PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-3 (3)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
Document Types
1.  Sequence-Specific Biosensors Report Drug-Induced Changes in Epigenetic Silencing in Living Cells 
DNA and Cell Biology  2012;31(Suppl 1):S-2-S-10.
Treatment with demethylating drugs can induce demethylation and reactivation of abnormally silenced tumor suppressor genes in cancer cells, but it can also induce potentially deleterious loss of methylation of repetitive elements. To enable the observation of unwanted drug effects related to loss of methylation of repetitive DNA, we have developed a novel biosensor capable of reporting changes in DNA accessibility via luminescence, in living cells. The biosensor design comprises two independent modules, each with a polydactyl zinc finger domain fused to a half intein and to a split-luciferase domain that can be joined by conditional protein splicing after binding to adjacent DNA targets. We show that an artificial zinc finger design specifically targeting DNA sequences near the promoter region of the L1PA2 subfamily of Line-1 retroelements is able to generate luminescent signals, reporting loss of epigenetic silencing and increased DNA accessibility of retroelements in human cells treated with the demethylating drugs decitabine or 5-azacytidine.
doi:10.1089/dna.2011.1537
PMCID: PMC3460616  PMID: 22313050
2.  Systematic Definition of Protein Constituents along the Major Polarization Axis Reveals an Adaptive Reuse of the Polarization Machinery in Pheromone-Treated Budding Yeast 
Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the ‘shmoo’) by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.
Upon sensing mating pheromone, budding yeast cells form a mating projection (the ‘shmoo’) that serves as a model for polarized cell growth, involving cytoskeletal/cytosolic restructuring and directed vesicular transport. We developed a cell microarray-based imaging assay for measuring localization of the yeast proteome during polarized growth. We find major organelles ordered along the polarization axis, localize 74 proteins to the growth tip, and observe adaptive reuse of general polarization machinery.
doi:10.1021/pr800524g
PMCID: PMC2651748  PMID: 19053807
Proteomics; polarized growth; subcellular localization; pheromone response; yeast
3.  Systematic profiling of cellular phenotypes with spotted cell microarrays reveals mating-pheromone response genes 
Genome Biology  2006;7(1):R6.
Spotted cell microarrays were developed for measuring cellular phenotypes on a large scale and used to identify genes involved in the response of yeast to mating pheromone.
We have developed spotted cell microarrays for measuring cellular phenotypes on a large scale. Collections of cells are printed, stained for subcellular features, then imaged via automated, high-throughput microscopy, allowing systematic phenotypic characterization. We used this technology to identify genes involved in the response of yeast to mating pheromone. Besides morphology assays, cell microarrays should be valuable for high-throughput in situ hybridization and immunoassays, enabling new classes of genetic assays based on cell imaging.
doi:10.1186/gb-2006-7-1-r6
PMCID: PMC1431703  PMID: 16507139

Results 1-3 (3)