本研究將針對Y染色體上AZFa區域的DBY基因進行啟動子位置的分析，將DBY基因coding region上游可能為轉錄啟動子區域，以PCR增幅放大後，構築到pGL3-basic載體中，與pGL3-SV40載體共轉殖到293T細胞（human embryonic kidney cell）、TM3（mouse leydig cell）、TM4（mouse sertoli cell）等細胞株中，以珊瑚蟲與螢光蟲等兩種luciferase的冷光強度分析此些區域轉錄表現情形，以確定DBY基因啟動子的正確位置及其對轉錄調控情形及在293T細胞（human embryonic kidney cell）、TM3（mouse leydig cell）、TM4（mouse sertoli cell）等三種細胞株的冷光活性表現，瞭解DBY基因啟動子在不同組織細胞中表現及調控情形。研究結果顯示，在293T細胞（human embryonic kidney cell）、TM3（mouse leydig cell）、TM4（mouse sertoli cell）等細胞株中，DBY基因coding region上游-316至轉譯起始點（start codon, +1）區域為啟動子最強的區域；在GC2-spd（mouse spermatocyte cell）細胞株中，DBY基因coding region上游-515至轉譯起始點（start codon）下游+634區域有較強啟動子活性，由以上結果推測，生殖spermatocyte細胞的轉錄調控方式與一般體細胞有所不同。針對DBY基因啟動子可能區域（-316~+1）進行轉錄因子分析，在-200、-100及-30區域分別有GC box’、CAT box及TATA box，取有SP1、AP-1及CBP等轉路因子結合位置，此區域（-316~+1）對轉錄作用是重要區域。 This study is focused on promoter analysis. In the promoter assay of DBY, the sequences of upstream of coding region for DBY will be available from genebank by bioinformatics search. The specific primers and PCR conditions for the upstream of start site for DBY gene will be designed and PCR product will be constructed into pGL3-basic vector. The constructer pGL3-basic vectors and pGL3-SV40 vector will be co-transfected to 293T（human embryonic kidney cell）、TM3（mouse leydig cell）、TM4（mouse sertoli cell）cells. The promoter region of DBY gene will be determinated by assaying the firefly and Renilla luciferase activity. The prediction promoter region of human DBY gene in -316 to +1, containing GC box、CAT box and TATA box at -200、-100 and -30 regions, that containing putative transcription factor binding site of SP1、AP-1 and CBP. Luciferase reporter analysis of a 3.0 kb 5’-flanking sequence (-2235 to +634 with respect to the translation start site). Serial deletions analysis promoter activity in 293T (Human embryonic kidney cell lines), TM3 (mouse Leydig cell), TM4 (mouse Sertoli cell) and GC-2spd(ts) (mouse spermatocyte) that revealed -316 to +1 fragment is core promoter region. The +1 to +634 fragment has strong promoter activity in 293T, TM3 and TM4, suggesting the region has presence of positive regulator element.
