Browsing by Subject "Yeast fungi--Genetics"
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Item Development of imaging-based high-throughput genetic assays and genomic evaluation of yeast gene function in cell cycle progression(2007-12) Niu, Wei; Marcotte, Edward M.Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.Item Genetic interactors of the Cdc42 GTPase effectors Gic1 and Gic2: their identification and functions in budding yeast cell polarity(2004) Gandhi, Meghal Kanaiyalal; Chan, Clarence S. M.Gic1 and Gic2 are structurally and functionally related effectors of the evolutionarily conserved Cdc42 GTPase in Saccharomyces cerevisiae. Like many other effectors of Cdc42, Gic1 and Gic2 function in the process of polarized cell growth. In the absence of both Gic1 and Gic2, yeast cells exhibit depolarized actin cytoskeleton and polarized growth defects at elevated temperatures. To obtain further insight into the biological role of Gic1 and Gic2, genetic approaches were used to identify functionally interacting partners of these proteins. A screen for multi-copy suppressors of the temperature-sensitivity of gic1 gic2 cells identified many genes (including AXL2, BNI1, CLN2, MSB1, MSB2, RSR1 and STE20) that have known roles in polarized cell growth. In addition, two pairs of structurally related genes - VHS2 and MLF3, MGC1 and TOS2 - with no previously reported functions were also identified. Functional characterization of VHS2 and MLF3 revealed their role in a pathway that affects the actin cytoskeleton organization and cell wall integrity. This pathway is functionally redundant to that mediated by GIC1 and GIC2. Functional characterization of MGC1 and TOS2 indicated that these genes function in the process of polarized growth, particularly in the process of cytokinesis. A genome-wide Synthetic Genetic Analysis identified more than 30 nonessential genes as those whose function overlaps with that of GIC1 and GIC2. Mutation in each of these genes exacerbates the growth defect of gic1 gic2 cells. As expected, some of these genes are involved in polarity-related functions, such as actin cytoskeleton organization, bud-site selection and cell wall biosynthesis. Others participate in a variety of biological processes, including organelle biogenesis, secretion and vesicular transport. The latter finding suggests that GIC1 and GIC2 may have function outside the scope of actin cytoskeleton organization. Taken together, the work presented here has uncovered the function of four previously uncharacterized genes in polarized cell growth. It has also provided hints to additional potential functions of GIC1 and GIC2. Further exploration of these functions might provide important links between Cdc42 signaling and cellular processes such as organelle biogenesis, secretion and vesicular transport, all of which need to be executed coordinately during polarized cell growth.