Functional Genomics of Dictyostelium Development

盘基网柄菌发育的功能基因组学

• 批准号: 6701768
• 负责人: WILLIAM F LOOMIS
• 金额: $ 28.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6701768
• 关键词: Dictyostelium; biotechnology; cell type; computer assisted sequence analysis; cyclic AMP; developmental genetics; functional /structural genomics; fungal genetics; gene expression; gene mutation; microarray technology; nucleic acid sequence; polymerase chain reaction; protein kinase A; transcription factor

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.

使用携带5、569个cDNA的微阵列以及几百个先前定义的dictyostelium基因，我们可以在整个24小时周期中检查几乎所有发育基因的表达模式。基因将根据其活性并用于定义发育阶段的时间和细胞类型聚类，并深入了解生理上显着的分化。该系统中开发的同步是我们具有统计学意义的2小时时间分辨率。通过微阵列分析在特定发育基因中携带零突变的菌株的微阵列分析，可以进一步定义在发育过程中协调和调节细胞功能的遗传网络。内部cAMP和cAMP依赖性蛋白激酶PKA的相对发育作用将由缺乏一个或多个负责积累cAMP反应的基因的菌株的微阵列分析来确定。我们有4个一般目标：1）将针对NC44和AX4菌株建立详细的基因表达发展模式，并带有整个靶标的微阵列。基因将基于其细胞类型的特异性和表达谱。 2）微阵列表达分析将对具有改变PKA调节的菌株进行，包括那些突变，这些突变影响了这些成分的腺苷酸酶，营地磷酸二酯酶，cAMP受体，PKA和调节剂。这些突变中有几个会影响发展速度。它们对表达概况的时间后果将为聚类增加另一个维度。 3）转录因子MYBB，GBF，STAT1和MYBC的全部发育作用将通过比较缺乏这些因素的突变菌株中的簇谱与野生型菌株中的群集谱进行评估。 4）各种特定发育基因中零突变对基因簇表达的影响将用于构建可能解释该系统中时间和细胞类型特定区别的因果事件网络。在扩展微阵列分析以包括进一步的发育突变体时，将测试从此类网络中得出的预测。