Large‐scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP‐fusion genomic DNA library

Ding, Da‐Qiao; Tomita, Yoshihiko; Yamamoto, Ayumu; Chikashige, Yuji; Haraguchi, Tokuko; Hiraoka, Yasushi

doi:10.1046/j.1365-2443.2000.00317.x

Cited by 140 publications

(114 citation statements)

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“…Strains used in this study are ura4-D18 and leu1-32 unless otherwise stated: PR109, wild-type (h Ϫ ); PR110, wild-type (h ϩ ); PS2343, wild-type (h Ϫ smt0); SC3250, rqh1::ura4 (h Ϫ ); SC3240, (Ding et al, 2000).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

“…If this model was correct, we would expect most of the Slx1-generated Rad22-YFP foci to occur in the DAPI-staining region of the nucleus that extends into the nucleolus. To test this hypothesis, we scored the location of Rad22-RFP foci while using a GFP fusion protein of Rrn5, an rDNA transcriptional activator, as a nucleolar marker (Figure 2A) (Ding et al, 2000). Quantification of Rad22-RFP foci was performed in wild-type, slx1⌬, and rqh1⌬ cells transformed with the plasmid expressing the Rrn5-GFP fusion protein.…”

Section: Nucleolar Localization Of Slx1-dependent Rad22 Focimentioning

confidence: 99%

“…Cells that had genomic rad22-RFP and plasmid pTA57 encoding for the Rrn5-GFP fusion protein were grown in EMM medium at 25°C until mid-log phase. Rrn5-GFP binds to rDNA and is used in this study as a marker of nucleolus (Ding et al, 2000). (A) Top, costaining of Rrn5-GFP (green, nucleolus region) and DAPI staining (blue, nonnucleolus region).…”

Section: Expression Of Slx1-r34a Dominant Negative Allele In a Rqh1⌬ mentioning

confidence: 99%

“…Identical quantification has been performed for Rad22-RFP foci. Nucleolus and nonnucleolus have been discriminated using plasmid pTA57 encoding for the Rrn5-GFP fusion protein (Ding et al, 2000). Rrn5-GFP binds to rDNA and is used as a marker of nucleolus.…”

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confidence: 99%

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Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Coulon

Noguchi

et al. 2006

MBoC

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Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate ϳ50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number. INTRODUCTIONWhile replicating the genome, DNA polymerases will sometimes stall when they encounter DNA lesions, protein complexes bound to DNA, or nucleotide starvation. Stalled forks pose serious threats to genomic integrity because they are prone to collapse or rearrangement (McGlynn and Lloyd, 2002). In contrast, there are many examples of natural replication pause and termination sites in a variety of organisms (Rothstein et al., 2000). These sites are often called replication fork barriers (RFBs). It is somewhat paradoxical that in some cases these "programmed" RFBs are thought to play important roles in maintaining genome integrity.The best-characterized RFBs occur in the ribosomal DNA genes (rDNA), which are present in long tandem repeats at one or a few chromosomal loci in most eukaryotic species, including yeasts and humans (Brewer and Fangman, 1988;Linskens and Huberman, 1988;Little et al., 1993;Gerber et al., 1997;Sanchez et al., 1998). The budding yeast Saccharomyces cerevisiae carries ϳ150 copies of the rDNA genes. The RFB is located in the nontranscribed sequence (NTS) between the 3Ј end of the 35S rRNA gene and the autonomously replicating sequence (ARS). The RFB inhibits replication fork progression in the direction opposite to rDNA transcription. This mechanism helps to prevent collisions between replication forks and the transcription machinery (Takeuchi et al., 2003). The protein fork blocking 1 (Fob1) binds DNA in the RFB region and is essential for RFB activity (Kobayashi and Horiuchi, 1996).The size of the rDNA array in S. cerevisiae is not static. For example, it will contract upon inactivation of RNA polymerase I and expands after RNA polymerase I activity is restored (Kobayashi et al., 1998). Fob1 is required for the mechanisms that control the copy number of rDNA repeats (Kobayashi et al., 2001;Johzuka and Horiuchi, 2002). The current model for the regulation of the rDNA copy number involv...

