Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion.
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.
Kiechle
Markus
M
Department of Pathology, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.
Manivasakam
Palaniyandi
P
Eckardt-Schupp
Friederike
F
Schiestl
Robert H
RH
Friedl
Anna A
AA
D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression / Q000528:radiation effects; D016681:Genome, Fungal; D007763:Lac Operon / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000379:methods; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000528:radiation effects*; D014170:Transformation, Genetic
D005656:Fungal Proteins