ANALYSES OF FUNCTIONS OF DNA REPLICATION REGULATORY FACTORS BY THE DROSOPHILA EYE-IMAGINAL DISC SPECIFIC EXPRESSION SYSTEM

果蝇眼成象盘特异性表达系统DNA复制调控因子的功能分析

基本信息

批准号：
09680640
负责人：
YAMAGUCHI Masamitsu
金额：
$ 2.37万
依托单位：
AICHI CANCER CENTER
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680640/
关键词：
DROSOPHILA TRANSGENICFLY DREF E2F GAL4 TRANSCRIPTION FACTOR DNA複製複眼原基 Pエレメント形態形成アポトーシス Gal4

项目摘要

Transgenic Drosophila lines expressing GAL4 specifically in the eye-imaginal discs were established. DREF cDNA and its fragments containing conserved regions 1,2 or 3 (CR1, CR2 or CR3) were ligated to the promoter containing GAL4-binding sites (UAS) in the P-element vector, and transgenic fly lines were established with these plasmid DNAs. These trnsgenic lines were utilized to examine effects of overexpression of DREF and its derivatives on eye development.Overexpression of the wild type DREF in eye imaginal discs caused abnormal eye morphology (rough eye phenotype). The rough eye phenotype is likely caused by the ectopic induction of DNA synthesis and apoptosis in the eye-imaginal disc cells. Since it is reported that overexpression of the transcription factor E2F can induce ectopic DNA synthesis, we have crossed the DREF-overexpressing flies with the E2F mutant flies. Half-reduction of the E2F gene copy number effectively suppressed the rough eye phenotype induced by overexpression of DREF, suggesting that DREF functions upstream of the E2F gene. Furthermore, we have cloned the E2F gene and found the three DRE-related sequences in the promoter region of the E2F gene. DREF specifically binds to these DRE-related sequences of the E2F gene promoter in vitro. Detailed analyses in vitro and in vivo demonstrated that DREF can activate the E2F gene promoter.In addition, overexpression of CRland CR3 of DREF in the eye imaginal discs also caused severe rough eye phenotype, In the eye discs of these flies, cells behind the morphogenetic furrow appeared to be arrested in G1 phase. Probably, CRland CR3 inhibited function of endogenous DREF in a dominant negative fashion to inhibit cells entering S phase.

建立了在眼睛构想中特别表达GAL4的转基因果蝇线。将含有保守区域1,2或3（CR1，CR2或CR3）的DREF cDNA及其片段连接到P元素中含有GAL4结合位点（UAS）的启动子，并用这些质粒DNA建立转基因飞行线。这些TRNSGENIC线被用来检查DREF及其衍生物对眼睛发育的过表达的影响。眼睛想象盘中野生型DREF导致眼睛形态异常（粗眼表型）。粗糙的眼表型可能是由眼睛构想细胞中DNA合成和凋亡的异位诱导引起的。据报道，转录因子E2F的过表达可以诱导异位DNA合成，因此我们已将过表达的果蝇与E2F突变蝇跨过。 E2F基因拷贝数的半还原有效地抑制了DREF过表达引起的粗糙眼表型，这表明DREF在E2F基因的上游功能。此外，我们已经克隆了E2F基因，并在E2F基因的启动子区域中发现了三个与DRE相关的序列。 DREF在体外特异性结合了E2F基因启动子的这些DRE相关序列。在体外和体内进行了详细的分析表明，DREF可以激活E2F基因启动子。在另外，眼睛想象盘中DREF的Crland Cr3的过表达也导致了这些苍蝇的严重粗糙眼盘，在这些蝇的眼盘，形态学犁沟后面的细胞似乎在G1相中似乎在G1阶段被捕。 Crland CR3可能以主要的负面方式抑制内源性干燥的功能，以抑制进入S相的细胞。