Summary
We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a λ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.
All insertions inactivated the LYS2 gene and were able to revert with low (about 10-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.
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References
Albertini AM, Hofer M, Calos MP, Miller JH (1982) On the formation of spontaneous deletions: The importance of short sequence homologies in the generation of large deletions. Cell 29:319–328
Auerswald EA, Ludwig G, Schaller H (1981) Structural analysis of Tn5. Cold Spring Harbor Symp Quant Biol 45:107–113
Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336
Berg DE, Davies J, Allet B, Rochaix JD (1975) Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci USA 73:3628–3632
Berg DE, Lodge J, Sasakawa C, Nag DK, Pladnis SH, Weston-Hafer K, Carle GF (1984) Transposon Tn5: specific sequence recognition and conservative transposition. Cold Spring Harbor Symp Quant Biol 49:215–226
Broach JR (1981) The yeast plasmid 2 μm circle. In: Strathern G et al. (eds) Molecular Biology of Yeast Saccharomyces, vol 1. Life Cycle and Inheritance. Cold Spring Harbor Laboratory Press, New York, pp 445–470
Broach JR, Hicks JB (1980) Replication and recombination functions associated with the yeast plasmid, 2 μm circle. Cell 21:501–508
Chernoff YO, Gordenin DA (1987) The expression of yeast transposon insertion in LYS2 gene and of deletions resulting from transposon imprecise excision. Genetika (in Russian) 23:31–41
Chernoff YO, Kidgotko OV, Demberelijn O, Luchnikova IL, Soldatov SP, Glazer VM, Gordenin DA (1984) Mitotic intragenic recombination in the yeast Saccharomyces cerevisiae: marker-effects on conversion and reciprocity of recombination. Curr Genet 9:31–37
Chernoff YO, Kidgotko OV, Demberelijn O, Gordenin DA (1986) Mapping of mutations in LYS2 gene of yeast Saccharomyces. In: Inge-Vechtomov SG (ed) Issledovania po genetike (Researches in genetics; in Russian). Leningrad, vol 10, pp 92–104
Clarke L, Carbon J (1980) Isolation of yeast centromere and a construction of a functional small circular chromosomes. Natue 287:504–509
Collins J, Volkaert J, Nevers P (1982) Precise and nearly precise excision of the symmetrical inverted repeats of Tn5; common features of recA-independent deletion in E. coli. Gene 19:139–146
de Bruijn FJ, Lupsky JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids —a review. Gene 27:131–149
Efstratiadis A, Posakony JW, Maniatis T, O'Connel G, Spritz RA, De Reil JK (1980) The structure and evolution of the human β-globin gene family. Cell 21:653–668
Egner C, Berg DE (1981) Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5. Proc Natl Acad Sci USA 78:459–463
Eibel H, Philippsen P (1983) Identification of the cloned Saccharomyces cerevisiae LYS2 gene by an integrative transformation approach. Mol Genet 91:66–73
Eibel H, Philippsen P (1984) Preferential integration of yeast transposable element Ty into a promoter region. Nature 307:386–388
Foster TJ, Lundblad V, Hanley-Way S, Halling SM, Kleckner N (1981) Three Tn10-associated excision events: relationship to transposition and role of direct and inverted repeats. Cell 23:215–227
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of yeast cells treated with alkali cations. J Bacteriol 153:163–168
Kleckner N (1981) Transposable elements in prokaryotes. Annu Rev Genet 15:341–404
Lundblad V, Kleckner N (1985) Mismatch repair mutations of Escherichia coli K12 enhance transposon excision. Genetics 109:3–19
Lundblad V, Taylor AF, Smith GR, Kleckner N (1984) Unusual alleles of recB and recC stimulate excision of inverted repeat transposons Tn10 and Tn5. Proc Natl Acad Sci USA 81:824–828
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Mazodier P, Cossart P, Giraud E, Gasser F (1985) Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes. Nucleic Acids Res 13:195–205
Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78
Sherman F, Fink GR, Lawrence CW (1979) Methods in yeast genetics. Cold Spring Harbor Laboratory Pess, New York, p 239
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Gordenin, D.A., Trofimova, M.V., Shaburova, O.N. et al. Precise excision of bacterial transposon Tn 5 in yeast. Mol Gen Genet 213, 388–393 (1988). https://doi.org/10.1007/BF00339607
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DOI: https://doi.org/10.1007/BF00339607