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

Dictyosteelium discoideum多细胞组织形成后，分化出前柄细胞和前孢子细胞，为了了解前孢子特异基因表达的分子机制，Dp 87和Sp 96（编码孢子外壳蛋白Sp 96）是很好的靶基因，因为'run on'实验表明它们只在前孢子细胞中转录。我们通过制备包括与报告基因缀合的不同长度的5 '上游区的嵌合基因并检测报告基因产物在转化细胞中的表达，鉴定了Dp 87和SP 96基因中每一个正确转录的至少一个重要顺式调节区。Sp 96基因的区域与特别出现在蛞蝓阶段的DNase-I超敏感位点之一吻合得很好。在Dp 87基因的情况下，我们还确定了一个可能的反式作用因子，它特异性地结合顺式区，该因子只在蛞蝓细胞核中发现。使用该区域的西南分析表明，在核提取物中有三条蛋白带。通过DNA酶I足迹法确定结合位点。现在我们正在寻找这种结合蛋白的cDNA。为了了解Dp 87和Sp 9 G基因的表达是否受已知控制前孢子细胞分化的cAMP调节，我们检查了这些基因在从蛞蝓解聚的细胞中的转录。北方分析和“运行”测定的结果表明，Dp 87和Sp 96基因在解聚后很快停止其转录，并且其已经存在的mRNA迅速降解。另一方面，向解聚的细胞中加入外源性cAMP通过信号转导诱导这些基因的转录立即恢复，并且合成的mRNA被新合成的蛋白质稳定。

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).