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Zhang H, Zhang L, Chen S, Yao M, Ma Z, Yuan Y. Yeast-based evolutionary modeling of androgen receptor mutations and natural selection. PLoS Genet 2022; 18:e1010518. [PMID: 36459502 PMCID: PMC9718406 DOI: 10.1371/journal.pgen.1010518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2022] [Accepted: 11/10/2022] [Indexed: 12/05/2022] Open
Abstract
Cancer progression is associated with the evolutionary accumulation of genetic mutations that are biologically significant. Mutations of the androgen receptor (AR) are associated with the development of prostate cancer (PCa) by responding to non-androgenic hormones, and the lack of annotations in their responsiveness to hormone ligands remains a daunting challenge. Here, we have used a yeast reporter system to quickly evaluate the responsiveness of all fifty clinical AR mutations to a variety of steroidal ligands including dihydrotestosterone (DHT), 17β-estradiol (E2), progesterone (PROG), and cyproterone acetate (CPA). Based on an AR-driven reporter that synthesizes histidine, a basic amino acid required for yeast survival and propagation, the yeast reporter system enabling clonal selection was further empowered by combining with a random DNA mutagenesis library to simulate the natural evolution of AR gene under the selective pressures of steroidal ligands. In a time-frame of 1-2 weeks, 19 AR mutants were identified, in which 11 AR mutants were validated for activation by tested steroidal compounds. The high efficiency of our artificial evolution strategy was further evidenced by a sequential selection that enabled the discovery of multipoint AR mutations and evolution directions under the pressure of steroidal ligands. In summary, our designer yeast is a portable reporter module that can be readily adapted to streamline high-throughput AR-compound screening, used as a PCa clinical reference, and combined with additional bioassay systems to further extend its potential.
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Affiliation(s)
- Haoran Zhang
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, People’s Republic of China
- School of Chemical Engineering and Technology, Tianjin University, Tianjin, People’s Republic of China
| | - Lu Zhang
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, People’s Republic of China
- School of Chemical Engineering and Technology, Tianjin University, Tianjin, People’s Republic of China
| | - Shaoyong Chen
- Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America
| | - Mingdong Yao
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, People’s Republic of China
- School of Chemical Engineering and Technology, Tianjin University, Tianjin, People’s Republic of China
| | - Zhenyi Ma
- School of Basic Medical Sciences, Tianjin Medical University, Tianjin, People’s Republic of China
| | - Yingjin Yuan
- Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, People’s Republic of China
- School of Chemical Engineering and Technology, Tianjin University, Tianjin, People’s Republic of China
- * E-mail:
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Hu Y, Yu Q, Chen Y, Zhang L, Fu X, Ding L, Liu Y, Guan Z, Fang W, Chen F, Song A. Labour-saving construction of a target protein interaction network by selective culture and high-throughput sequencing. Biotechnol J 2021; 16:e2100204. [PMID: 34328672 DOI: 10.1002/biot.202100204] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2021] [Revised: 07/27/2021] [Accepted: 07/28/2021] [Indexed: 12/14/2022]
Abstract
Yeast two-hybrid (Y2H) is a classic method of screening for protein-protein interactions. However, the operation process is labor intensive and time consuming, and there is a high possibility of false positives and false negatives. Combined with wet lab operation and bioinformatics analysis, we developed a novel method of Y2H library screening using Chrysanthemum morifolium CmMPK3 as an example. The protocol can not only greatly simplify the steps of traditional Y2H library screening but also identify as many interacting proteins as possible. Furthermore, this protocol is applicable to any species, even if no genomic information is available yet.
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Affiliation(s)
- Yueheng Hu
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Qi Yu
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Yu Chen
- College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, China
| | - Luyao Zhang
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Xianrong Fu
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Lian Ding
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Ye Liu
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Zhiyong Guan
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Weimin Fang
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Fadi Chen
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
| | - Aiping Song
- State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing, China
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Moosavi B, Mousavi B, Yang WC, Yang GF. Yeast-based assays for detecting protein-protein/drug interactions and their inhibitors. Eur J Cell Biol 2017. [PMID: 28645461 DOI: 10.1016/j.ejcb.2017.06.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Understanding cellular processes at molecular levels in health and disease requires the knowledge of protein-protein interactions (PPIs). In line with this, identification of PPIs at genome-wide scale is highly valuable to understand how different cellular pathways are interconnected, and it eventually facilitates designing effective drugs against certain PPIs. Furthermore, investigating PPIs at a small laboratory scale for deciphering certain biochemical pathways has been demanded for years. In this regard, yeast two hybrid system (Y2HS) has proven an extremely useful tool to discover novel PPIs, while Y2HS derivatives and novel yeast-based assays are contributing significantly to identification of protein-drug/inhibitor interaction at both large- and small-scale set-ups. These methods have been evolving over time to provide more accurate, reproducible and quantitative results. Here we briefly describe different yeast-based assays for identification of various protein-protein/drug/inhibitor interactions and their specific applications, advantages, shortcomings, and improvements. The broad range of yeast-based assays facilitates application of the most suitable method(s) for each specific need.
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Affiliation(s)
- Behrooz Moosavi
- Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079, PR China.
| | - Bibimaryam Mousavi
- Laboratory of Organometallics, Catalysis and Ordered Materials, State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, Wuhan, 430070, PR China
| | - Wen-Chao Yang
- Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079, PR China
| | - Guang-Fu Yang
- Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079, PR China.
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Fatima S, Wagstaff KM, Lieu KG, Davies RG, Tanaka SS, Yamaguchi YL, Loveland KL, Tam PP, Jans DA. Interactome of the inhibitory isoform of the nuclear transporter Importin 13. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2017; 1864:546-561. [DOI: 10.1016/j.bbamcr.2016.12.017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Revised: 11/11/2016] [Accepted: 12/15/2016] [Indexed: 10/20/2022]
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Frey S, Lahmann Y, Hartmann T, Seiler S, Pöggeler S. Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora. Mol Microbiol 2015; 97:676-97. [PMID: 25989468 DOI: 10.1111/mmi.13054] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/05/2015] [Indexed: 01/06/2023]
Abstract
The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility.
