[33] High-throughput methods for the large-scale analysis of gene function by transposon tagging

Kumar, Anuj; Etages, Shelley Ann des; Coelho, P. S. R.; Roeder, G. Shirleen; Snyder, M

doi:10.1016/s0076-6879(00)28418-8

Cited by 36 publications

(26 citation statements)

References 24 publications

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“…The resulting kinase-V5-His6X fusion protein has a His6X tag for protein purification and a 14-amino-acid V5 epitope tag (GKPIPNPLLGLDST) for antibody detection. Sequence-verified plasmids were transformed into yeast (Y258: MATa, pep4-3, his4-580, ura3-52, leu2-3, 112) using a standard lithium acetate transformation protocol as described previously (Kumar et al 2000), and the resulting strains were used to purify the V5-kinase fusion proteins (see Supplemental Table S7 for strains used in this study). Yeast cultures were grown overnight at 30°C in 6 mL of Sc-Ura/2% dextrose and diluted to OD600 = 0.1 the next day in 400 mL of Sc-Ura/2% raffinose.…”

Section: Kinase-v5 Probe Preparationmentioning

confidence: 99%

Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes

Fasolo¹,

Sboner²,

Sun³

et al. 2011

Genes Dev.

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Protein kinases are key regulators of cellular processes. In spite of considerable effort, a full understanding of the pathways they participate in remains elusive. We globally investigated the proteins that interact with the majority of yeast protein kinases using protein microarrays. Eighty-five kinases were purified and used to probe yeast proteome microarrays. One-thousand-twenty-three interactions were identified, and the vast majority were novel. Coimmunoprecipitation experiments indicate that many of these interactions occurred in vivo. Many novel links of kinases to previously distinct cellular pathways were discovered. For example, the well-studied Kss1 filamentous pathway was found to bind components of diverse cellular pathways, such as those of the stress response pathway and the Ccr4-Not transcriptional/translational regulatory complex; genetic tests revealed that these different components operate in the filamentation pathway in vivo. Overall, our results indicate that kinases operate in a highly interconnected network that coordinates many activities of the proteome. Our results further demonstrate that protein microarrays uncover a diverse set of interactions not observed previously.

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Section: Kinase-v5 Probe Preparationmentioning

confidence: 99%

Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes

Fasolo¹,

Sboner²,

Sun³

et al. 2011

Genes Dev.

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“…Protein products were subsequently localized by indirect immunofluorescence using monoclonal antibodies directed against the V5 epitope. To accommodate higher throughput, yeast cells were prepared for immunofluorescence analysis in a 96-well format as described (Kumar et al 2000b).…”

Section: Genome-wide Epitope-tagging and Large-scale Immunolocalizationmentioning

confidence: 99%

Subcellular localization of the yeast proteome

et al. 2002

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Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability-a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. A global understanding of the molecular mechanisms underpinning cell biology necessitates an understanding not only of an organism's genome but also of the protein complement encoded within this genome (the proteome). In the past, data regarding an organism's proteome have typically been accumulated piecemeal through studies of a single protein or cell pathway. Genomic methodologies have altered this paradigm: a variety of approaches are now in place by which proteins may be directly analyzed on a proteome-wide scale. Chromatography-coupled mass spectrometry (Gygi et al. 1999;Washburn et al. 2001), large-scale two-hybrid screens (Uetz et al. 2000;Ito et al. 2001;Tong et al. 2002), immunoprecipitation/mass spectrometric analysis of protein complexes (Gavin et al. 2002;Ho et al. 2002), and protein microarray technologies (MacBeath and Schreiber 2000;Zhu et al. 2000Zhu et al. , 2001 are yielding unprecedented quantities of protein data. Recent genomic techniques combining microarray technologies with either chromatin immunoprecipitation (Ren et al. 2000;Iyer et al. 2001) or targeted DNA methylation (van Steensel et al. 2001) have been used to globally map binding sites of chromosomal proteins in vivo. Initiatives are even underway to automate and industrialize processes by which protein structures may be solved, potentially providing a library of structural...

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“…The approach is cost-effective and applicable to a wide variety of microbes (3,4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5).…”

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confidence: 99%

Comprehensive transposon mutant library of Pseudomonas aeruginosa

Jacobs

Alwood

Thaipisuttikul

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

1,038

949

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We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300 -400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.hole-genome sequences provide the foundation for the creation of relatively complete collections of strains carrying defined mutations in individual genes. Such libraries should facilitate the comprehensive identification of genes required for a wide range of biological processes. A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach (1). Other projects, also following a strategy of gene-by-gene disruption, are underway for Escherichia coli (E. coli genome project, www. genome.wisc.edu͞functional͞tnmutagenesis.htm), and have recently been completed for Bacillus subtilis (2).An alternative strategy for generating mutant libraries consists of ''random'' whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites. The approach is cost-effective and applicable to a wide variety of microbes (3, 4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5). Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth, although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined (6)(7)(8). In this report, we describe the generation and initial phenotypic analysis of a near-saturation library of transposon insertion mutants of the opportunistic pathogen Pseudomonas aeruginos...

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[33] High-throughput methods for the large-scale analysis of gene function by transposon tagging

Cited by 36 publications

References 24 publications

Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes

Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes

Subcellular localization of the yeast proteome

Comprehensive transposon mutant library of Pseudomonas aeruginosa

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