Functional Genomics of Dictyostelium Development

使用携带5,569个cDNA的微阵列以及先前定义的数百个网柄基底菌基因，我们可以检查几乎所有发育基因在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分类，用于定义发育阶段以及洞察生理上显著的分化。在这个系统中发展的同步性是这样的，我们有统计上显着的2小时的时间分辨率。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携带特定发育基因零突变的菌株的微阵列分析来进一步定义。内部cAMP和cAMP依赖的蛋白激酶PKA的相对发育作用将通过对缺乏一个或多个负责积累对cAMP反应的基因的菌株的微阵列分析来确定。我们有4个总体目标：1)通过携带全套靶点的微阵列，为NC44和AX4菌株建立详细的基因表达发育模式。基因将根据其细胞类型特异性和表达谱进行分类。2)将对PKA调节改变的菌株进行微阵列表达分析，包括那些影响腺苷环化酶、cAMP磷酸二酯酶、cAMP受体、PKA和这些成分的调节子的突变菌株。这些突变中有几个会影响发育速度。它们对表达特征的时间影响将为集群增加另一个维度。3)通过比较缺乏转录因子MybB、GBF、STAT1和MybC的突变株和野生型菌株的簇谱，可以评估这些转录因子在整个发育过程中的作用。4)各种特定发育基因零突变对基因簇表达的影响将被用来构建因果事件网络，这可能解释该系统中时间和细胞类型的特定分化。当微阵列分析扩展到包括进一步的发育突变时，从这种网络得出的预测将得到检验。

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.