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Affiliation(s)
- Stefan Frey
- Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University, Göttingen, Germany
| | - Yasmine Lahmann
- Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University, Göttingen, Germany
| | - Thomas Hartmann
- Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, Göttingen, Germany
| | - Stephan Seiler
- Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, Göttingen, Germany.,Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University, Göttingen, Germany
| | - Stefanie Pöggeler
- Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August-University, Göttingen, Germany.,Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University, Göttingen, Germany
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Kalhorzadeh P, Hu Z, Cools T, Amiard S, Willing EM, De Winne N, Gevaert K, De Jaeger G, Schneeberger K, White CI, De Veylder L. Arabidopsis thaliana RNase H2 deficiency counteracts the needs for the WEE1 checkpoint kinase but triggers genome instability. THE PLANT CELL 2014; 26:3680-92. [PMID: 25217508 PMCID: PMC4213155 DOI: 10.1105/tpc.114.128108] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.
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Affiliation(s)
- Pooneh Kalhorzadeh
- Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), B-9052 Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium
| | - Zhubing Hu
- Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), B-9052 Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium
| | - Toon Cools
- Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), B-9052 Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium
| | - Simon Amiard
- Génétique, Reproduction et Développement, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6293-Clermont Université-Institut National de la Santé et de la Recherche Médicale U1103, F-63177 Aubière, France
| | - Eva-Maria Willing
- Department for Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany
| | - Nancy De Winne
- Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), B-9052 Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium
| | - Kris Gevaert
- Department of Medical Protein Research, Flanders Institute for Biotechnology (VIB), B-9000 Ghent, Belgium Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium
| | - Geert De Jaeger
- Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), B-9052 Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium
| | - Korbinian Schneeberger
- Department for Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany
| | - Charles I White
- Génétique, Reproduction et Développement, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6293-Clermont Université-Institut National de la Santé et de la Recherche Médicale U1103, F-63177 Aubière, France
| | - Lieven De Veylder
- Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), B-9052 Ghent, Belgium Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium
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7
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Davies RG, Wagstaff KM, McLaughlin EA, Loveland KL, Jans DA. The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2013; 1833:3436-3444. [DOI: 10.1016/j.bbamcr.2013.05.015] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Revised: 05/10/2013] [Accepted: 05/13/2013] [Indexed: 01/07/2023]
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Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. Microbiol Mol Biol Rev 2012; 76:331-82. [PMID: 22688816 DOI: 10.1128/mmbr.05021-11] [Citation(s) in RCA: 135] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli, but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays.
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9
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Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R. Interaction trap/two-hybrid system to identify interacting proteins. ACTA ACUST UNITED AC 2012; Chapter 17:17.3.1-17.3.35. [PMID: 22161546 DOI: 10.1002/0471143030.cb1703s53] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
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10
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Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R. Interaction trap/two-hybrid system to identify interacting proteins. ACTA ACUST UNITED AC 2012; Chapter 4:Unit 4.4. [PMID: 21462161 DOI: 10.1002/0471142301.ns0404s55] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
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Affiliation(s)
- Erica A Golemis
- Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA. EA
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11
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Roberts GG, Parrish JR, Mangiola BA, Finley RL. High-throughput yeast two-hybrid screening. Methods Mol Biol 2012; 812:39-61. [PMID: 22218853 DOI: 10.1007/978-1-61779-455-1_3] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Charting the interactions among proteins is essential for understanding biological processes. While a number of complementary technologies for detecting protein interactions are available, the yeast two-hybrid system is one of the few that have been successfully scaled up. Two-hybrid screens have been used to construct extensive protein interaction maps for humans and several model organisms, and these maps have proven invaluable for studies on a variety of biological systems. These maps, however, have not come close to covering all proteins or interactions detectable by yeast two-hybrid. This is due in part to the difficulty of using library screening methods to sample all possible binary combinations of proteins. Ideally, every binary pair of proteins would be tested individually to ensure that every detectable interaction is identified. For organisms with large proteomes, however, this is not economically feasible and instead efficient pooling schemes must be implemented. The high-throughput two-hybrid screening methods presented here are designed to efficiently maximize coverage for selected sets of proteins or entire proteomes. We present two high-throughput screening protocols. Both methods are designed to identify interactors for any number of bait proteins expressed as DNA-binding domain (BD) fusions. The choice of which protocol to use depends largely on the nature of the available library of proteins fused to an activation domain (AD). The first protocol is appropriate for screening a library of AD clones, such as a cDNA library, a domain library, or a large pool of AD clones. By contrast, the second protocol is appropriate for screening a large array of individual sequence-verified AD clones. This protocol screens small pools of AD clones from the array in a two-phase scheme. Although the methods presented were developed using the LexA version of the yeast two-hybrid system, we include notes as appropriate to accommodate users of other versions.
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Affiliation(s)
- George G Roberts
- Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA
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12
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Yan X, Hu S, Guan YX, Yao SJ. Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli. Appl Microbiol Biotechnol 2011; 93:1065-74. [DOI: 10.1007/s00253-011-3599-2] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2011] [Revised: 08/25/2011] [Accepted: 09/20/2011] [Indexed: 11/29/2022]
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13
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Reiterative Recombination for the in vivo assembly of libraries of multigene pathways. Proc Natl Acad Sci U S A 2011; 108:15135-40. [PMID: 21876185 DOI: 10.1073/pnas.1100507108] [Citation(s) in RCA: 87] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.
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Charette JM, Baserga SJ. The DEAD-box RNA helicase-like Utp25 is an SSU processome component. RNA (NEW YORK, N.Y.) 2010; 16:2156-69. [PMID: 20884785 PMCID: PMC2957055 DOI: 10.1261/rna.2359810] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/14/2023]
Abstract
The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A₀, A₁, and A₂. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature-sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.
