有以前关于多个转录起始位点的报道。此外，我们在水牛 ESC 样细胞中鉴定到四个假基因的表达。在所鉴定的多个转录本中，我们发现四个 cDNA 克隆（1083bp）来源于 ESC 样细胞，与母基因具有 96.9-99.3%的序列同源性，并具有编码 139、206、206 和 324 个氨基酸的截断蛋白的能力。已经提出了 OCT4 的多个假基因，这可能导致在表达研究中错误检测到该基因。然而，只有少数被报道转录，并且在干细胞中没有报道翻译。使用 ESC 样细胞对 OCT4 蛋白进行 Western blot 分析显示出多个条带，表明一些假设的假基因正在被翻译。这些新的假基因或其蛋白产物可能具有一些重要的调节功能。
