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

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.

Ionizing Radiation Induces Microhomology-Mediated End Joiningin transin Yeast and Mammalian Cells

“…Spun-down yeast cells were exposed to 50 Gy, which resulted in about 60% survival (34), 3 h before the heat-shock step of the transformation procedure (41). …”

“…Previous studies have shown that ionizing radiation enhances the frequency of nonhomologous integration in yeast and mammalian cells (33, 34). In addition, other damaging agents, including camptothecin, VP16, UV radiation and hydrogen peroxide, increase nonhomologous integration of transfected plasmids in mammalian cells (35-37).…”

Hypotonic stress-induced calcium signaling in Saccharomyces cerevisiae involves TRP-like transporters on the endoplasmic reticulum membrane

“…The method described by Kiechle et al [33] was used to measure the ␤-galactosidase activity in exponentially growing cells. Briefly, 200 l of the cell suspension ('exp', for exponentially growing treatment) were incubated at room temperature for 1.5 h and then collected by centrifugation at 13,000 rpm for 2 min, resuspended in 550 l of pre-cold Z-buffer (75 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 ) and quickly frozen in dry ice and conserved at −80 • C.…”

Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene

“…The phenotype of E2 which appeared as PHOT-negative in the first round of screening was probably based on epigenetic gene silencing effects (e.g., co-suppression and/or antisense RNA). Moore 1988; Kiechle et al, 2002), and bacteria (Golub and LOW 1983;Asai et al, 1993), we irradiated the cells with 354 nm UV light before transformation. This treatment reduced the GTR, resulting in fewer homologous recombinants and an increased number of transformants with non-homologous integrated aphVIII.…”