尋找與細胞凋亡相關的新基因

Abstract

細胞凋亡（apoptosis）是大部分多細胞動物在發育及維持生理平衡上所不可或缺的一種機制。在線蟲（Caenorhabditis elegans）先前的研究中已發現超過20個與此機制相關的基因，其中4個基因：egl-1(BH3-only), ced-9(Bcl-2), ced-4(APAF1) and ced-3(Caspase) 組成了細胞凋亡的核心路徑. 這些研究指引了細胞凋亡在其他物種中的研究，並促成了包含果蠅，老鼠，人類及其他物種中類似基因的發現。線蟲中的相關研究仍有很大的發展空間；舉例而言，在線蟲所有進行細胞凋亡的細胞中，我們僅知道少數幾顆細胞啟動egl-1的機制；ced-3 的下游受質也有待補齊。若能在線蟲中建立細胞凋亡的完整藍圖，對於此領域的發展將有莫大的助益。在本研究中，我根據先前實驗的觀察結果－「grp-1這個與不對稱細胞分裂相關的基因，在同時與一些促進細胞凋亡的基因發生突變時（double mutant）會影響線蟲尾部特定細胞的命運，使其逃過死劫進而分化成為hypodermal cell導致其尾部出現特定型態上的異常。」來進行enhancer screen，在grp-1的背景下尋找促使細胞凋亡 (pro-apoptosis) 的未知基因。在本實驗中我依據線蟲尾部型態之異常分離了116個突變品系，我挑選其中71個具有較高外顯率的突變品系做進一步的分析，並依照其細胞凋亡發生的情形將其分為四群。互補試驗顯示其中6個突變是發生於已知與此機制相關的基因，包含了ced-3，ced-4以及ced-8。我目前正藉由遺傳以及性狀來分析tp6與tp7這兩個突變。這兩個突變都會導致線蟲胚胎中細胞殘骸數目的顯著減少，而其位置也暗示它們可能是此領域中未曾發現的新基因。

Apoptosis is an essential process for both development and homeostasis of multi-cellular animals. Previous studies of the nematode Caenorhabditis elegans have identified more than 20 genes involved in this process and placed them into a genetic pathway with four genes: egl-1(BH3-only), ced-9(Bcl-2), ced-4(APAF1) and ced-3(Caspase) that constitutes the core-cell death machinery. These works led to the finding of similar genes that control apoptosis in other organisms, including flies, mice and humans. However, there is still plenty more to be learnt; for example the mechanisms that controls egl-1’s expression in most cells doomed to die are unknown; and only few substrates of CED-3 has been found. In this study, I base on the previous observation that the double mutations in grp-1 (GTP exchange factor for ARFs, a gene involved in asymmetric cell divisions) and a pro-apoptotic gene could switch the apoptosis fate of specific cells in the tail to hypodermal cell fate and cause an arch-like morphological defect to undertake a genetic enhancer screen for new pro-apoptotic genes in the grp-1 background. From this screen, I isolated 116 mutant strains based on the “tail-defect” phenotype. Seventy one of these mutants with higher penetrance were further analyzed and put into four groups according to the pattern of their embryonic cell deaths. The complementation tests show that four mutations are alleles of previously identified pro-apoptotic genes, including ced-3,ced-4 and ced-8. I am genetically and phenotypically characterizing two mutants tp6 and tp7. They display reduced numbers of cell corpses during embryogenesis in the absence of the grp-1 mutation. On the basis of their mapping positions tp6 and tp7 likely define new pro-apoptotic genes.