Del Vecchio Igor, Azzalin Alberto, Guidi Elena, Amati Giuseppe, Caramori Tiziana, Uboldi Cristina, Comincini Sergio, Ferretti Luca
Università degli Studi di Pavia, Dipartimento di Genetica e Microbiologia, Pavia, Italy.
Gene. 2005 Aug 15;356:101-8. doi: 10.1016/j.gene.2005.04.028.
The PRND gene encodes Doppel (Dpl), a protein that is strongly expressed in testis and at much lower levels in other tissues. Despite the recent discovery of Dpl involvement in spermiogenesis and in apoptotic death of cerebellar neurons, respectively in wild type and transgenic mice, the physiological role of this prion-like protein remains unknown. To better understand which factors may contribute to the modulation of PRND activity, a study of the bovine promoter region was performed. First, the transcription start site of PRND mRNA was identified using an innovative fluorescently labelled oligonucleotide extension (FLOE) method. The initiation site mapped 129 nt upstream of the protein coding sequence and represents a refinement of a previous assignment based on RACE. Second, deletion mutants of the 4530 nt encompassing 2704 nt 5' of the bovine PRND, exon 1, intron 1, and the first 6 nt of exon 2, have been investigated with CAT-reporter assays in order to identify critical elements for the activation of the gene. The results showed that the region -323/+32 (+1 is the transcription start site mapped by FLOE) represents the promoter region and contains positive cis-acting elements (CCAAT and E box) confirming previous reports with the mouse gene. Additional regulatory elements, including binding sites for repressor molecules, have been identified upstream of that region and in the first portion of intron 1, suggesting a complex tissue-specific regulation of Doppel gene expression.
PRND基因编码多配体蛋白（Doppel，Dpl），该蛋白在睾丸中强烈表达，在其他组织中的表达水平则低得多。尽管最近分别在野生型和转基因小鼠中发现Dpl参与精子发生和小脑神经元的凋亡死亡，但这种朊病毒样蛋白的生理作用仍然未知。为了更好地了解哪些因素可能有助于调节PRND活性，对牛启动子区域进行了研究。首先，使用创新的荧光标记寡核苷酸延伸（FLOE）方法确定了PRND mRNA的转录起始位点。起始位点位于蛋白质编码序列上游129个核苷酸处，是基于RACE的先前定位的改进。其次，为了确定该基因激活的关键元件，用氯霉素乙酰转移酶（CAT）报告基因分析法研究了包含牛PRND 5'端2704个核苷酸、外显子1、内含子1和外显子2前6个核苷酸的4530个核苷酸的缺失突变体。结果表明，-323/+32区域（+1是由FLOE定位的转录起始位点）代表启动子区域，并含有阳性顺式作用元件（CCAAT和E盒），证实了先前关于小鼠基因的报道。在该区域上游和内含子1的第一部分还鉴定出了其他调控元件，包括阻遏分子的结合位点，这表明多配体蛋白基因表达存在复杂的组织特异性调控。
