臺灣博碩士論文加值系統

在T細胞急性白血病(T cell Acute Lymphoblastic Leukemia，T-ALL)中，發現約有30%的病患，在其癌細胞中有TAL1基因重組現象，並因此導致TAL1基因在T細胞的不正常表現，干擾T細胞之發育，而促進腫瘤形成。 TAL1蛋白是帶有bHLH功能區之轉錄調節因子，其正常功能在調控早期造血系統、血球分化和胚胎期的血管生成。TAL1蛋白本質上不具DNA結合能力，但藉著與常在性表現之E2A基因的產物，亦即屬於Class A bHLH蛋白的E12和E47等，形成異雙體，而與特定的E-box (CANNTG) DNA序列結合。一般認為在T-ALL癌細胞中，TAL1蛋白異常表現，可能藉由其轉錄活性活化了下游基因，而促進腫瘤的形成。 然而，關於有那些基因是受TAL1致癌蛋白所調控的標的基因，卻尚未被確認。 為了找尋TAL1蛋白的標的基因，以瞭解白血病的致病分子機制及複雜的造血機制，由一種紅血球性白血病細胞K562中，以TAL1單株抗體及E12多株抗體，分離出TAL1-E12異雙體結合之染色質(chromatin)，經選殖及定序而得到6個帶有E-box(CANNTG)序列之DNA片段。 本研究中，將這些DNA片段分別植入到報導基因載體pGL3中，並以細胞轉染方式，分別送到COS1細胞中，以檢視這些 DNA片段之轉錄活性。 分析結果顯示，有兩個 DNA片段(K34及K94)具有2倍的轉錄活性，在有SV40促進子存在下，則其轉錄活性會被提升到約5倍以上，而在有TAL1及E12之表現質體共同轉染時，則轉錄活性又可進一步提升到10倍以上。 此外，將此二種報導質體轉染到K562細胞中時，其轉錄活性亦可見到有約10倍提升，顯示此二個DNA片段可能帶有受TAL1-E12異雙體調控之潛在標的基因之啟動子。

Abstract：
TAL1 gene was originally discovered as a result of its activation by chromosome rearrangements in T cell acute lymphoblastic leukaemia(T-ALL). Further studies have shown that TAL1 expression is aberrantly activated by several mechanisms including chromosome translocations, interstitial deletion and transactivation without detectable chromosomal alteration.
TAL1 gene encodes a bHLH transcription factor, that is essential for the development of all haematopoietic lineages and its expression is maintained during differentiation along erythroid, mast and megakaryocytic cell lineages, but not in normal peripheral T-lymphocytes. The bHLH motif of these protein is responsible for DNA binding and dimerization with other bHLH proteins involved in transcription regulation. TAL1 protein is able to form heterodimers with the ubiquitously expressed E2A gene products, E47 and E12, and the heterodimers bind to E-box motif with the general sequence CANNTG. But the target genes for TAL1 oncoprotein have not yet been identified.
We have previously isolated TAL1/E12 heterodimer bound genomic fragments by chromatin immunoprecipitation from K562 cells, and selected 6 fragments with one to four E-box CANNTG sequences. In order to determine if these fragments could be the regulatory elements of potential target genes of TAL1 oncoprotein, we inserted these 6 DNA fragments individually into pGL3 to generate recombinant reporter plasmids. The transfection experiments indicated that K34 and K94 DNA fragments behaved as a transcriptional transactivating sequence, and TAL1 and E12 proteins are required for efficient transcriptional activity. We also showed that transfection of these two recombinant constructs into K562 cells generated positive transcriptional activity, in a level similar to that in TAL1 and E12 co-transfected COS1 cells. These results established that both K34 and K94 DNA fragments are likely to contain a promoter of potential TAL1 target genes.

目錄
中文摘要…………………………………………………….. 1
英文摘要…………………………………………………….. 3
背景介紹…………………………………………………….. 5
材料與方法………………………………………………….. 8
結果…………………………………………………………..22
討論…………………………………………………………..26
參考文獻……………………………………………………..30
圖表…………………………………………………………..39

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