Functional Genomics of Dictyostelium Development

利用携带5569个cDNA以及几百个先前定义的网骨藻基因的微阵列，我们可以检查几乎所有发育基因在整个24小时周期中的表达模式。基因将根据它们活跃的时间和细胞类型进行聚类，并用于定义发育阶段以及深入了解生理学上的显著分化。该系统中的发育同步性使得我们具有统计学显著的2小时时间分辨率。在发育过程中协调和调节细胞功能的遗传网络可以通过对特定发育基因中携带无效突变的菌株进行微阵列分析来进一步定义。内部cAMP和cAMP依赖性蛋白激酶PKA的相对发育作用将通过缺乏一个或多个负责cAMP应答积累的基因的菌株的微阵列分析来确定。我们有四个目标：1）将用携带全套靶标的微阵列建立菌株NC 44和AX 4的基因表达的详细发育模式。基因将根据其细胞类型特异性和表达谱进行聚类。2)将对PKA调节改变的菌株进行微阵列表达分析，包括具有影响腺苷酸环化酶、cAMP磷酸二酯酶、cAMP受体、PKA和这些组分的调节剂的突变的菌株。其中一些突变影响发育速度。它们对表达谱的时间影响将为聚类增加另一个维度。3)转录因子MybB、GBF、STAT1和MybC的完整发育作用将通过比较缺乏这些因子的突变株与野生型株中的簇谱来评估。4)各种特定发育基因中的无效突变对基因簇表达的影响将用于构建因果事件网络，这些因果事件可能解释该系统中的时间和细胞类型特异性分化。当微阵列分析扩展到包括进一步的发育突变体时，将对来自这种网络的预测进行测试。

Using microarrays carrying 5, 569 cDNAs as well as several hundred previously defined Dictyostelium genes, we can examine the expression patterns of almost all developmental genes throughout the 24 hour cycle. Genes will be clustered on the basis of the time and cell type in which they are active and used to define developmental stages as well as give insight into physiologically significant differentiations. The synchrony of development in this system is such we have statistically significant 2 hour temporal resolution. The genetic networks that coordinate and modulate cellular functions during development can be further defined by microarray analyses of strains carrying null mutations in specific developmental genes. The relative developmental roles of internal cAMP and the cAMP dependent protein kinase PKA will be determined by microarray analyses of strains lacking one or more of the genes responsible for accumulation of response to cAMP. We have 4 general aims: 1) Detailed developmental patterns of gene expression will be established for strains NC44 and AX4 with microarrays carrying the full set of targets. Genes will be clustered on the basis of their cell-type specificity and expression profiles. 2) Microarray expression analyses will be carried out on strains with altered PKA regulation including those with mutations affecting adenyly cyclases, cAMP phosphodiesterases, cAMP receptors, PKA and modulators of these components. Several of these mutations affect the rate of development. Their temporal consequences to expression profiles will add another dimension to clustering. 3) The full developmental roles of the transcription factors MybB, GBF, STAT1, and MybC will be assessed by comparing cluster profiles in mutant strains lacking these factors to those in wild type strains. 4)The effects of null mutations in various specific developmental genes on the expression of clusters of genes will be used to construct networks of causal events that may account for temporal and cell-type specific differentiations in this system. Predictions derived from such networks will be tested when microarray analyses are extended to include further developmental mutants.