Biology subjects

Pandita, M.

Publications and source records attributed to Pandita, M..

3 recordsLinked to original sources

bioRxiv2020

An Assay for Apoptosis detection based on Quantification of Multi nuclei feature and Nucleus to Cytoplasm ratio in S. cerevisiae cells treated with Acetic Acid and Hydrogen peroxide

The programmed cell death, apoptosis is a complex universal biological process in all types of eukaryotes ranging from single cell to multi-cellular organisms. The markers for apoptosis have been studied by assays based on both biochemical as well as microscopy however most assays are not affordable for many smaller labs. Acetic acid and hydrogen peroxide both induce apoptosis at higher concentrations in S. cerevisiae. Here we describe an assay system for the detection of apoptosis features based on DAPI staining followed by fluorescence microscopy in the cells treated with apoptosis inducing concentration of acetic acid and hydrogen peroxide. In this assay both untreated and cells treated with acetic acid and hydrogen peroxide were stained with DAPI and observed for the late stage apoptosis feature, Nuclear DNA fragmentation based multi nuclei centers and increase in the nuclear region enlargement. Further the multi nuclei feature and enlarged nuclei region as nucleus to cytoplasm ratio was quantified using Image J software. We report that S. cerevisiae strain BY4741 cells when treated with apoptosis inducing doses of acetic acid (140mM) and hydrogen peroxide (10mM) for 200 minutes, showed apoptosis marker feature such as nuclear region enlargement with multi-nuclei feature due to nuclear DNA fragmentation and increased nucleus to cytoplasm ratio when compared with untreated cells. We propose that this assay can be utilized for scoring the quantitative apoptotic feature as increase in multi-nuclei centers due to DNA fragmentation and nucleus to cytoplasm ratio as an indicator of apoptosis in S. cerevisiae upon treatment with apoptosis inducing agents. The assay system described here is easy to perform and affordable for the smaller lab to analyze the apoptotic features in S. cerevisiae cells which can be applied to other system as well.

genetics↗

bioRxiv2020

Deletion of Autophagy gene ATG1 and F-box motif encoding gene YDR131C together leads to Synthetic Growth Defects and Flocculation behaviour in Saccharomyces cerevisiae

F-box motif encoding YDR131C is functionally uncharacterized gene which forms the complex with the SCF-E3 ligase. The F-box motif containing proteins are involved in substrate recruitment for the ubiquitination and subsequent degradation through 26S proteasome. Autophagy gene, ATG1 (ULK1in human) is a well conserved serine-threonine kinase, required for vesicle formation and cytoplasm to vacuole targeting pathway. Atg1p forms the complex with Atg13p and Atg17p during autophagy. The understanding of crosstalk between ubiquitin and autophagy pathways is crucial for synthetic lethality screen and drug targeting. Here we have conducted the study for genetic interaction between uncharacterized YDR131C and ATG1 gene representing both specific and non-specific protein degradation pathways. The single and double gene knockout strains of YDR131Cand ATG1 genes were constructed in the BY4741 genetic background and analysed for growth fitness. The strains were also evaluated for cellular growth response in presence of hydroxyurea (HU), methyl methane sulfonate (MMS), and hydrogen peroxide (H2O2) stress causing agents by spot assay. The ydr131c{Delta}atg1{Delta} showed the synthetic growth defect phenotype with floc formation in rich medium which showed floc disruption in presence of EDTA. The ydr131c{Delta}atg1{Delta} cells showed the sensitivity to stress agents HU, MMS, and H2O2 when compared with ydr131c{Delta}, atg1{Delta}, and WT cells.. Based on the observations, we report that YDR131C and ATG1 functions in parallel pathways for growth fitness and cellular growth response to stress agents. Interestingly this study also revealed the crosstalk between ubiquitination and autophagy pathways. The defects in both the pathways could lead to synthetic growth defects which may have implication for the precision medicine initiatives.

genetics↗

bioRxiv2020

Deletion of Autophagy gene ATG1 and Glyoxylate cycle Regulator encoding gene UCC1 together leads to Synthetic Growth Defects and Sensitivity to Genotoxic agents in Saccharomyces cerevisiae

Atg1 of S. cerevisiae is a key component of autophagy encoded by ATG1 gene, involved in the process of degradation of cytosolic components through autophagy. UCC1, an F-box encoding gene is involved in the negative regulation of glyoxylate pathway via degradation of Cit2 enzyme by ubiquitin proteasome system. We investigated the genetic interaction between ATG1 and UCC1 using the gene deletion approach. The atg1{Delta}ucc1{Delta} cells showed the synthetic growth defects with abnormal budding and sensitivity to genotoxic and oxidative stress agents. Based on the observations, we report that ATG1 and UCC1 interact genetically to regulate the cell growth fitness and function in parallel pathway in cellular response to the genotoxic stress agents. The present investigation also revealed the cross talks among autophagy, ubiquitin proteasome system, and glyoxylate pathways.

genetics↗