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Nucleic Acids Research, 2002, Vol. 30, No. 24 e136
© 2002 Oxford University Press

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

Markus Kiechle1,2, Palaniyandi Manivasakam1, Friederike Eckardt-Schupp2, Robert H. Schiestl1 and Anna A. Friedl*,2,3

1 Department of Pathology, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA, 2 Institute of Molecular Radiation Biology, GSF-Research Center, D-85764 Neuherberg, Germany and 3 Radiobiological Institute, University of Munich, D-80336 Munich, Germany

*To whom correspondence should be addressed at Radiobiological Institute, University of Munich, Schillerstrasse 42, 80336 Munich, Germany. Tel: +49 89 5996807; Fax: +49 89 5996 840; Email: anna.friedl{at}lrz.uni-muenchen.de

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°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.


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C. Y. Chan, M. Kiechle, P. Manivasakam, and R. H. Schiestl
Ionizing radiation and restriction enzymes induce microhomology-mediated illegitimate recombination in Saccharomyces cerevisiae
Nucleic Acids Res., August 1, 2007; 35(15): 5051 - 5059.
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