Regulation of expression of cell-type-specific genes of Dictyostelium discoideum

盘基网柄菌多细胞组织形成后，前柄细胞和前孢子细胞在其中分化。为了了解前孢子特异基因表达的分子机制，Dp87和Sp96(编码孢子外壳蛋白Sp96)是很好的靶基因，因为Run On分析表明它们只在前孢子细胞中转录。我们通过使含有不同长度的5‘上游区的嵌合基因与报告基因结合，并检测报告基因产物在转化细胞中的表达，至少确定了Dp87和SP96基因正确转录的一个重要顺式调控区。Sp96基因的区域与一个DNase-I敏感部位非常吻合，该部位专门出现在鼻涕虫阶段。在Dp87基因的例子中，我们还发现了一个可能的与顺式区域特异结合的转录因子，该因子仅在鼻涕虫细胞核中发现。使用该区域的西南分析表明，在核提取物中有三条蛋白条带。我们通过DNase-I足印确定了结合部位。现在我们正在寻找这种结合蛋白的cDNA.为了了解Dp87和Sp9G基因的表达是否受到控制前孢子细胞分化的cAMP的调控，我们检测了这些基因在鼻涕虫分解的细胞中的转录。Northern分析和Run On分析的结果表明，Dp87和Sp96基因在解聚后不久就停止转录，它们已经存在的mRNAs迅速降解。另一方面，外源性cAMP加入到分解的细胞中，通过信号转导立即恢复这些基因的转录，合成的mRNAs被新合成的蛋白质稳定(S)。

After formation of multicellular tissue of Ddictyostelium discoideum, prestalk and prespore cells differentiate within it. To understand the molecular mechanisms of expression prespore specific genes, Dp87 and Sp96 (encoding spore coat protein Sp96) are good target genes because the 'run on' assays showed that they transcribed only in prespore cells. We identified at least one of the important cis-regulating regions for correct transcription of each of Dp87 and SP96 gene, by making chimeric genes including different lengths of 5'upstream regions conjugated with reporter genes and examining the expression of the reporter gene products in transformed cells. The region for Sp96 gene coincided well with one of the DNase-I hypersensitive sites that specifically appear at the slug stage. In the case of Dp87 gene, we also identified a possible transacting factor which specifically bind the cis-region and the factor was found only in slug cell nuclei. South-western analyses using this region indicated that there were three protein bands in the nuclear extract. We decided the binding site by DNase-I foot printing. Now we are looking for the cDNA of this binding protein. To understand whether the expression of Dp87 and Sp9G genes are regulated by cAMP which is known to control prespore cell differentiation, we examined the transcription of these genes in cells disaggregated from slugs. The results of northern analyses and 'run on' assays indicate that both Dp87 and Sp96 genes stop their transcription soon after disaggregation and their mRNAs already present are degraded rapidly. On the other hand, addition of exogenous cAMP to disaggregated cells induces immediate resumption of transcription of these genes through transduction of signals and synthesized mRNAs are stabilized by newly synthesized protein(s).