Cluster analysis of dynamic parameters of gene expression

Cluster analysis has proven to be a valuable statistical method for analyzing whole genome expression data. Although clustering methods have great utility, they do represent a lower level statistical analysis that is not directly tied to a specific model. To extend such methods and to allow for more sophisticated lines of inference, we use cluster analysis in conjunction with a specific model of gene expression dynamics. This model provides phenomenological dynamic parameters on both linear and non-linear responses of the system. This analysis determines the parameters of two different transition matrices (linear and nonlinear) that describe the influence of one gene expression level on another. Using yeast cell cycle microarray data as test set, we calculated the transition matrices and used these dynamic parameters as a metric for cluster analysis. Hierarchical cluster analysis of this transition matrix reveals how a set of genes influence the expression of other genes activated during different cell cycle phases. Most strikingly, genes in different stages of cell cycle preferentially activate or inactivate genes in other stages of cell cycle, and this relationship can be readily visualized in a two-way clustering image. The observation is prior to any knowledge of the chronological characteristics of the cell cycle process. This method shows the utility of using model parameters as a metric in cluster analysis.     

Cold fission: splitting the pombe cell at room temperature

The mechanisms responsible for cytokinesis and its coordination with other events of the cell cycle are poorly understood. Genetic studies of cytokinesis in fission yeast are one useful approach to this problem. A number of conditional mutants of fission yeast that show defects in the formation of the septum of cytokinesis have been identified. Cloning of the genes affected in these mutants has begun to shed light upon the elements required to direct the construction of the division septum and also upon how the initiation of septum formation may be coordinated with mitosis.     

Histone gene transcription: A model for responsiveness to an integrated series of regulatory signals mediating cell cycle control and proliferation/differentiation interrelationships

Histone gene expression is restricted to the S-phase of the cell cycle. Control is at multiple levels and is mediated by the integration of regulatory signals in response to cell cycle progression and the onset of differentiation. The H4 gene promoter is organized into a series of independent and overlapping regulatory elements which exhibit selective, phosphorylation-dependent interactions with multiple transactivation factors. The three-dimensional organization of the promoter and, in particular, its chromatin structure, nucleosome organization, and interactions with the nuclear matrix may contribute to interrelationships of activities at multiple promoter elements. Molecular mechanisms are discussed that may participate in the coordinate expression of S-phase-specific core and H1 histone genes, together with other genes functionally coupled with DNA replication.     

Protein feature based identification of cell cycle regulated proteins in yeast

DNA microarrays have been used extensively to identify cell cycle regulated genes in yeast; however, the overlap in the genes identified is surprisingly small. We show that certain protein features can be used to distinguish cell cycle regulated genes from other genes with high confidence (features include protein phosphorylation, glycosylation, subcellular location and instability/degradation). We demonstrate that co-expressed, periodic genes encode proteins which share combinations of features, and provide an overview of the proteome dynamics during the cycle. A large set of novel putative cell cycle regulated proteins were identified, many of which have no known function.     

Systematic identification of novel, essential host genes affecting bromovirus RNA replication

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ～100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ～900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ～81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.     

An inducible expression vector for both fission and budding yeast

We have developed a vector system for inducible gene expression in both fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae). The autonomously replicating expression vector contains multiple glucocorticoid response elements, rendering a linked promoter inducible 20-70-fold by glucocorticoid hormones in the presence of the mammalian glucocorticoid receptor. A polylinker with several unique cloning sites allows insertion of cDNAs of interest. Glucocorticoids are gratuitous signalling molecules in yeast, exerting little or no effect on the expression of genes other than those fused to the regulated promoter.