Nucleic Acids Research, 1990, Vol. 18, No. 22 6485-6489
© 1990
MOLECULAR BIOLOGY |
High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe
Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University 3-1 Yamadaoka Suita, Osaka 565, Japan
*To whom correspondence should be addressed
Received October 1, 1990. Accepted November 22, 1990.
We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 106 colonies per 108 cells transfected with 2 µg of library and 1 µg of vector, 50–60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz.pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast.
This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.
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H. Yamamoto, K. Tsukahara, Y. Kanaoka, S. Jinno, and H. Okayama Isolation of a Mammalian Homologue of a Fission Yeast Differentiation Regulator Mol. Cell. Biol., May 1, 1999; 19(5): 3829 - 3841. [Abstract] [Full Text] [PDF]
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C. Affourtit, M. S. Albury, K. Krab, and A. L. Moore Functional Expression of the Plant Alternative Oxidase Affects Growth of the Yeast Schizosaccharomyces pombe J. Biol. Chem., March 5, 1999; 274(10): 6212 - 6218. [Abstract] [Full Text] [PDF]
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P. Feldmann, E. N. Eicher, S. J. Leevers, E. Hafen, and D. A. Hughes Control of Growth and Differentiation by Drosophila RasGAP, a Homolog of p120 Ras-GTPase-Activating Protein Mol. Cell. Biol., March 1, 1999; 19(3): 1928 - 1937. [Abstract] [Full Text] [PDF]
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H. Tanaka, K. Tanaka, H. Murakami, and H. Okayama Fission Yeast Cdc24 Is a Replication Factor C- and Proliferating Cell Nuclear Antigen-Interacting Factor Essential for S-Phase Completion Mol. Cell. Biol., February 1, 1999; 19(2): 1038 - 1048. [Abstract] [Full Text] [PDF]
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L Cerutti and V Simanis Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast J. Cell Sci., January 7, 1999; 112(14): 2313 - 2321. [Abstract] [PDF]
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M. S. Albury, C. Affourtit, and A. L. Moore A Highly Conserved Glutamate Residue (Glu-270) Is Essential for Plant Alternative Oxidase Activity J. Biol. Chem., November 13, 1998; 273(46): 30301 - 30305. [Abstract] [Full Text] [PDF]
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K. Dimitrov and S. Sazer The Role of fnx1, a Fission Yeast Multidrug Resistance Protein, in the Transition of Cells to a Quiescent G0 State Mol. Cell. Biol., September 1, 1998; 18(9): 5239 - 5246. [Abstract] [Full Text]
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K. Tsukahara, H. Yamamoto, and H. Okayama An RNA Binding Protein Negatively Controlling Differentiation in Fission Yeast Mol. Cell. Biol., August 1, 1998; 18(8): 4488 - 4498. [Abstract] [Full Text]
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S. Tahara, K. Tanaka, Y. Yuasa, and H. Okayama Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2-Cdc10, Controlling the Cell Cycle "Start" Mol. Biol. Cell, June 1, 1998; 9(6): 1577 - 1588. [Abstract] [Full Text]
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K. Kitamura, H. Maekawa, and C. Shimoda Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase Mol. Biol. Cell, May 1, 1998; 9(5): 1065 - 1080. [Abstract] [Full Text]
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S. Horie, Y. Watanabe, K. Tanaka, S. Nishiwaki, H. Fujioka, H. Abe, M. Yamamoto, and C. Shimoda The Schizosaccharomyces pombe mei4+ Gene Encodes a Meiosis-Specific Transcription Factor Containing a forkhead DNA-Binding Domain Mol. Cell. Biol., April 1, 1998; 18(4): 2118 - 2129. [Abstract] [Full Text]
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A. Matynia, U. Mueller, N. Ong, J. Demeter, A. L. Granger, K. Hinata, and S. Sazer Isolation and Characterization of Fission Yeast sns Mutants Defective at the Mitosis-to-Interphase Transition Genetics, April 1, 1998; 148(4): 1799 - 1811. [Abstract] [Full Text] [PDF]
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H. Okada, K. Tada, T. Sekiya, K. Yokoyama, A. Takahashi, H. Tohda, H. Kumagai, and Y. Morikawa Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414 Appl. Envir. Microbiol., February 1, 1998; 64(2): 555 - 563. [Abstract] [Full Text]
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N. Okazaki, K. Okazaki, Y. Watanabe, M. Kato-Hayashi, M. Yamamoto, and H. Okayama Novel Factor Highly Conserved among Eukaryotes Controls Sexual Development in Fission Yeast Mol. Cell. Biol., February 1, 1998; 18(2): 887 - 895. [Abstract] [Full Text]
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M. Kimura, I. Kaneko, M. Komiyama, A. Takatsuki, H. Koshino, K. Yoneyama, and I. Yamaguchi Trichothecene 3-O-Acetyltransferase Protects Both the Producing Organism and Transformed Yeast from Related Mycotoxins. CLONING AND CHARACTERIZATION OF Tri101 J. Biol. Chem., January 16, 1998; 273(3): 1654 - 1661. [Abstract] [Full Text] [PDF]
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Isolation of a Schizosaccharomyces pombe rad21ts Mutant That Is Aberrant in Chromosome Segregation, Microtubule Function, DNA Repair and Sensitive to Hydroxyurea: Possible Involvement of Rad21 in Ubiquitin-Mediated Proteolysis Genetics, January 1, 1998; 148(1): 49 - 58.
