Genetic interaction between RLM1 and F-box motif encoding gene SAF1 contributes to stress response in Saccharomyces cerevisiae

Abstract

Background Stress response is mediated by the transcription of stress-responsive genes. The F-box motif protein Saf1p is involved in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1p upon nutrient stress. The four transcription regulators, BUR6, MED6, SPT10, SUA7, are listed for SAF1 in the genome database of Saccharomyces cerevisiae. Here in this study, we carried out an in-silico analysis of gene expression and transcription factor databases to understand the regulation of SAF1 expression during stress for hypothesis and experimental analysis. Result An analysis of the GEO profile database indicated an increase in SAF1 expression when cells were treated with stress agents such as Clioquinol, Pterostilbene, Gentamicin, Hypoxia, Genotoxic, desiccation, and heat. The increase in expression of SAF1 during stress conditions correlated positively with the expression of RLM1, encoding the Rlm1p transcription factor. The expression of AAH1 encoding Aah1p, a Saf1p substrate for ubiquitination, appeared to be negatively correlated with the expression of RLM1 as revealed by an analysis of the Yeastract expression database. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes together may contribute to stress tolerance. The experimental analysis of cellular growth response of cells lacking both SAF1 and RLM1 to selected stress agents such as cell wall and osmo-stressors, by spot assay indicated stress tolerance phenotype similar to parental strain however sensitivity to genotoxic and microtubule depolymerizing stress agents. Conclusions Based on in-silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.

Deletion of autophagy related, ATG1 and F‐box motif encoding YDR131C, together, lead to synthetic growth defects and flocculation behavior in Saccharomyces cerevisiae

“…To compare morphological features the method mentioned in the study was adopted. [ 23 ] For phase‐contrast microcopy, an aliquot of the log‐phase grown culture of each strain from liquid media was observed under Leica DM3000 microscope (Leica) using ×100 lens. The cells of each strain grown for growth kinetics were also observed using phase contrast filters.…”

“…For gene deletion, we adopted the method used in the studies. [21][22][23][24] The plasmids, pFA6a-KanMX6, and pFA6a-His3MX6 were used for amplification of deletion cassettes for YDR131C deletion. The sequences of primers are listed in Table 2.…”

“…To compare the growth rate between wildtype (WT) and deletion mutants (ydr131cΔ, atg1Δ, and ydr131cΔatg1Δ) on solid and liquid media the method adopted as mentioned in the studies. [21,[23][24][25] For comparative growth assessment on solid media, each strain was streaked on yeast extract-peptone-dextrose (YPD) + agar plates (yeast-1%, w/v peptone-2%, w/v dextrose-2%, and w/v agar-2%) and incubated for 36-48 h, at 30°C. For comparative growth kinetics, each strain was grown in triplicates in a YPD medium for 14 h. Optical density (OD) at 600 nm was measured for every 2 h and the average OD of triplicate cultures at each time point was plotted for comparative growth assessment.…”

“…For chitin distribution and nucleus status in WT and mutants, the method mentioned in the study was adopted. [21,23,24] WT and mutant strains were grown overnight at 30°C and transferred to fresh culture in a 1:10 ratio on the next day. Cells were grown till the log phase and collected by centrifugation and then suspended in 100 µl of the solution containing calcofluor white (CFW) (50 μg/ml) fluorescent dye.…”

“…To compare the cellular growth response of WT and derivative mutants (ydr131cΔ, atg1Δ, and ydr131cΔatg1Δ) in presence of stress agents, spot assay was performed as per the method mentioned in the study. [21,23,24] Briefly, WT and deletion derivatives (ydr131cΔ, atg1Δ, and ydr131c-Δatg1Δ) were grown till log phase (OD 600nm −0.8 to 1.0), culture concentration was adjusted and serially diluted. From each dilution, a 3 µl aliquot was spotted onto agar plates containing YPD and YPD + stress agents such as hydroxyurea (HU) (200 mM), MMS (0.035%), and hydrogen peroxide (4 mM), followed by incubation of the plates at 30°C for 2-3 days.…”