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Kiechle, M.* ; Manivasakam, P.* ; Eckardt-Schupp, F. ; Schiestl, R.H.* ; Friedl, A.A.*

Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion.

Nucleic Acids Res. 30:e136 (2002)

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Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by gamma-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30degreesC, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements.

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