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

建立了在果蝇眼盘中特异表达GAL 4的转基因果蝇品系。将DREF cDNA及其保守区1、2或3（CR 1、CR2或CR 3）片段与P元件载体中含有GAL 4结合位点（UAS）的启动子连接，用这些质粒DNA建立转基因果蝇品系。利用这些转基因株系研究了DREF及其衍生物过表达对眼发育的影响，野生型DREF在眼胚盘中过表达导致眼形态异常（粗眼表型）。粗糙眼表型可能是由眼-成虫盘细胞中DNA合成和凋亡的异位诱导引起的。由于有报道称转录因子E2 F的过表达可以诱导异位DNA合成，我们将DREF过表达果蝇与E2 F突变果蝇杂交。E2 F基因拷贝数的一半减少有效地抑制了由DREF过表达诱导的粗糙眼表型，表明DREF在E2 F基因上游起作用。此外，我们还克隆了E2 F基因，并在E2 F基因的启动子区发现了三个DREs相关序列。DREF在体外特异性结合E2 F基因启动子的这些DRE相关序列。体外和体内的详细分析表明，DREF可以激活E2 F基因启动子，此外，DREF的CR 1和CR 3在眼盘中的过表达也导致了严重的粗糙眼表型，在这些果蝇的眼盘中，形态发生沟后面的细胞似乎被阻滞在G1期。CR 1和CR 3可能以显性负性方式抑制内源性DREF的功能，从而抑制细胞进入S期。

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.