Yamaguchi M, Nishida Y, Matsukage A
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Nagoya, Japan.
Gene Expr. 1995;4(4-5):183-93.
The regulatory region of Drosophila proliferating cell nuclear antigen (PCNA) gene consists of a promoter region (-168 to +24 with respect to the transcription initiation site) and an upstream region containing three homeodomain protein binding sites (HDB) (-357 to -165). The PCNA gene regulatory regions with HDB (-607 to +137) or without HDB (-168 to +137) were fused with the lacZ and transgenic flies were established by P-element-mediated transformation. Male transgenic flies were crossed with wild-type females, and zygotic expression of the lacZ was monitored by quantitative beta-galactosidase assay, at various stages of development. Expression of the lacZ was high in embryos, first and second instar larvae, and adult females, and low at other stages of development. Only a marginal difference in expression was observed between flies carrying the homeodomain protein binding region and those not carrying it. Spatial pattern of the lacZ expression in the embryo visualized by immunostaining with the anti-lacZ antibody was similar to the distribution of the endogenous PCNA protein. Here, too, only a marginal difference was observed between transgenic flies carrying two different constructs of the PCNA lacZ. In genetic crossing experiments of transgenic flies with those carrying mutation in homeobox genes, no significant change in the lacZ expression pattern was observed. However, when male transgenic flies were crossed with female flies homozygous for a torso gain-of-function allele, repression of the lacZ expression was observed in the central region of the embryo. Because these local changes in the lacZ expression depend on the homeodomain protein binding region, unidentified homeodomain proteins are probably involved. Our results suggest that the promoter region is practically sufficient for expression of the PCNA gene and that the homeodomain protein binding region functions as a silencer when torso is activated ectopically.
果蝇增殖细胞核抗原（PCNA）基因的调控区域由一个启动子区域（相对于转录起始位点为-168至+24）和一个上游区域组成，该上游区域包含三个同源异型域蛋白结合位点（HDB）（-357至-165）。将带有HDB（-607至+137）或不带有HDB（-168至+137）的PCNA基因调控区域与lacZ融合，并通过P因子介导的转化建立转基因果蝇。雄性转基因果蝇与野生型雌性果蝇杂交，并通过定量β-半乳糖苷酶测定法在发育的各个阶段监测lacZ的合子表达。lacZ在胚胎、一龄和二龄幼虫以及成年雌性果蝇中表达较高，而在发育的其他阶段表达较低。在携带同源异型域蛋白结合区域的果蝇和不携带该区域的果蝇之间，仅观察到表达上的微小差异。用抗lacZ抗体进行免疫染色观察到的胚胎中lacZ表达的空间模式与内源性PCNA蛋白的分布相似。同样，在携带两种不同PCNA lacZ构建体的转基因果蝇之间也仅观察到微小差异。在转基因果蝇与携带同源异型盒基因突变的果蝇的遗传杂交实验中，未观察到lacZ表达模式的显著变化。然而，当雄性转基因果蝇与躯干功能获得性等位基因纯合的雌性果蝇杂交时，在胚胎的中央区域观察到lacZ表达受到抑制。由于lacZ表达的这些局部变化依赖于同源异型域蛋白结合区域，可能涉及未鉴定的同源异型域蛋白。我们的结果表明，启动子区域实际上足以驱动PCNA基因的表达，并且当躯干异位激活时，同源异型域蛋白结合区域起沉默子的作用。
