Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis

2011 ◽  
Vol 7 (9) ◽  
pp. 2589 ◽  
Author(s):  
Peter W. Bircham ◽  
David R. Maass ◽  
Christina A. Roberts ◽  
Poh Y. Kiew ◽  
Yee S. Low ◽  
...  
Keyword(s):  
High Throughput ◽  
Secretory Pathway ◽  
Synthetic Genetic Array ◽  
Array Analysis

scholarly journals Synthetic Genetic Array Analysis of the PtdIns 4-kinase Pik1p Identifies Components in a Golgi-specific Ypt31/rab-GTPase Signaling Pathway

2005 ◽  
Vol 16 (2) ◽  
pp. 776-793 ◽  
Author(s):  
Vicki A. Sciorra ◽  
Anjon Audhya ◽  
Ainslie B. Parsons ◽  
Nava Segev ◽  
Charles Boone ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Rab Gtpase ◽  
Secretory Function ◽  
Synthetic Genetic Array ◽  
Array Analysis ◽  
Biologically Relevant ◽  
Specific Subset ◽  
Associated Proteins

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.

scholarly journals High throughput comparative genomic hybridization array analysis of multifocal urothelial cancers

2006 ◽  
Vol 97 (8) ◽  
pp. 746-752 ◽  
Author(s):  
Hiroaki Kawanishi ◽  
Takeshi Takahashi ◽  
Masaaki Ito ◽  
Jun Watanabe ◽  
Shin Higashi ◽  
...  
Keyword(s):  
Comparative Genomic Hybridization ◽  
High Throughput ◽  
Comparative Genomic ◽  
Genomic Hybridization ◽  
Array Analysis ◽  
Comparative Genomic Hybridization Array

Synthetic Genetic Array Analysis

2016 ◽  
Vol 2016 (4) ◽  
pp. pdb.prot088807 ◽  
Author(s):  
Elena Kuzmin ◽  
Michael Costanzo ◽  
Brenda Andrews ◽  
Charles Boone
Keyword(s):  
Synthetic Genetic Array ◽  
Array Analysis

scholarly journals High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

2021 ◽  
Author(s):  
◽  
Liam D P Sampson
Keyword(s):  
Structure Analysis ◽  
Small Molecule ◽  
High Throughput ◽  
Gene Networks ◽  
Chemical Structure ◽  
Resistance Mutation ◽  
Synthetic Genetic Array ◽  
Compound Libraries ◽  
Drug Mechanism ◽  
A Genome

<p>The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation.</p>

scholarly journals High Throughput Drug Discovery in S. Cerevisiae: the Characterisation of FC-592 and FC-888

2021 ◽  
Author(s):  
◽  
Liam D P Sampson
Keyword(s):  
Structure Analysis ◽  
Small Molecule ◽  
High Throughput ◽  
Gene Networks ◽  
Chemical Structure ◽  
Resistance Mutation ◽  
Synthetic Genetic Array ◽  
Compound Libraries ◽  
Drug Mechanism ◽  
A Genome

<p>The discovery and characterisation of novel small molecule drug candidates is a medical priority. Recent advances in synthetic organic chemistry allow the de novo production of diversity oriented synthetic compound libraries and synthetic modification of natural products to provide candidate compounds for screening as potential therapeutics, bioactive agents or genetic probes. Small drugs function through interaction with complex genetic networks and pathways. However, it is difficult to characterise these interactions on a genome wide level to achieve understanding of drug mechanism. Here, discovery based approaches are utilised to achieve system wide parsing of biological mechanism, in an attempt to characterise the action of novel synthetic compounds and natural product derivatives. Chemical genomic analysis allows for such understanding by examining growth profiles of a genomic deletion library of Saccharomyces cerevisiae mutants in the presence of sub-inhibitory concentrations of drug. The gene targets of small molecule compounds can be identified by noting deletion strains which display increased sensitivity, indicating chemical interaction with the associated gene network. In addition, the development and characterisation of resistant mutants can be used to identify putative drug targets. In this strategy, characterisation of the mechanism of resistance gives insight into drug mode-of-action. This study develops a high throughput yeast inhibition assay to identify bioactive compounds from a synthetic organic compound library, and attempts to characterise mechanism of action by establishing a profile of each compound’s interaction with these gene networks; and mapping a resistance mutation to provide evidence of inhibitory mechanism. Two candidate compounds are identified, FC-592 and FC-888. FC-592 displayed cytostatic inhibition. Further, yeast tag microarray homozygous profiling (HOP), chemical structure analysis, and cell-cycle analysis via flow cytometry for this compound provided evidence for a mechanism of poor specificity that targets glycoprotein biosynthesis and the secretory (Sec) pathway, as well as the cell-division cycle (CDC) pathway. Attempts to characterise a mutant resistant to this compound via synthetic genetic array mapping were unsuccessful when the resistance mutation proved to mediate a slow growth phenotype, abrogating the Synthetic Genetic Array Mapping approach utilised. Pending further analysis, it is suggested that this compound could have a role as a genetic probe in future exploration of the Sec and CDC pathways. Chemical structure analysis and a non-specific HOP screen chemigenomic profile suggested that FC-888 is an alkylating agent with a broad affinity for cellular nucleophiles. The compound demonstrates cytotoxic activity, and its efflux is not mediated by the pleiotropic drug resistance (PDR) network. It is suggested that the compound could find utility as a probe dissecting processes related to cellular defence against non-DNA specific alkylation.</p>

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