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Section: Strains and Plasmidsmentioning

confidence: 99%

Section: Nucleolar Localization Of Slx1-dependent Rad22 Focimentioning

confidence: 99%

Section: Expression Of Slx1-r34a Dominant Negative Allele In a Rqh1⌬ mentioning

confidence: 99%

mentioning

confidence: 99%

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Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Coulon

Noguchi

et al. 2006

MBoC

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“…Similar strategies for particular human and mouse cell types are currently under way in several laboratories. Even though subcellular localization has not yet been used as a major technique to identify modularity, the first inroads into this challenging experimental problem have been made from datasets that were created using tagged yeast proteins [18,19] and a small subset of green-fluorescent-protein (GFP)-conjugated human proteins with unknown function [20]. A systematic liveand fixed-cell microscopy effort to measure the localization of all signaling proteins in a mouse B-cell model is currently under way in our laboratory under the umbrella of the Alliance for Cell Signaling (http://www.afcs.org/).…”

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confidence: 99%

Fluorescence imaging of signaling networks

Meyer¹,

Teruel²

2003

Trends in Cell Biology

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Receptor-triggered signaling processes exhibit complex cross-talk and feedback interactions, with many signaling proteins and second messengers acting locally within the cell. The flow of information in this inputoutput system can only be understood by tracking where and when local signaling activities are induced. Systematic strategies are therefore needed to measure the localization and translocation of all signaling proteins, and to develop fluorescent biosensors that can track local signaling activities in individual cells. Such a biosensor tool chest can be based on two types of green fluorescent protein constructs that either translocate or undergo fluorescence-resonance-energy transfer when local signaling occurs. Broad strategies to measure quantitative, dynamic parameters in signaling networks, together with perturbation approaches, are needed to develop comprehensive models of signaling networks * .Recent technical developments have made it possible to ask how information flows from many different cellularreceptor inputs to a diverse set of physiological cell functions (outputs). Expression profiling [1], proteomics [2] and evolutionary comparison with known signaling proteins [3] can now be used to identify all predicted signaling proteins in a particular cell type. Although studies in yeast and other model organisms have been leading the way, these approaches can now be applied to obtain a list of relevant signaling proteins in a particular mouse or human cell. Given our current knowledge, one can expect a typical mammalian cell to contain somewhere between several hundred and a few thousand different signaling proteins, and more than ten second messengers. These signaling proteins and second messengers are organized in a network-like fashion, and it is likely that some of the principles of metabolic networks [4] and transcriptional networks [5,6] also apply to mammalian signaling networks.To discuss some of the unique features of the cell signaling problem, an idealized signaling network is shown in Fig. 1. As a working hypothesis, the internal structure of this input -output system can be described using three concepts: signaling pathways, signaling modules and signaling nodes. For signaling pathways, the main flow of information is sequential and goes through a linear chain of intermediate steps from the receptor to a particular cell function. This idea has been extremely powerful and has shaped our current view of signal transduction. For example, tyrosine kinase and other receptor stimuli turn on a multistep mitogenactivated-protein-kinase pathway to activate specific genes that upregulate cell growth [7].The second concept of signaling modules can be subdivided into modules that are connected by feedback involving a diffusion step (diffusible feedback systems) and modules that are physically linked complexes of signaling proteins and/or scaffolding proteins [8,9]. An example of a diffusible feedback module can be seen in the positive and negative feedback loops by which calcium and the inositol...

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The beauty of the yeast: Live cell microscopy at the limits of optical resolution

Kohlwein

2000

Microsc. Res. Tech.

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The yeast Saccharomyces cerevisiae is a very powerful system for cell biological research. Recent advances in electronic light microscopy together with the application of green fluorescent protein and other in vivo staining techniques have allowed novel and exciting insights into structural organization and dynamics of cells as small as yeast. Methods for staining yeast for microscopic inspection and for introducing tags for localization studies of proteins in living or fixed cells are summarized. Electronic light microscopy, video/deconvolution methods, and confocal laser scanning microscopy as novel tools for structural analyses, and their practical applications in yeast, are discussed. Microsc. Res. Tech. 51:511–529, 2000. © 2000 Wiley‐Liss, Inc.

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Large‐scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP‐fusion genomic DNA library

Abstract: Background: Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green¯uorescent protein (GFP)-fusion genomic DNA library of S. pombe.

Cited by 140 publications

References 74 publications

Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Fluorescence imaging of signaling networks

The beauty of the yeast: Live cell microscopy at the limits of optical resolution

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Large‐scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP‐fusion genomic DNA library

Abstract: Background: Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green¯uorescent protein (GFP)-fusion genomic DNA library of S. pombe.

Cited by 140 publications

References 74 publications

Rad22Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Rad22Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Fluorescence imaging of signaling networks

The beauty of the yeast: Live cell microscopy at the limits of optical resolution

Contact Info

Product

Resources

About

Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease

Rad22^Rad52-dependent Repair of Ribosomal DNA Repeats Cleaved by Slx1-Slx4 Endonuclease