Applications of Functional Genomics in Studies of Yeast Signaling Networks and Genome Structure.

Abstract

Genomic and proteomic approaches combined with traditional methods in molecular and cell biology have been applied to the model organism Saccharomyces cerevisiae, resulting in the identification of links between nitrogen-response pathways and the discovery of previously overlooked genes in the yeast genome. Pseudohyphal growth and autophagy have been studied separately as nitrogen stress response pathways. Pseudohyphal growth refers to a developmental transition in yeast, wherein nitrogen stress results in the formation of branched and elongated filaments, called pseudohyphae. Autophagy is a stress response induced by conditions of nitrogen deprivation in which proteins are trafficked to the vacuole for degradation and recycling. Our studies using microarray-based expression profiling revealed extensive upregulation of the components within the autophagy pathway during early pseudohyphal growth. While both pathways are activated upon nitrogen stress, the inhibition of autophagy results in increased pseudohyphal growth. This result suggests a model in which autophagy mitigates nutrient stress, delaying the onset of pseudohyphal growth; conversely, inhibition of autophagy exacerbates nitrogen stress, resulting in precocious and overactive pseudohyphal growth. Further phenotypic analysis of pseudohyphal growth upon overexpression of autophagy-related (ATG) genes shows that overexpression of several ATG genes inhibits pseudohyphal growth. Since overexpression of ATG genes does not significantly affect autophagic activity or cellular nitrogen stress, this result suggests that additional undefined regulatory mechanism regulate the interrelationship between these processes. In a separate study, a collection of 276 genes encoding caboxy-terminal fusions to yellow fluorescent protein has been constructed. This plasmid-based collection consisting of genes functioning as kinases, transcription factors and signaling proteins serves as a toolkit for future large-scale localization studies. In the last part of my thesis, the identification and characterization of a novel nested anstisense gene, NAG1, is described. NAG1 is entirely within the coding sequence of YGR031W in an antisense orientation on the opposite strand. Further analysis shows that NAG1 plays a role in cell wall biogenesis and is under control of the cell wall integrity pathway. Beyond its function in cell wall biogenesis, NAG1 is noteworthy in that it represents the first example of a nested protein-coding gene in the yeast genome.