DBY基因的啟動子及睪丸組織的轉錄調控分析

CNU IR > College of Smart Living > Dept. of Childhood Education and Nursery > MOST Project > Item 310902800/2404

Please use this identifier to cite or link to this item: https://ir.cnu.edu.tw/handle/310902800/2404

Title:	DBY基因的啟動子及睪丸組織的轉錄調控分析 The promoter assays and transcription regulation in testiss for DBY gene
Authors:	鄧燕妮
Contributors:	嬰幼兒保育系
Keywords:	DBY 基因啟動子冷光 DBY promoter luciferase
Date:	2005
Issue Date:	2008-07-31 14:37:53 (UTC+8)
Publisher:	台南縣：嘉南藥理科技大學嬰幼兒保育系
Abstract:	DBY 基因在睪丸組織表現特定長度2319 bps 的轉錄產物，在其他器官則表現較長的轉錄產物（4416 bps），推測DBY 在睪丸組織有特定轉錄機制進行調控，因此本研究針對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 載體共轉染到293T 細胞（human embryonic kidney cell）、TM3（mouse leydig cell）、TM4（mouse sertoli cell ）和GC-2spd(ts) （ mouse spermatocyte cell）等四種細胞株中，以螢光蟲發出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）和GC-2spd(ts)等四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的差異。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 基因轉錄起始點上游-316 至轉錄起始點（start codon, +1）區域為啟動子最強的區域；但在GC2-spd（mouse spermatocyte cell）細胞株中，DBY 基因轉錄起始點上游-515至轉錄起始點（start codon）下游+634 區域有較強啟動子活性，轉錄起始點上游-316 至轉錄起始點（start codon, +1）區域的啟動子活性則與其他區域沒有明顯差異。由以上研究結果推測，生殖spermatocyte 細（如： mouse GC2-spd spermatocyte cell）的轉錄調控方式與一般體細胞（somatic cell）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 等轉錄因子結合位置，此區域（-316~+1）在體細胞是轉錄作用重要區域。而在生殖細胞轉錄起始點（start codon）+1 至下游+634 區域應有可促進轉錄活性的因子調控，以加強轉錄作用的進行。 This study is focused on promoter analysis of DBY gene. In the promoter assay, 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） and GC2-spd（mouse spermatocyte cell） cells. The promoter region of DBY gene will be determinated by assaying the firefly 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 transcription 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 GC2-spd （ mouse spermatocyte cell）but not in 293T（human 2 embryonic kidney cell）、TM3（mouse leydig cell）、TM4（mouse sertoli cell）, suggesting the region has presence of positive regulator element. The transcription regulation and gene expression of germ cells are different from somatic cells.
Relation:	計畫編號：NSC94-2320-B-041-002
Appears in Collections:	[Dept. of Childhood Education and Nursery] MOST Project

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