| Abstract: | p73是一個p53同源物，它具有與p53的DNA結合、轉活化及寡聚物聚合部位非常高度的序列相似性。研究顯示p73具有與p53相似的寡聚合及轉活化能力，並且p73可活化p53-感應基因如p21，並經由引發細胞凋亡來抑制細胞生長。在之前的研究中，我們發現p73基因在肝細胞癌中並無突變，而且p73基因表達的量在癌組織中總是比在相對的正常組織中高。在本研究中，我們著重探討p73基因的表達調控機制，包括甲基化及驅動子等之分析。首先我們分析在癌組織和正常組織中p73基因在驅動子及第一外顯子的甲基化模式。結果顯示，不管在癌組織或正常組織p73基因都沒有甲基化。其次，我們選殖了一段包含p73基因驅動子區域的1.3 kb DNA片段，並建構一系列連接GFP或luciferase報導基因的刪除序列。轉殖分析的結果顯1.3 kb的片段含有最高的驅動子活性，但是136 bp的片段即已含有基本的活性。另外，我們也從斑馬魚的卵巢RNA選殖出p73 cDNA。此cDNA 包含一個可轉譯出640個胺基酸的ORF (1923 bp)，它與鯰魚(Barbul barbus)、老鼠(Mus musculus)、人(Homo sapiens)、猿猴(Cercopithecus aethiops)及蝸牛(Mya arenaria)的p73相似度分別為95%、71﹪、71%、70%、32%。由 RT-PCR結果得知，p73的表現局限在皮膚、魚鰭、腦、卵巢、睪丸，與p53的廣泛表現顯著不同。在胚胎發育過程中p73 RNA在受精3小時後即可偵測到表現，直到120小時。利用全體原位雜交及免疫化學染色方法，發現p73也在頭部的嗅球及卵巢和睪丸的特定細胞上有表現。斑馬魚p63基因也已以同樣的方式選殖出。這些結果顯示p73可能參與卵巢和睪丸的某些特定功能，也可能與感覺器官的訊息傳遞有關。未來進一步實驗我們將使用morpholino antisense oligo來探討p73在斑馬魚胚胎發 育時所扮演的角色。
p73, the p53 homologue, was shown to have remarkable sequence similarity to the DNA-binding, transactivation, and oligomeri- zation domains of p53. It possess oligomerization and transactivation properties similar to p53 and can activate p53-responsive genes such as p21, and suppress cell growth by inducing apoptosis. In the previous study, we found that p73 was not mutated in hepatocellular carcinomas and that the levels of p73 were elevated in the cancerous tissues as compared to their normal counterparts. In this study, we have worked on exploring the mechanisms regulating p73 gene expression, including methylation and promoter/enhancer studies. First, we analyzed the methylation patterns in the promoter and exon 1 regions of p73 gene in cancerous and normal tissues. Results indicate that there are no methylations associated with either cancerous or normal samples. Secondly, we cloned a 1.3 kb DNA fragment of the p73 promoter region and made a series of deletion constructs fused to either GFP or luciferase reporter gene. Transient transfection analysis revealed that the 1.3 kb fragment had the highest promoter activity compared to the other shorter constructs but p-136 (-136 to exon 1) already had the basal promoter activity. Finally, we cloned the p73 cDNA from zebrafish ovary RNA. The consensus open reading frame (1923 bp) encodes a polypeptide of 640 amino acids which shares 95, 71, 71, 70 and 32% identity to the p73 of barbel, mouse, human, Cercopithecus aethiops (African green monkey) and Mya arenaria(sftshell), respectively. RT-PCR analysis revealed that zebrafish p73 was expressed in restricted tissues such as skin, fin, brain, ovary and testis, in contrast to the more ubiquitous expression of zebrafish p53. During embryonic development, expression of p73 was detected at 3 hours post fertilization (hpf), and thereafter through 120 hpf. By wholemount in situ hybridization and immunohistochemical staining, p73 was found expressed in the olfactory bulb like region and in the granulosa/theca cell layer of the ovary and in the Leydig/Sertoli cells of the testis. Zebrafish p63 homologue has been cloned using the same strategy and expression profile also examined. These results indicated that p73 may participate in certain aspect of ovary/testis function, as well as in the signaling in the sensory organ. Further experiment using morpholino antisense oligo of p73 will be conducted to dissect the role of p73 in zebrafish development. |
