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研究生:葉季昀
研究生(外文):Yeh, Jih-Yun
論文名稱:果蠅GATA轉錄因子之遺傳學研究
論文名稱(外文):Genetic studies of drosophila dGTA-II gene
指導教授:蔡世峰蔡世峰引用關係
指導教授(外文):Tsai, Shih-Feng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:1994
畢業學年度:82
語文別:中文
中文關鍵詞:遺傳果蠅
外文關鍵詞:HEREDITY
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近年來之研究顯示,GATA-1轉錄因子在調控血紅素基因組織特異性表現及血球分化上,扮演著重要的角色。目前為止,在哺乳類至少有四種GATA因子,它們皆是藉由指狀區域構造 (Zincfinger domain)與目標核酸序列(T/A)GATA(A/G)結合。本實驗室先前由果蠅分離出二種GATA基因,分別命名為dGATA-I與dGATA-II。本論文承續先前研究,分析果蠅GATA-II基因結構,並進一步以遺傳學方法,探討dGATA-II基因之功能與調控機制。
利用引子延伸(primer extension)與5'端互補核酸序列定序(5' cDNA sequencing),我們得以精確地定位dGATA-II基因之轉錄起始點。由此得知dGATA-II基因由九個表現子 (exon)所組成,且整個基因大約橫跨了36kb。所有表現子與內含子 (intron)的交界序列(boundary sequences)也因此確定。此外,藉由果蠅唾腺巨大染色體圖譜定位(polytene chromosomemapping)與P1殖株的南方墨跡介析,我們進一步將dGATA-II基因定位於84F1-2區間。
基於此圖譜訊息,我們從G.Rubin實驗室獲得P元素(P-element)嵌插於84F區域之果蠅株。經南方墨跡分析,確實有二株之P元素嵌插於dGATA-II基因5'端上游區域。利用抗β-半乳糖甘酵素之單株抗體,對其中1(3)530果蠅株 ,進行全胚胎原位偵測 (whole mount in situ detection),顯示其lacZ報導基因(reporter gene)表現形式,與本實驗室先前所做dGATA-II基因轉錄形式(RNA in situ hybridizatiOn)相符。β-半乳糖甘酵素明顯地表現於中樞神經系統,中腸道前端 (anterior part ofmidgut)與後呼吸孔(posterior spiracle)。最後,我們構築了二個帶有dGATA-II基因5'端上游序列的構體(constructs),偵測其所驅動lacZ報導基因在組織的表現形式,以進一步界定dGATA-II基因的調控區域。
GATA transcription factors are a class of DNA binding proteins recognize the consensus sequence, (T/A)GATA(A/G), found in the regulatory regions of target genes. At least four mammalian GATA binding proteins have been identified and each plays a central role in determining tissue-specific functions. We previously isolated two cDNA and genomic clones for the GATA homologous proteins from Drosophila melanogaster, designated, dGATA-I and dGATA-II. In this study, we determined the organization of dGATA-II gene and the exon/intron boundary sequences. The dGATA-II gene consists of nine exons and the entire genomic sequence covers nearly 36kb. We also performed primer extension and 5' RACE procedure to determine the precise transcription initiation site. In situ hybridization with the salivary gland polytene chromosome has shown that dGATA-ll is located in the 84F region on the third chromosome. P1 clones corresponding to the 84F region were analyzed by Southern analysis to refine the chromosomal position of dGATA-II gene to 84F1-2. To obtain mutants for dGATA-II gene, two strategies were used simultaneously. We designed and tested a scheme of P element mutagenesis, followed by PCR detection with target-site specific primers. In addition, we searched the database for P element insertion strains that mapped to 84F region. Southern analysis was used to confirm that two strains ( l/(3)5930 and #P812 ) carry P element insertion at the 5' upstream regions of the dGATA-II gene.Whole mount in situ detection of l/(3)5930 strain embryos with anti B- galactosidase antibodies gave an expression pattern similar to that obtained for the endogenous dGATA-II transcripts. This result suggests that the reporter gene is under the control of dGATA-II regulatory sequence. We also constructed two dGATA-II 5' genomic fragments to drive the β-galactosidase reporter gene. One germ line transformant was isolated and analyzed for its gene expression pattern.



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