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Affiliation(s)
- J Michael Charette
- Department of Molecular Biophysics & Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA
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Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R. Interaction trap/two-hybrid system to identify interacting proteins. ACTA ACUST UNITED AC 2009; Chapter 19:19.2.1-19.2.35. [PMID: 19688737 DOI: 10.1002/0471140864.ps1902s57] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
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16
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Wanke D, Harter K. Analysis of plant regulatory DNA sequences by the yeast-one-hybrid assay. Methods Mol Biol 2009; 479:291-309. [PMID: 19083180 DOI: 10.1007/978-1-59745-289-2_19] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Regulatory DNA sequences harbor the essential information to control specific gene expression changes and integrate information derived from upstream signaling cascades. This regulatory potential is mediated by direct binding of proteins, e.g., transcription factors, to defined stretches of DNA motifs in regulatory regions. The analysis of these DNA regions, at which several signaling pathways could merge to orchestrate gene expression, is still a challenging task. To date, the combination of functional approaches in the laboratory and computer aided sequence evaluation is frequently used for regulatory sequence analysis. The yeast-one-hybrid method is a possible approach to test for direct binding of plant proteins to DNA in a heterologous system. Moreover, it is the most frequently used method for the identification of DNA-binding proteins targeting a given DNA sequence by screening a cDNA library.
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Affiliation(s)
- Dierk Wanke
- ZMBP Pflanzenphysiologie, Universität Tübingen, Tübingen, Germany
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17
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Plate I, Hallwyl SCL, Shi I, Krejci L, Müller C, Albertsen L, Sung P, Mortensen UH. Interaction with RPA is necessary for Rad52 repair center formation and for its mediator activity. J Biol Chem 2008; 283:29077-85. [PMID: 18703507 PMCID: PMC2570898 DOI: 10.1074/jbc.m804881200] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2008] [Revised: 08/12/2008] [Indexed: 11/06/2022] Open
Abstract
Homologous recombination (HR) is a major DNA repair pathway and therefore essential for maintaining the integrity of the genome. HR is catalyzed by proteins encoded by genes of the RAD52 epistasis group, including the recombinase Rad51 and its mediator Rad52. HR proteins fused with green fluorescent protein form foci at damaged DNA reflecting the assembly of repair centers that harbor a high concentration of repair proteins. Rad52 mediates the recruitment of Rad51 and other HR proteins to DNA damage. To understand the mechanism for the assembly of Rad52-dependent DNA repair centers, we used a mutational strategy to identify a Rad52 domain essential for its recruitment to DNA repair foci. We present evidence to implicate an acidic domain in Rad52 in DNA repair focus formation. Mutations in this domain confer marked DNA damage sensitivity and recombination deficiency. Importantly, these Rad52 mutants are specifically compromised for interaction with the single-stranded DNA-binding factor RPA. Based on these findings, we propose a model where Rad52 displaces RPA from single-stranded DNA using the acidic domain as a molecular lever.
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Affiliation(s)
- Iben Plate
- Center for Microbial Biotechnology, Technical University of Denmark, Lyng by 2800, Denmark
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18
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Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R. Interaction trap/two-hybrid system to identify interacting proteins. ACTA ACUST UNITED AC 2008; Chapter 20:Unit 20.1. [PMID: 18425763 DOI: 10.1002/0471142727.mb2001s82] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
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19
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Dirnberger D, Messerschmid M, Baumeister R. An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor. Nucleic Acids Res 2008; 36:e37. [PMID: 18319286 PMCID: PMC2330230 DOI: 10.1093/nar/gkm1163] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
The yeast split-ubiquitin system has previously been shown to be suitable to detect protein interactions of membrane proteins and of transcription factors in vivo. Therefore, this technology complements the classical split-transcription factor based yeast two-hybrid system (Y2H). Success or failure of the Y2H depends primarily on the ability to avoid false-negative and false-positive hits that become a limiting factor for the value of the system, especially in large scale proteomic analyses. We provide here a systematic assessment of parameters to help improving the quality of split-ubiquitin cDNA-library screenings. We experimentally defined the optimal 5-fluoroorotic acid (5-FOA) concentration as a key parameter to increase the reproducibility of interactions and, at the same time, to keep non-specific background growth low. Furthermore, we show that the efficacy of the 5-FOA selection is modulated by the plating density of the yeast clones. Moreover, a reporter-specific class of false-positive hits was identified, and a simple phenotypic assay for efficient de-selection was developed. We demonstrate the application of this improved system to identify novel interacting proteins of the human Frizzled 1 receptor. We identified several novel interactors with components of the Wnt-Frizzled signalling pathways and discuss their potential roles as direct mediators of Frizzled receptor signalling. The present work is the first example of a split-ubiquitin interaction screen using an in-situ expressed receptor of the serpentine class, emphasizing the suitability of the described improvements in the screening protocol.
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Affiliation(s)
- Dietmar Dirnberger
- Bio3/Bioinformatics and Molecular Genetics (Faculty of Biology), Center for Biochemistry and Molecular Cell Research (ZBMZ, Faculty of Medicine), University of Freiburg, ZBSA (Center for Systems Biology), Martinsried, Germany
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20
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Bickle MBT, Dusserre E, Moncorgé O, Bottin H, Colas P. Selection and characterization of large collections of peptide aptamers through optimized yeast two-hybrid procedures. Nat Protoc 2007; 1:1066-91. [PMID: 17406388 DOI: 10.1038/nprot.2006.32] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Peptide aptamers are combinatorial proteins that specifically bind intracellular proteins and modulate their function. They are powerful tools to study protein function within complex regulatory networks and to guide small-molecule drug discovery. Here we describe methodological improvements that enhance the yeast two-hybrid selection and characterization of large collections of peptide aptamers. We provide a detailed protocol to perform high-efficiency transformation of peptide aptamer libraries, in-depth validation experiments of the bait proteins, high-efficiency mating to screen large numbers of peptide aptamers and streamlined confirmation of the positive clones. We also describe yeast two-hybrid mating assays, which can be used to determine the specificity of the selected aptamers, map their binding sites on target proteins and provide structural insights on their target-binding surface. Overall, 12 weeks are required to perform the protocols. The improvements on the yeast two-hybrid method can be also usefully applied to the screening of cDNA libraries to identify protein interactions.
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Affiliation(s)
- Marc B T Bickle
- Aptanomics, 181-203, Avenue Jean Jaurès, 69007 Lyon, France.
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21
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Abstract
This chapter aims to describe methods to identify and characterize protein-protein interactions that were developed during our studies on translation initiation factor complexes. Methods include the two-hybrid assay, the GST pull-down assay, and the coimmunoprecipitation (co-IP) assay. The two-hybrid assay provides for a convenient start to find the minimal interaction domains, which generally produce well-behaved recombinant proteins suited for various in vitro interaction assays. Emphasis is placed on demonstrating physiological relevance of identified interactions. The effective strategy is to find mutations that reduce the interaction by genetic or site-directed mutational approaches and obtain correlations between their effects in vitro (GST pull down) and effects in vivo (co-IP).
