These cells did not appear to be true pseudohyphae, as they had a highly aberrant and variable morphology, similar
to that seen in Candida Ivacaftor concentration albicans strains defective in cell cycle progression. The numbers of cells with normal and abnormal morphology were quantitated and are shown in Table 1 and Figure 2c and 2d. When compared to wildtype, log phase cultures of the rad54Δ/rad54Δ strain had far fewer normal budding yeast cells, and a large increase in the number of cells exhibiting the abnormal morphology shown in Figure 2b. The elongated pseudohyphal cells displayed an aberrant nuclear morphology with a preponderance of the pseudohyphal cells having an elongated single DAPI staining body stuck in the neck between the two
cell bodies (Figure 2c). Additional nuclear morphologies included apparent anucleate cells (two selleck chemical cells with only one nucleus), cells with a nucleus EPZ5676 chemical structure in each bud where one nucleus is elongated, and cells with multiple nuclei (Figure 2c). Regarding the pseudohyphal cells, in the single nucleate cells, 9/14 had an elongated single nucleus, and in the cells with two nuclei, 10/20 had one or two elongated nuclei. Table 1 Log phase morphology of Candida albicans mutants Strain Unbudded Budded Abnormal/Pseudohyphae Total Wildtype 108 191 1 300 rdh54Δ/RDH54 111 187 2 300 rdh54Δ/rdh54Δ 78 221 1 300 rad54Δ/RAD54 71 227 1 300 rad54Δ/rad54Δ-1 92 143 65 300 rad54Δ/RAD54(+) 108 191 1 300 DAPI staining of cells also showed additional defects in chromosome segregation in the rad54Δ/rad54Δ strain. There was an increase in G2 doublet cells that have a single nucleus at the neck (Figure 2d). This morphology is suggestive of a DNA damage checkpoint arrest in Saccharomyces cerevisiae [25] and could apply to Candida albicans [26]. These phenotypes were not seen in the rdh54Δ/rdh54Δ strain, showing that these Morin Hydrate two genes have
different roles in vivo. Additionally, neither the wildtype strain nor the RAD54 reintegration strain showed these aberrant nuclear morphologies. Sensitivity to DNA damage is increased in the Candida albicans rad54Δ/rad54Δ mutant In Saccharomyces cerevisiae, deletion of RDH54 and RAD54 leads to increased sensitivity to DNA damage. The Saccharomyces cerevisiae haploid rad54Δ is highly sensitive to methyl methanesulfonate (MMS) [19], but the Saccharomyces cerevisiae RDH54 gene does not appear to have as strong of a role in haploid cells, as deletion of RDH54 only increases MMS sensitivity in diploids at normal MMS concentrations [27]. To test the effect of deletion of Candida albicans RAD54 and RDH54 on MMS and menadione sensitivity, spot dilution assays were performed on YPD agar plates containing a range of MMS concentrations from 0.0025% to 0.02%, or menadione concentrations from 0.05 mM to 0.5 mM.