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S. Yamaguchi, H. Murakami, and H. Okayama A WD Repeat Protein Controls the Cell Cycle and Differentiation by Negatively Regulating Cdc2/B-Type Cyclin Complexes Mol. Biol. Cell, December 1, 1997; 8(12): 2475 - 2486. [Abstract] [Full Text]
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M. Bezanilla, S. L. Forsburg, and T. D. Pollard Identification of a Second Myosin-II in Schizosaccharomyces pombe:. Myp2p Is Conditionally Required for Cytokinesis Mol. Biol. Cell, December 1, 1997; 8(12): 2693 - 2705. [Abstract] [Full Text]
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T. Kodaki and S. Yamashita Cloning, Expression, and Characterization of a Novel Phospholipase D Complementary DNA from Rat Brain J. Biol. Chem., April 25, 1997; 272(17): 11408 - 11413. [Abstract] [Full Text] [PDF]
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Z Hao, A Furunobu, A Nagata, and H Okayama A zinc finger protein required for stationary phase viability in fission yeast J. Cell Sci., January 10, 1997; 110(20): 2557 - 2566. [Abstract] [PDF]
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H. B. Lieberman, K. M. Hopkins, M. Nass, D. Demetrick, and S. Davey A human homolog of the Schizosaccharomyces pombe rad9+ checkpoint control gene PNAS, November 26, 1996; 93(24): 13890 - 13895. [Abstract] [Full Text] [PDF]
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M. S. Albury, P. Dudley, F. Z. Watts, and A. L. Moore Targeting the Plant Alternative Oxidase Protein to Schizosaccharomyces pombe Mitochondria Confers Cyanide-insensitive Respiration J. Biol. Chem., July 19, 1996; 271(29): 17062 - 17066. [Abstract] [Full Text] [PDF]
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T. G. Turi, U. W. Mueller, S. Sazer, and J. K. Rose Characterization of a Nuclear Protein Conferring Brefeldin A Resistance in Schizosaccharomyces pombe J. Biol. Chem., April 12, 1996; 271(15): 9166 - 9171. [Abstract] [Full Text] [PDF]
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K. Akasaka, M. Tamada, F. Wang, K.-i. Kariya, F. Shima, A. Kikuchi, M. Yamamoto, M. Shirouzu, S. Yokoyama, and T. Kataoka Differential Structural Requirements for Interaction of Ras Protein with Its Distinct Downstream Effectors J. Biol. Chem., March 8, 1996; 271(10): 5353 - 5360. [Abstract] [Full Text] [PDF]
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H Bastians and H Ponstingl The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation J. Cell Sci., January 12, 1996; 109(12): 2865 - 2874. [Abstract] [PDF]
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P Dal Santo, B Blanchard, and C. Hoffman The Schizosaccharomyces pombe pyp1 protein tyrosine phosphatase negatively regulates nutrient monitoring pathways J. Cell Sci., January 7, 1996; 109(7): 1919 - 1925. [Abstract] [PDF]
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H. Izumiya and M. Yamamoto Cloning and Functional Analysis of the ndk1 Gene Encoding Nucleoside-diphosphate Kinase in Schizosaccharomyces pombe J. Biol. Chem., November 17, 1995; 270(46): 27859 - 27864. [Abstract] [Full Text] [PDF]
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K Takegawa, D. DeWald, and S. Emr Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology J. Cell Sci., January 12, 1995; 108(12): 3745 - 3756. [Abstract] [PDF]
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K Labib, S Moreno, and P Nurse Interaction of cdc2 and rum1 regulates Start and S-phase in fission yeast J. Cell Sci., January 10, 1995; 108(10): 3285 - 3294. [Abstract] [PDF]
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J. Sakakibara, R. Watanabe, Y. Kanai, and T. Ono Molecular Cloning and Expression of Rat Squalene Epoxidase J. Biol. Chem., January 6, 1995; 270(1): 17 - 20. [Abstract] [Full Text] [PDF]
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