Abstract
Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.
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References
Levin HL, Moran JV (2011) Dynamic interactions between transposable elements and their hosts. Nat Rev Genet 12(9):615–627. doi:10.1038/nrg3030, nrg3030 [pii]
Beck CR, Garcia-Perez JL, Badge RM, Moran JV (2011) LINE-1 elements in structural variation and disease. Annu Rev Genomics Hum Genet 12:187–215. doi:10.1146/annurev-genom-082509-141802
Singleton TL, Levin HL (2002) A long terminal repeat retrotransposon of fission yeast has strong preferences for specific sites of insertion. Eukaryot Cell 1:44–55
Bowen NJ, Jordan I, Epstein J, Wood V, Levin HL (2003) Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements derived from the complete genome sequence of Schizosaccharomyces pombe. Genome Res 13(Sept):1984–1997
Leem YE, Ripmaster TL, Kelly FD, Ebina H, Heincelman ME, Zhang K, Grewal SIS, Hoffman CS, Levin HL (2008) Retrotransposon Tf1 is targeted to pol II promoters by transcription activators. Mol Cell 30(1):98–107
Guo Y, Levin HL (2010) High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe. Genome Res 20(2):239–248
Feng G, Leem YE, Levin HL (2013) Transposon integration enhances expression of stress response genes. Nucleic Acids Res 41(2):775–789. doi:10.1093/nar/gks1185
Chatterjee AG, Esnault C, Guo Y, Hung S, McQueen PG, Levin HL (2014) Serial number tagging reveals a prominent sequence preference of retrotransposon integration. Nucleic Acids Res 42(13):8449–8460. doi:10.1093/nar/gku534
Atwood A, Choi J, Levin HL (1998) The application of a homologous recombination assay revealed amino acid residues in an LTR-retrotransposon that were critical for integration. J Virol 72(2):1324–1333
Levin HL (1995) A novel mechanism of self-primed reverse transcription defines a new family of retroelements. Mol Cell Biol 15:3310–3317
Levin HL, Boeke JD (1992) Demonstration of retrotransposition of the Tf1 element in fission yeast. EMBO J 11:1145–1153
Boeke JD, Garfinkel DJ, Styles CA, Fink GR (1985) Ty elements transpose through an RNA intermediate. Cell 40(491):491–500
Forsburg SL, Rhind N (2006) Basic methods for fission yeast. Yeast 23(3):173–183
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Sangesland, M., Atwood-Moore, A., Rai, S.K., Levin, H.L. (2016). Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe . In: Garcia-Pérez, J. (eds) Transposons and Retrotransposons. Methods in Molecular Biology, vol 1400. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3372-3_8
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DOI: https://doi.org/10.1007/978-1-4939-3372-3_8
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