Abstract
Thanks to the convenience and flexibility of the multicopy plasmid-based approach for heterologous gene expression, this technique has long been used for biological studies, especially in prokaryotes and lower eukaryotes. For better understanding of biological mechanisms, however, there are increasing demands on the experimental technologies enabling fine-tuned expression of introduced heterologous genes or serving conditions that are closer to the physiological conditions. For this purpose, the use of direct tagging of a chromosomal gene has been gradually increasing, although the use conditions of this approach are relatively limited compared to plasmid-based methods. Expression of a cloned gene using chromosomal integration has a property intermediate between multicopy plasmid-based method and direct tagging of an endogenous gene. Here, we describe the principle and methods of introduction of a cloned gene into the targeting loci of the chromosome in fission yeast.
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References
Maundrell K. (1993) Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123, 127–130.
Matsuyama A, Shirai A, Yashiroda Y, et al. (2004) pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast. Yeast 21, 1289–1305.
Matsuyama A, Shirai A, Yoshida M. (2008) A novel series of vectors for chromosomal Âintegration in fission yeast. Biochem. Biophys. Res. Commun. 374, 315–319.
Bach ML. (1987) Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe. Curr. Genet. 12, 527–534.
Maundrell K, Hutchison A, Shall S. (1988) Sequence analysis of ARS elements in fission yeast. EMBO J. 7, 2203–2209.
Kikuchi Y, Kitazawa Y, Shimatake H, Yamamoto M. (1988) The primary structure of the leu1 + gene of Schizosaccharomyces pombe. Curr. Genet. 14, 375–379.
Bähler J, Wu JQ, Longtine MS, et al. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943–951.
Goldstein AL, McCusker JH. (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.
Kimura M, Kamakura T, Tao QZ, et al. (1994) Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae. Mol. Gen. Genet. 242, 121–129.
Matsuyama A, Yoshida M. (2009) Systematic cloning of an ORFeome using the Gateway system. Methods Mol. Biol. 577, 11–24.
Keeney JB, Boeke JD. (1994) Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136, 849–856.
Basi G, Schmid E, Maundrell K. (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123, 131–136.
Acknowledgments
This work was supported by CREST Research Project, Japan Science and Technology Agency.
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Matsuyama, A., Yoshida, M. (2012). Heterologous Gene Expression by Chromosomal Integration in Fission Yeast. In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_23
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DOI: https://doi.org/10.1007/978-1-61779-433-9_23
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