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Affiliation(s)
- Chingakham Ranjit Singh
- Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, USA
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22
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Parrish JR, Gulyas KD, Finley RL. Yeast two-hybrid contributions to interactome mapping. Curr Opin Biotechnol 2006; 17:387-93. [PMID: 16806892 DOI: 10.1016/j.copbio.2006.06.006] [Citation(s) in RCA: 167] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2006] [Revised: 06/01/2006] [Accepted: 06/15/2006] [Indexed: 11/23/2022]
Abstract
Interactome mapping, the systematic identification of protein interactions within an organism, promises to facilitate systems-level studies of biological processes. Using in vitro technologies that measure specific protein interactions, static maps are being generated that include many of the protein networks that occur in vivo. Most of the binary protein interaction data currently available was generated by large-scale yeast two-hybrid screens. Recent efforts to map interactions in model organisms and in humans illustrate the promise and some of the limitations of the two-hybrid approach. Although these maps are incomplete and include false positives, they are proving useful as a framework around which to elaborate and model the in vivo interactome.
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Affiliation(s)
- Jodi R Parrish
- Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA
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23
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Abstract
The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.
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Affiliation(s)
- Zairong Li
- Department of Neuroscience, Mount Sinai School of Medicine, New York, New York 10029, USA
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24
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Semple JI, Prime G, Wallis LJ, Sanderson CM, Markie D. Two-hybrid reporter vectors for gap repair cloning. Biotechniques 2005; 38:927-34. [PMID: 16018554 DOI: 10.2144/05386rr03] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Yeast two-hybrid analysis is a valuable approach to the discovery and characterization of protein interactions. We have developed vectors that can indicate the presence of an insert when used in two-hybrid bait and prey construction by gap repair cloning. The strategy uses a recombination cloning site flanked by sequences encoding the GAL4 activation and binding domains. After gap repair cloning in standard hosts carrying an ADE2 reporter gene, disruption of GAL4 by an insert can be identified by the development of red colony color, while empty vector plasmids produce white colonies. Function in yeast two-hybrid applications was initially validated using known interacting proteins in pair-wise analyses, and subsequently, the bait vectors were used in library screens with the mouse Mad212 and human Mccd1 proteins, identifying a number of putative new interactions for these proteins. These vectors should facilitate high-throughput yeast two-hybrid screens in which large numbers of bait and prey constructs may be required.
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25
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Figueroa P, Gusmaroli G, Serino G, Habashi J, Ma L, Shen Y, Feng S, Bostick M, Callis J, Hellmann H, Deng XW. Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo. THE PLANT CELL 2005; 17:1180-95. [PMID: 15772280 PMCID: PMC1087995 DOI: 10.1105/tpc.105.031989] [Citation(s) in RCA: 137] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2005] [Accepted: 02/19/2005] [Indexed: 05/18/2023]
Abstract
Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.
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Affiliation(s)
- Pablo Figueroa
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Conecticut 06520-8104, USA
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26
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Shor E, Weinstein J, Rothstein R. A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3 and CSM2: four genes involved in error-free DNA repair. Genetics 2005; 169:1275-89. [PMID: 15654096 PMCID: PMC1449555 DOI: 10.1534/genetics.104.036764] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Helicases of the RecQ family and topoisomerase III are evolutionarily conserved proteins important for maintenance of genome stability. In Saccharomyces cerevisiae, loss of the TOP3 gene, encoding topoisomerase III, results in a phenotype of slow growth, DNA damage sensitivity, meiotic defects, and hyperrecombination. The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, and the two proteins are thought to act in concert in vivo. Much recent genetic and biochemical evidence points to the role of RecQ helicases and topoisomerase III in regulating homologous recombination (HR) during DNA replication. Previously, we found that mutations in HR genes partially suppress top3 slow growth. Here, we describe the analysis of four additional mutational suppressors of top3 defects: shu1, shu2, psy3, and csm2. These genes belong to one epistasis group and their protein products interact with each other, strongly suggesting that they function as a complex in vivo. Their mutant phenotype indicates that they are important for error-free repair of spontaneous and induced DNA lesions, protecting the genome from mutation. These mutants exhibit an epistatic relationship with rad52 and show altered dynamics of Rad52-YFP foci, suggesting a role for these proteins in recombinational repair.
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Affiliation(s)
- Erika Shor
- Department of Genetics and Development, Columbia University College of Physicians & Surgeons, New York, New York 10032-2704, USA
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27
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Bilsland E, Molin C, Swaminathan S, Ramne A, Sunnerhagen P. Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance. Mol Microbiol 2004; 53:1743-56. [PMID: 15341652 DOI: 10.1111/j.1365-2958.2004.04238.x] [Citation(s) in RCA: 152] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We demonstrate a role in oxidative and metal stress resistance for the MAPK-activated protein kinases Rck1 and Rck2 in Saccharomyces cerevisiae. We show that Hog1 is robustly phosphorylated in a Pbs2-dependent way during oxidative stress, and that Rck2 also is phosphorylated under these circumstances. Hog1 concentrates in the nucleus in oxidative stress. Hog1 localization is partially dependent on Rck2, as rck2 cells have more nuclear Hog1 than wild-type cells. We find several proteins with a role in oxidative stress resistance using Rck1 or Rck2 as baits in a two-hybrid screen. We identify the transcription factor Yap2 as a putative target for Rck1, and the Zn2+ transporter Zrc1 as a target for Rck2. Yap2 is normally cytoplasmic, but rapidly migrates to the nucleus upon exposure to oxidative stress agents. In a fraction of untreated pbs2 cells, Yap2 is nuclear. Zrc1 co-immunoprecipitates with Rck2, and ZRC1 is genetically downstream of RCK2. These data connect activation of the Hog1 MAPK cascade with effectors having a role in oxidative stress resistance.
