The photosynthetic cyanobacterium Synechocystis sp. strain PCC 6803 uses a complex genetic program to control its physiological response to alternating light conditions. To study this regulatory program time-series experiments were conducted by exposing Synechocystis sp. to serial perturbations in light intensity. In each experiment whole-genome DNA microarrays were used to monitor gene transcription in 20-min intervals over 8-and 16-h periods. The data was analyzed using time-lagged correlation analysis, which identifies genetic interaction networks by constructing correlations between time-shifted transcription profiles with different levels of statistical confidence. These networks allow inference of putative cause-effect relationships among the organism's genes. Using light intensity as our initial input signal, we identified six groups of genes whose time-lagged profiles possessed significant correlation, or anti-correlation, with the light intensity. We expanded this network by using the average profile from each group of genes as a seed, and searching for other genes whose time-lagged profiles possessed significant correlation, or anti-correlation, with the group's average profile. The final network comprised 50 different groups containing 259 genes. Several of these gene groups possess known light-stimulated gene clusters, such as Synechocystis sp. photosystems I and II and carbon dioxide fixation pathways, while others represent novel findings in this work.The DNA microarray has become an established tool for the parallel monitoring of gene expression profiles. Most common experimental design strategies observe static gene expression differences between conditions, such as disease versus nondisease case comparisons. While such experiments generate information for diagnostic applications, they are not well suited for uncovering the roles of these genes in the larger context of cellular regulation.Dynamic transcriptional data allow the formation of gene clusters with similar temporal expression profiles. The various forms of clustering Alter et al. 2000;Holter et al. 2000) employed to date have produced valuable information, including potential gene relationships and the identity of transcription factor binding motifs. These methods, however, are limited in their ability to infer causality or directional relationships between genes. The results of clustering algorithms often yield relations such as "gene A is a good predictor of gene B," which is an equivalent statement to "gene B is a good predictor of gene A." Neither Bayesian networks (Friedman et al. 2000), nor information theory-based approaches (Somogyi and Fuhrman 1997) have made use of the sequential nature of time-series data in current applications. Conversely, when enough time points are available to prevent over fitting the data and find statistically significant correlations, a discovery method to uncover potential causal relationships among genes may be attempted. Directionality can be added to these probabilistic networks by determining the...

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.

Cole BK, Kuhn NS, Green-Mitchell SM, Leone KA, Raab RM, Nadler JL, Chakrabarti SK. 12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response.