| Summary: | 碩士 === 國立陽明大學 === 遺傳學研究所 === 83 === dGATA-II核酸結合蛋白是本實驗室先前由果蠅分離出的轉錄因子，其具二0個指狀區域功能基 (Zinc finger dolmain)，可識別目標核酸保留序列－－W／GATA／R並與之結合。由於過去研究，使 dGATA－II基因結構已被清楚了解，本論文承續前者，對dGATA－II轉錄因子在果蠅胚胎時期，促使細胞發育達到特定形態的功能進行探討。
利用 RNA全埋在位染色法 (RNA whole mount in situ hybri－dization)，我們得知 dGATA－II RNA在胚胎發育早期即在未來發育為頭部、中樞神經、中腸和後呼吸孔等區域組織表現，且隨該組織發育仍有持續表現，顯示dCATA－II可能參與這些組織功能特異性的分化過程。而在幼蟲時期，受 dGATA－II基因驅動之報導基因表現於腦、中樞神經與胸節復屬肢的imaginal discs中。此外，使用多種分析方法，仍無證據顯示dGATA－II基因在ovary中有表現。
為了進一步對dGATA－II基因在果蠅胚胎發育過程參與的角色進行了解，我們分析二P元素嵌插株：l(3)5930和 P812，兩者P元素嵌插點都位於dGATA－II基因轉錄啟始點前的5’端控制區域內，且同型台子死亡。本艾利用遺傳雜交實驗，認定 l(3)5930和 P812之同型合子是因P元素嵌插所致死，而發育停滯在胚胎晚期。吾人嘗試提供轉位酉每(transposase)至P812嵌插株中，以獲得復原體果蠅株，但分析其子代發現皆為不完整跳離，因此無法回復野生種外表型。
本研究確認dGATA－II基因之表現形式及分析二株突變種之dGATA－II基因結構，將有助於後續對功能之瞭解。
dGATA-D transcription factor is a Drosophila DNA-binding protein with two zinc finger domains recognizmg the consensus sequence, W/GATA/R, found in the regulatory regions of the target genes. In this study, we used RNA whole mount in situ hybridization to analyze the expression pattern of the dGATA-II gene in the embryonic stage, and found it expresses in the brain, central nervous system, midgut and posterior spiracles at different stages. At imaginal disc stage, the lacZ reporter gene driven by the dGATA-It promoter was expressed in brain, CNS and the appendixes of the thoracic segments. In addition, we apphed RT-PCR, in-situ hybridization and X-gal staining to determine whether dGATA-II gene is expressed in the ovary. Our results do not provide evidence that GATA-II gene is expressed materally.
To confirm the critical role of dGATA-II factor for Droso-philn embryonic development, we analyzed l(3)5930 and P812, two P elelment insertion strains with the homozygous lethal pheno-types. Both P elements insertion sites locate at the dGATA-II 5' regulatory region just upstream from the dGATA-II transcription start site. We determined that both strains are lethal at late embryonic stage and the lethal phenotypes are resulted from the P element insertion. To directly prove that P-element insertion at the dGATA-II promoter causes embryonic lethal phenotype, we carried out P element transposition in P812 to obtain precise excision. Unfortunately, all the hues carry an incomplete excision, and fail to give a revertant.
In this study, we confirm that dGATA-II gene expression pattern and analyze the genomic structures of the two P-element insertion strains. These will be useful to the subsequent functional studies about dGATA-II gene.
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