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Affiliation(s)
- Elizabeth Bilsland
- Department of Cell and Molecular Biology, Lundberg Laboratory, Göteborg University, PO Box 462, SE-405 30 Göteborg, Sweden
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28
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Sundaram S, Brand JH, Hymes MJ, Hiremath S, Podila GK. Isolation and analysis of a symbiosis-regulated and Ras-interacting vesicular assembly protein gene from the ectomycorrhizal fungus Laccaria bicolor. THE NEW PHYTOLOGIST 2004; 161:529-538. [PMID: 33873504 DOI: 10.1046/j.1469-8137.2003.00935.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/12/2023]
Abstract
• A yeast two-hybrid library prepared from Laccaria bicolor × Pinus resinosa mycorrhizas was screened using a LbRAS clone, previously characterized, as a bait to isolate LbRAS interacting signaling-related genes from L. bicolor. • Using this method, a novel line of Ras-interacting yeast two-hybrid mycorrhizal (Rythm) clones were isolated and analysed for their symbiosis-regulation. One such clone identified (RythmA) had homology to Ap180-like vesicular proteins. • Sequence homology and parsimony-based phylogenetic analysis showed its relatedness to Ap180-like proteins from other systems. DNA analysis suggested that L. bicolor had one or two copies of the RythmA gene. • An RNA analysis showed that the expression of RythmA could be detected 36 h after interaction with the host, which follows the expression of Lbras. Immunolocalization of LbRAS near dolipore septum of the fungal cells in the Hartig net area suggests that RythmA protein may be involved in the transport of signaling proteins such as LbRAS.
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Affiliation(s)
- Sathish Sundaram
- Department of Biological Sciences, Michigan Tech University, Houghton, MI 49931, USA
- Present address: Vattikudi Urology Institute, Henry Ford Medical Center, Detroit, MI 48202, USA
| | - Joshua H Brand
- Department of Biological Sciences, Michigan Tech University, Houghton, MI 49931, USA
| | - Matthew J Hymes
- Department of Biological Sciences, Michigan Tech University, Houghton, MI 49931, USA
| | | | - Gopi K Podila
- Department of Biological Sciences, University of Alabama, Huntsville, AL 35899, USA
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29
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Haney SA, Keeney D, Chen L, Moghazeh S, Projan S, Rasmussen B. Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli. BMC Genomics 2003; 4:36. [PMID: 12964949 PMCID: PMC212392 DOI: 10.1186/1471-2164-4-36] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2003] [Accepted: 09/10/2003] [Indexed: 11/26/2022] Open
Abstract
BACKGROUND Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library. RESULTS Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. CONCLUSION Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.
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Affiliation(s)
- Steven A Haney
- Department of Infectious Disease, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
| | - David Keeney
- Department of Infectious Disease, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
| | - Lei Chen
- Department of Infectious Disease, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
| | - Soraya Moghazeh
- Department of Infectious Disease, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
| | - Steve Projan
- Department of Infectious Disease, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
| | - Beth Rasmussen
- Department of Infectious Disease, Wyeth Research, 401 N. Middletown Rd., Pearl River, NY 10965, USA
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Pornillos O, Higginson DS, Stray KM, Fisher RD, Garrus JE, Payne M, He GP, Wang HE, Morham SG, Sundquist WI. HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein. J Cell Biol 2003; 162:425-34. [PMID: 12900394 PMCID: PMC2172688 DOI: 10.1083/jcb.200302138] [Citation(s) in RCA: 198] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.
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Affiliation(s)
- Owen Pornillos
- Department of Biochemistry, University of Utah, School of Medicine, Salt Lake City, UT 84132, USA.
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31
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Won J, Kim DY, La M, Kim D, Meadows GG, Joe CO. Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis. J Biol Chem 2003; 278:19347-51. [PMID: 12657644 DOI: 10.1074/jbc.m213098200] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).
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Affiliation(s)
- Jungyeon Won
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Korea
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32
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33
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Serino G, Su H, Peng Z, Tsuge T, Wei N, Gu H, Deng XW. Characterization of the last subunit of the Arabidopsis COP9 signalosome: implications for the overall structure and origin of the complex. THE PLANT CELL 2003; 15:719-31. [PMID: 12615944 PMCID: PMC150025 DOI: 10.1105/tpc.009092] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/05/2002] [Accepted: 12/15/2002] [Indexed: 05/18/2023]
Abstract
The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.
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Affiliation(s)
- Giovanna Serino
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104, USA
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34
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Li K, Wang L, Cheng J, Lu YY, Zhang LX, Mu JS, Hong Y, Liu Y, Duan HJ, Wang G, Li L, Chen JM. Interaction between hepatitis C virus core protein and translin protein- a possible molecular mechanism for hepatocellular carcinoma and lymphoma caused by hepatitis C virus. World J Gastroenterol 2003; 9:300-3. [PMID: 12532453 PMCID: PMC4611333 DOI: 10.3748/wjg.v9.i2.300] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the interaction between hepatitis C virus core protein and translin protein and its role in the pathogenensis of hepatocellular carcinoma and lymphoma.
METHODS: With the components of the yeast two hybrid system 3,“ bait” plasmids of HCV core the gene was constructed. After proving that hepatitis C virus core protein could be firmly expressed in AH109 yeast strains, yeast two- hybrid screening was performed by mating AH109 with Y187 that transformed with liver cDNA library plasmids - pACT2 and then plated on quadrople dropout (QDO) medium and then assayed for α-gal activity. Sequencing analysis of the genes of library plasmids in yeast colonies that could grow on QDO with α-gal activity was performed. The interaction between HCV core protein and the protein we obtained from positive colony was further confirmed by repeating yeast two - hybrid analysis and coimmunoprecipitation in vitro.
RESULTS: A gene from a positive colony was the gene of translin, a recombination hotspot binding protein. The interaction between HCV core protein and translin protein could be proved not only in yeast, but also in vitro.
CONCLUSION: The core protein of HCV can interact with translin protein. This can partly explain the molecular mechanism for hepatocellular carcinoma and lymphoma caused by HCV.
