Reference: Kiechle M, et al. (2002) Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion. Nucleic Acids Res 30(24):e136

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Abstract


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 (30 degrees C, 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.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Kiechle M, Manivasakam P, Eckardt-Schupp F, Schiestl RH, Friedl AA
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