Genome analysis of the cellular slime molds to understand the genetic system controlling cell differentiation

细胞黏菌盘基网柄菌被用来研究发育和分化的遗传控制。这种生物通常以单细胞阿米巴虫的形式繁殖，但在饥饿时聚集并构建多细胞结构。首先，进行大规模的cDNA分析以建立遗传资源。从5个发育阶段的细胞构建了14个cDNA文库，测定了95,884个克隆的核苷酸序列，其中包括72,703个全长克隆。这些序列被组装成6790个独立的基因，相当于预测的12,500个基因的54%。这些资源随后被用于反向遗传和生物信息学分析。取得了以下研究成果：1.获得了正常发育过程中的基因表达模式，显示了细胞聚集后表达基因的整体剧烈变化。整装原位杂交显示，在茎前区域形成了细小的花纹。提取了260个基因编码可能的转录因子，并通过实时定量聚合酶链式反应获得了其中40个基因的精确表达模式。13个基因被打乱。根据野生型和中断突变体在发育过程中的基因表达模式，利用S系统计算得到了9个可能的转录因子之间的遗传网络。通过比对全长cDNA和基因组序列获得上游启动子序列，并参照表达特异性提取候选顺式调控元件。

The cellular slime mold, Dictyostelium discoideum, was used to understand the genetic control of development and differentiation. This organism normally proliferates as unicellular amoebae but gathers and constructs multicellular structures upon starvation. First, large scale analysis of cDNAs was performed to establish the genetic resources. Fourteen cDNA libraries were constructed from the cells at 5 developmental stages, and the nucleotide sequences of 95,884 clones, including 72,703 full-length ones, were determined. The sequences were assembled into 6,790 independent genes, which corresponded to 54% of the predicted 12,500 genes. These resources were then used for reverse-genetic and bioinformatic analyses. The following results were obtained.1. Gene expression patterns during normal development were obtained showing occurrence of the overall drastic changes in expressed genes after cell aggregation. Whole mount in situ hybridization revealed the fine pattern formation in the prestalk region.2. Two hundred and sixty genes were extracted to encode probable transcription factors, and precise expression patterns were obtained by real-time PCR for 40 of them. Thirteen genes were disrupted.3. The Genetic network among 9 probable transcription factors was obtained using the S-system calculation based on the gene expression patterns during development in the wild type and disruption mutants.4. Upstream promoter sequences were obtained by aligning the full-length cDNA and genome sequences, and candidate cis regulatory elements were extracted referring to the expression specificities.