Isolation of KlCMD1 as an extra-genic suppressor of H2O2 sensitivity. (A) Genetic screen to identify suppressor(s) able to rescue the growth defect of Kloch1-1 cells. The KlCMD1 gene, responsible to allow again the growth of the mutant cells in medium containing the hydrogen peroxide was subcloned into centromeric and multicopy plasmids, CpKlCMD1 and MpKlCMD1 respectively. The growth at 28°C was monitored after 3 d; three independent transformants have been checked, obtaining identical results. (B) Comparison of transcript levels of calmodulin in parental and mutant strains by Northern blot analysis. RNAs were extracted from cells after growth for 48 h in SD minimal medium. The same amount of total RNA (40 μg) from the strains was loaded on each lane; the ethidium bromide-stained gel of the autoradiogram is shown in the bottom part of the panel and the mRNA loading was normalized using the 26S rRNA bands. Quantification, by densitometric analysis, of the radiolabeled signal on the blot is shown in the right part of the panel. The hybridization signal for wild type strain was set as 1.
Zanni et al. BMC Cell Biology 2009 10:86 doi:10.1186/1471-2121-10-86