The most enriched cluster upon Etoposide treatment is the p53-signaling pathway, followed by the functional categories of nuclear envelope and cell cycle regulation (Table 4).
The differences among tree topologies based on these functional categories can be caused by various factors, including different selective constraints imposed by the functional categories that were involved in various metabolic networks [ 26- 31].
The resulting tree topology was clearly dominated by the functional categories that are known for C domains (as described in the previous section), rather than species phylogeny or substrate specificity alone.
Here, accumulated expression changes in predefined gene sets (as given by the functional categories in Cyanobase or KEGG pathways) were assessed for upregulation or downregulation at a given time point.
We used clones from normalized and subtracted cDNA libraries as well as genes selected by the functional categories of Gene Ontology for inclusion onto a microarray targeted at characterizing transcriptome responses to environmental stressors.
To check for such a bias, we verified whether the results were affected by the functional categories of the gene surveyed and by codon usage, which can be used as a proxy for expression levels as well as selective constraints on the protein (Stoletzki et al. 2005; Bierne and Eyre-Walker 2006; Sauvage et al. 2007).
bThe probability (P) of observing a given number of isotigs within the functional category by chance using the hypergeometric distribution.
In addition to the increased statistical power by grouping shRNAs according to the functional category of their target genes, this method also drastically reduces the problem of off-target effects, as it is unlikely that multiple shRNAs have a similar off-target effect in a specific GO group.
Then we determined the functional category of each gene by using the functional category scheme (version 2.1) retrieved from FuncatDB.
By contrast, the functional categories enriched during wake included cell metabolism (Adcy10, Foxf2, Cox8a, Cox19 and Ppp1r3c), nucleotide binding (Gem, Pak3, Rhoh, Trio, Kif15 and Ttl), anatomical structure development (Klf4, Cdh6, Ptp4a1, Uhrf1, Trp53, Eif2b5 and Hoxc6), endocytosis (Mrc2, Ehd2, Hip1 and Mertk), and ion binding (Kcnmb2, Kcnh5, Kcnj1 and Cacna1a).
