FUNCTION AND EXPRESSION-REGULATION OF DROSOPHILA DNA REPLICATION ENZY ME GENE

1.利用转基因文件对果蝇增殖细胞核抗原基因转录调控元件进行分析：我们建立了150多个独立的携带有不同突变的果蝇增殖细胞核抗原启动子-LacZ融合基因的转基因文件。对这些转基因株系在发育过程中LacZ表达的检测使我们能够鉴定出三个转录调控元件(Ure：upstrem调节元件，Dre：DNA复制相关元件和E2F-识别位点)。结果表明，在整个发育过程中，E2F识别位点对增殖细胞核抗原基因的表达起决定性作用。DRE在胚胎和幼虫中是必需的。尿素酶在各种幼虫组织中都是必需的。利用Gal4靶向表达系统分析DNA复制酶基因调控因子的功能：Dre结合因子在果蝇眼想象盘中的过表达导致ADOLT Fly复杂眼的形态发生异常。这很可能是由于异位诱导眼成像盘中的细胞增殖所致，尽管详细的机制目前正在调查中。唾液腺中显性-负性DRFE的过度表达抑制了染色体的多角化。这些结果表明，DREF在内化循环中起着重要的调节作用。果蝇以外物种DREF同源基因的鉴定：家蚕的免疫筛选。使用抗果蝇DREF抗体的cDNA文库使我们能够挑选出几个家蚕DEEF同源物的候选克隆，我们正在分析这些候选克隆的核苷酸序列。比较这两个物种DREF的结构，有望为我们鉴定DREF在哺乳动物中的同源物提供线索。

1. Analyzes of transcriptional regulatory elements of Drosophila PCNA gene using transgenic files : We have established more than 150 independent lines of transgenic files carrying PCNA promoter-lacZ fusion genes having various mutations in the PCNA promoter. Examination of lacZ expression from these transgenic lines during development allowed us to identify three transcriptional regulatory elements (URE : upstrem regulatory elemet, DRE : DNA replication-related element and E2F-recongnition sites). The results indicate that E2F-recognition sites are eddectial for the PCNA gene expression throughout development. DRE is essential in embryos and larvae. URE is essential in various larval tissues.2. Analyzes of functions of regulatory factors for DNA replication enzyme genes using Gal4-targetted expression system : Overexpression of DREF (DRE-binding factor) in Drosophila eye imaginal discs results in abnormal morphogenesis of complex eyes in adolt fly. This is likely caused by ectopic induction of cell proliferation in eye imaginal discs, although detailed machanisms are now under investigation. Overexpression of dominant-negtive form of DRFE in salivary glands inhibited the polytenization of chromosomes. The results indicate that DREF plays an important role in regulation of endoreplization cycles.3. Identification of DREF homologue in species other than Drosophila : Immunoscreeming of Bombyx mori. cDNA library using anti-Drosophila DREF antibody allowed us to pick up several candidate clones for silk worm homologue of DEEF.We are now analyzing nucleotide sequences of these candiate clones. Comparison of structures of DREF between these two species would hopefuly prvide us a clue to identify mammalian homologue of DREF.