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Affiliation(s)
- Ke Li
- Jun Cheng Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, Beijing 100039, China
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Abstract
With the sequencing of many genomes now completed, biologists are faced with the challenge of deciphering the function and association of an immense number of predicted proteins. Comprehension of the networks of proteins and chemicals that comprise the cells and tissues of an organism, and the specific roles of proteins in these networks, will be a necessary next step to understanding cellular function in healthy and diseased states. In the past decade, the budding yeast Saccharomyces cerevisiae has emerged as an important tool for large-scale functional genomics analyses. This review describes the use of yeast cell-based assays in the post-genomic era, focusing on high-throughput functional genomics and drug discovery.
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Affiliation(s)
- Chandra L Tucker
- Dept of Genome Sciences, University of Washington, Box 357730, Seattle, WA 98195, USA.
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36
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Poirey R, Despons L, Leh V, Lafuente MJ, Potier S, Souciet JL, Jauniaux JC. Functional analysis of the Saccharomyces cerevisiae DUP240 multigene family reveals membrane-associated proteins that are not essential for cell viability. MICROBIOLOGY (READING, ENGLAND) 2002; 148:2111-2123. [PMID: 12101299 DOI: 10.1099/00221287-148-7-2111] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The DUP240 gene family of Saccharomyces cerevisiae is composed of 10 members. They encode proteins of about 240 amino acids which contain two predicted transmembrane domains. Database searches identified only one homologue in the closely related species Saccharomyces bayanus, indicating that the DUP240 genes encode proteins specific to Saccharomyces sensu stricto. The short-flanking homology PCR gene-replacement strategy with a variety of selective markers for replacements, and classical genetic methods, were used to generate strains deleted for all 10 DUP240 genes. All of the knock-out strains were viable and had similar growth kinetics to the wild-type. Two-hybrid screens, hSos1p fusions and GFP fusions were carried out; the results indicated that the Dup240 proteins are membrane associated, and that some of them are concentrated around the plasma membrane.
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Affiliation(s)
- Rémy Poirey
- Laboratoire de Génétique et Microbiologie, UPRES-A 7010 ULP/CNRS, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France2
- Angewandte Tumorvirologie, Abteilung F0100 and Virologie Appliquée à l'Oncologie (Unité INSERM 375), Deutsches Krebsforschungszentrum, P. 1011949, D-69009 Heidelberg, Germany1
| | - Laurence Despons
- Laboratoire de Génétique et Microbiologie, UPRES-A 7010 ULP/CNRS, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France2
| | - Véronique Leh
- Laboratoire de Génétique et Microbiologie, UPRES-A 7010 ULP/CNRS, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France2
| | - Maria-Jose Lafuente
- Angewandte Tumorvirologie, Abteilung F0100 and Virologie Appliquée à l'Oncologie (Unité INSERM 375), Deutsches Krebsforschungszentrum, P. 1011949, D-69009 Heidelberg, Germany1
| | - Serge Potier
- Laboratoire de Génétique et Microbiologie, UPRES-A 7010 ULP/CNRS, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France2
| | - Jean-Luc Souciet
- Laboratoire de Génétique et Microbiologie, UPRES-A 7010 ULP/CNRS, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg cedex, France2
| | - Jean-Claude Jauniaux
- Angewandte Tumorvirologie, Abteilung F0100 and Virologie Appliquée à l'Oncologie (Unité INSERM 375), Deutsches Krebsforschungszentrum, P. 1011949, D-69009 Heidelberg, Germany1
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37
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de Soultrait VR, Caumont A, Durrens P, Calmels C, Parissi V, Recordon P, Bon E, Desjobert C, Tarrago-Litvak L, Fournier M. HIV-1 integrase interacts with yeast microtubule-associated proteins. BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1575:40-8. [PMID: 12020817 DOI: 10.1016/s0167-4781(02)00241-5] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The human immunodeficiency virus type 1 (HIV-1) integrase (IN) mediates the insertion of viral DNA into the human genome. In addition to IN, cellular and viral proteins are associated to proviral DNA in the so-called preintegration complex (PIC). We previously reported that the expression of HIV-1 IN in yeast leads to the emergence of a lethal phenotype. This effect may be linked to the IN activity on infected human cells where integration requires the cleavage of genomic DNA. To isolate and characterize potential cellular partners of HIV-1 IN, we used it as a bait in a two-hybrid system with a yeast genomic library. IN interacted with proteins belonging to the microtubule network, or involved in the protein synthesis apparatus. We focused our interest on one of the selected inserts, L2, which corresponds to the C-end half of the yeast STU2p, a microtubule-associated protein (MAP). STU2p is an essential component of the yeast spindle pole body (SPB), which is able to bind microtubules in vitro. After expressing and purifying L2 as a recombinant protein, we showed its binding to IN by ELISA immunodetection. L2 was also able to inhibit IN activity in vitro. In addition, the effect of L2 was tested using the "lethal yeast phenotype". The coexpression of IN and the L2 peptide abolished the lethal phenotype, thus showing important in vivo interactions between IN and L2. The identification of components of the microtubule network associated with IN suggest a role of this complex in the transport of HIV-1 IN present in the PIC to the nucleus, as already described for other human viruses.
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Affiliation(s)
- Vaea Richard de Soultrait
- UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, BP 103, Bat. 3A-3 Etage, 146 rue Léo Saignat, 33076 Bordeaux X Cedex, France
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38
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de Soultrait VR, Caumont A, Parissi V, Morellet N, Ventura M, Lenoir C, Litvak S, Fournier M, Roques B. A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase. J Mol Biol 2002; 318:45-58. [PMID: 12054767 DOI: 10.1016/s0022-2836(02)00033-5] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.
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39
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Krogsdam AM, Nielsen CAF, Neve S, Holst D, Helledie T, Thomsen B, Bendixen C, Mandrup S, Kristiansen K. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation. Biochem J 2002; 363:157-65. [PMID: 11903058 PMCID: PMC1222462 DOI: 10.1042/0264-6021:3630157] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains of PPAR gamma and PPAR alpha were significantly weaker. PPAR-NCoR interactions were antagonized by ligands in the two-hybrid system, but were ligand-insensitive in in vitro pull-down assays. Interaction between PPAR delta and NCoR was unaffected by coexpression of retinoid X receptor (RXR) alpha. The PPAR delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well with interaction domains I and II of NCoR. In transient transfection experiments, NCoR and the related silencing mediator for retinoid and thyroid hormone receptor (SMRT) were shown to exert a marked dose-dependent repression of ligand-induced PPAR delta-mediated transactivation; in addition, transactivation induced by the cAMP-elevating agent forskolin was efficiently reduced to basal levels by NCoR as well as SMRT coexpression. Our results suggest that the transactivation potential of liganded PPAR delta can be fine-tuned by interaction with NCoR and SMRT in a manner determined by the expression levels of corepressors and coactivators.
