Cloning of Drosophila Gene which Code for UV-damaged DNA Binding Protein.

通过凝胶电泳DNA带移分析，我们在果蝇胚胎细胞中鉴定出两个与紫外线照射的双链DNA具有高亲和力的DNA结合蛋白复合体。通过对DNA修复缺陷的果蝇突变体的筛选，发现了5个缺失这两种蛋白质复合体的突变体。四个切除修复缺陷突变体(Mus201、phr、Mus308和Mus205)缺少一个蛋白质复合体(因子2)。在复制后修复缺陷突变体Mus104中未检测到另一种蛋白复合体(因子1)。这些发现表明基因产物可能参与了紫外光诱导的光产物的损伤识别和DNA修复。利用肝素琼脂糖柱层析，我们可以将每个因子分离成不同的组分。含有因子2的部分，即有能力形成带2的部分，通过紫外光照射的DNA亲和柱层析进一步纯化。在具有第二带形成能力的组分中，在银染的SDS-PAGE凝胶中只有一条蛋白质带。推测其分子质量在45K-50K道尔顿之间。我们假设这个蛋白质是因子2。

Using a gel electrophoresis DNA band shift assay, we have identified two DNA binding protein complex in Drosophila embryonic cell which have a high affinity for UV-irradiated, double-stranded DNA. Screening Drosophila mutants which are deficient in DNA repair has identified five mutants which lack either of the two protein complex. Four excision repair deficient mutants (mus201, phr, mus308 and mus205) lack one protein complex (Factor 2). Another protein complex (Factor 1) was not detectable in post-replication repair deficient mutant mus104. These findings suggest a possible involvement of t he gene product in lesion recoghition and DNA repair of UV-light induced photoproduct.Using heparin agarose column chromatography, we can separate each factor into different fraction. The fraction which contain Factor 2, that is the fraction which have ability to form band 2, was further purified by UV-irradiated DNA affinity column chromatography. In the fraction which has ability to form band 2, we can identify only one protein band in silver stained SDS-PAGE gel. The molecular mass was assummed to be between 45k and 50k dalton. We assumed this protein is Factor 2.