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Affiliation(s)
- Anne-M Krogsdam
- Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, Campusvej 55, DK-5230 Odense M, Denmark
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40
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Finley RL, Zhang H, Zhong J, Stanyon CA. Regulated expression of proteins in yeast using the MAL61-62 promoter and a mating scheme to increase dynamic range. Gene 2002; 285:49-57. [PMID: 12039031 DOI: 10.1016/s0378-1119(02)00420-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The ability to express heterologous genes in yeast has become indispensable for many biological research techniques. Expression systems that can be regulated are particularly useful because they allow an experimenter to control the timing and levels of gene expression. Despite their many advantages, however, surprisingly few conditional expression systems are available for yeast. Moreover, of those that have been described, many are not ideal either because they have high background expression levels, low induced levels, or because they require restrictive growth conditions. Here we describe a conditional expression system that takes advantage of the yeast MAL62 promoter (MAL62p), which can be controlled by adding maltose or glucose to the growth medium to induce or repress transcription, respectively. In addition, we use a mating scheme to dramatically increase the dynamic range of expression levels possible. We show that MAL62p background activity can be effectively eliminated by maintaining expression constructs in a mal(-) yeast strain. High-level expression can be induced in diploids formed by mating the mal(-) strain with a MAL(+) strain. A similar mating scheme may be useful for other conditional expression systems as well. Among other uses, this approach should aid high throughput yeast two-hybrid assays, which rely on maintaining large libraries of expression strains, which are eventually mated to conduct assays for protein interactions. We demonstrate a two-hybrid system in which MAL62p is used in conjunction with the yeast GAL1 promoter to independently regulate expression of both hybrid proteins, and to allow detection of interactions involving toxic proteins.
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Affiliation(s)
- Russell L Finley
- Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA.
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41
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Ma D. Applications of yeast in drug discovery. PROGRESS IN DRUG RESEARCH. FORTSCHRITTE DER ARZNEIMITTELFORSCHUNG. PROGRES DES RECHERCHES PHARMACEUTIQUES 2002; 57:117-62. [PMID: 11728000 DOI: 10.1007/978-3-0348-8308-5_3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/09/2023]
Abstract
The yeast Saccharomyces cerevisiae is perhaps the best-studied eukaryotic organism. Its experimental tractability, combined with the remarkable conservation of gene function throughout evolution, makes yeast the ideal model genetic organism. Yeast is a non-pathogenic model of fungal pathogens used to identify antifungal targets suitable for drug development and to elucidate mechanisms of action of antifungal agents. As a model of fundamental cellular processes and metabolic pathways of the human, yeast has improved our understanding and facilitated the molecular analysis of many disease genes. The completion of the Saccharomyces genome sequence helped launch the post-genomic era, focusing on functional analyses of whole genomes. Yeast paved the way for the systematic analysis of large and complex genomes by serving as a test bed for novel experimental approaches and technologies, tools that are fast becoming the standard in drug discovery research
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Affiliation(s)
- D Ma
- Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
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42
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Sims TL, Ordanic M. Identification of a S-ribonuclease-binding protein in Petunia hybrida. PLANT MOLECULAR BIOLOGY 2001; 47:771-783. [PMID: 11785938 DOI: 10.1023/a:1013639528858] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBPI may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.
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Affiliation(s)
- T L Sims
- Department of Biological Sciences and Plant Molecular Biology Center, Northern Illinois University, DeKalb, 60115-2861, USA.
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43
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Peng Z, Serino G, Deng XW. Molecular characterization of subunit 6 of the COP9 signalosome and its role in multifaceted developmental processes in Arabidopsis. THE PLANT CELL 2001; 13:2393-407. [PMID: 11701877 PMCID: PMC139460 DOI: 10.1105/tpc.010248] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/20/2001] [Accepted: 08/16/2001] [Indexed: 05/18/2023]
Abstract
The COP9 signalosome is a highly conserved protein complex initially identified as a repressor of photomorphogenesis. Here, we report that subunit 6 of the Arabidopsis COP9 signalosome is encoded by a family of two genes (CSN6A and CSN6B) located on chromosomes V and IV, respectively. The CSN6A and CSN6B proteins share 87% amino acid identity and contain a MPR1p and PAD1p N-terminal (MPN) domain at the N-terminal region. The CSN6 proteins share homology with CSN5 and belong to the Mov34 superfamily of proteins. CSN6 proteins present only in the complex form and coimmunoprecipitate with other known subunits of the COP9 signalosome. Partial loss-of-function strains of the COP9 signalosome created by antisense and cosuppression with CSN6A exhibit diverse developmental defects, including homeotic organ transformation, symmetric body organization, and organ boundary definition. Protein blot analysis revealed that the defective plants accumulate significant amounts of ubiquitinated proteins, supporting the conclusion that the COP9 signalosome regulates multifaceted developmental processes through its involvement in ubiquitin/proteasome-mediated protein degradation.
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Affiliation(s)
- Z Peng
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104, USA
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44
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Georgakopoulos T, Koutroubas G, Vakonakis I, Tzermia M, Prokova V, Voutsina A, Alexandraki D. Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w ORF family suggests relations to mitochondrial/peroxisomal functions and amino acid signalling pathways. Yeast 2001; 18:1155-71. [PMID: 11536337 DOI: 10.1002/yea.764] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Saccharomyces cerevisiae YFR021w, YGR223c and YPL100w are paralogous ORFs of unknown function. Phenotypic analysis of overexpression, single-, double- and triple-ORF deletion strains under various growth conditions indicated mitochondria-related functions for all three ORFs. Two-hybrid screens of a yeast genomic library identified potentially interacting proteins for the three ORFs. Among these, the transcriptional activator Rtg3p interacted with both Yfr021wp and Ypl100wp and both ORF single deletions reduced the constitutive expression of the RTG-regulated CIT2 and DLD3 genes and caused typical retrograde response of CIT2 and DLD3 under growth conditions requiring functional mitochondria, indicating that YFR021w and YPL100w are also involved in unidentified mitochondrial functions. Ptr3p, a component of the amino acid sensor Ssy1p/Ptr3p, was also found as a two-hybrid interactant of Yfr021wp. Of the three single-ORF deletions, ypl100w Delta exhibited ptr3 Delta-similar phenotypes. These findings, combined with the fact that RTG-dependent expression is modulated by specific amino acids, suggested possible relations of Yfr021wp and Ypl100wp to amino acid signalling pathways. Under most conditions examined, the effects of the single- and double-ORF deletions indicated that YFR021w, YPL100w and YGR223c are not parts of the same pathway. We found no unique phenotype attributed to the deletion of YGR223c. However, its function interferes with the function of the other two ORFs, as revealed by the effects of double- and triple-ORF deletions.
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Affiliation(s)
- T Georgakopoulos
- Foundation for Research and Technology-HELLAS, Institute of Molecular Biology and Biotechnology, PO Box 1527, Heraklion 711 10, Crete, Greece
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45
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Nagengast AA, Salz HK. The Drosophila U2 snRNP protein U2A' has an essential function that is SNF/U2B" independent. Nucleic Acids Res 2001; 29:3841-7. [PMID: 11557816 PMCID: PMC55907 DOI: 10.1093/nar/29.18.3841] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Recruitment of the U2 snRNP to the pre-mRNA is an essential step in spliceosome assembly. Although the protein components of the U2 snRNP have been identified, their individual contributions to function are poorly defined. In vitro studies with the Drosophila and human proteins suggest that two of the U2 snRNP-specific proteins, U2A' and U2B", function exclusively as a dimer. In Drosophila the presence of the U2B" counterpart, Sans-Fille (SNF), in the U2 snRNP is dispensable for viability, suggesting that SNF is not necessary for U2 snRNP function in vivo. With the identification of a single U2A'-like protein in the Drosophila genome, we can now investigate the relationship between SNF and its putative binding partner in vivo. Here we show that Drosophila U2A' protein interacts with SNF in vivo and, like its human counterpart, is U2 snRNP specific. Unexpectedly, however, we find that loss of function causes lethality, suggesting that U2A', but not SNF, is critical for U2 snRNP function. Moreover, although we demonstrate that several domains in the SNF protein are important for the interaction with the Drosophila U2A' protein, including a redundant domain at the normally dispensable C-terminus, we find that U2A' does not require heterodimer formation for either its vital function or U2 snRNP assembly. Thus together these data demonstrate that in Drosophila U2A' has an essential function that is unrelated to its role as the partner protein of SNF/U2B".
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Affiliation(s)
- A A Nagengast
- Department of Genetics, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA
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46
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Chen BS, Sun ZW, Hampsey M. A Gal4-sigma 54 hybrid protein that functions as a potent activator of RNA polymerase II transcription in yeast. J Biol Chem 2001; 276:23881-7. [PMID: 11313364 DOI: 10.1074/jbc.m102893200] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The bacterial final sigma(54) protein associates with core RNA polymerase to form a holoenzyme complex that renders cognate promoters enhancer-dependent. Although unusual in bacteria, enhancer-dependent transcription is the paradigm in eukaryotes. Here we report that a fragment of Escherichia coli final sigma(54) encompassing amino acid residues 29-177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain. Activation by Gal4-final sigma(54) is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-final sigma(54) functions by a normal mechanism of transcriptional activation. Surprisingly, deletion of the AHC1 gene, which encodes a polypeptide unique to the ADA coactivator complex, stimulates Gal4-final sigma(54)-mediated activation and enhances the toxicity of Gal4-final sigma(54). Accordingly, the SAGA and ADA complexes, both of which include Gcn5 as their histone acetyltransferase subunit, exert opposite effects on transcriptional activation by Gal4-final sigma(54). Gal4-final sigma(54) activation and toxicity are also dependent upon specific final sigma(54) residues that are required for activator-responsive promoter melting by final sigma(54) in bacteria, implying that activation is a consequence of final sigma(54)-specific features rather than a structurally fortuitous polypeptide fragment. As such, Gal4-final sigma(54) represents a novel tool with the potential to provide insight into the mechanism by which natural activators function in eukaryotic cells.
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Affiliation(s)
- B S Chen
- Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635, USA
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47
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Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R. Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins. ACTA ACUST UNITED AC 2001; Chapter 19:Unit19.2. [DOI: 10.1002/0471140864.ps1902s14] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
| | | | | | | | | | - Roger Brent
- The Molecular Sciences Institute Berkeley California
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48
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Golemis EA, Serebriiskii I, Finley RL, Kolonin (hunt by interaction mating MG, Gyuris J, Brent R. Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins. ACTA ACUST UNITED AC 2001; Chapter 20:Unit 20.1. [DOI: 10.1002/0471142727.mb2001s46] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Affiliation(s)
| | | | | | | | | | - Roger Brent
- The Molecular Sciences Institute Berkeley California
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49
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Golemis EA, Serebriiskii I, Finley RL, Kolonin MG, Gyuris J, Brent R. Interaction trap/two-hybrid system to identify interacting proteins. CURRENT PROTOCOLS IN CELL BIOLOGY 2001; Chapter 17:Unit 17.3. [PMID: 18228339 PMCID: PMC4095973 DOI: 10.1002/0471143030.cb1703s08] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
This unit presents protocols designed to detect interacting proteins. Using yeast as a "test tube" and transcriptional activation of a reporter system, interacting proteins can be identified. The system can also be used to test complex formation for proteins for which there exists a reason to expect interaction.
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Affiliation(s)
- E A Golemis
- Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA
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50
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Abstract
Two-hybrid schemes for detecting protein-protein interactions have deepened our understanding of biology by allowing scientists to identify individual important proteins. Recent developments will allow biologists to chart regulatory networks and to rapidly generate hypotheses for the function of genes, allelic variants, and the connections between proteins that make up these networks. Future developments will allow biologists to test inferences about the function of network elements, and allow global approaches to questions of biological function.
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Affiliation(s)
- R Brent
- Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
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