Biology subjects

Bairwa, N. K.

Publications and source records attributed to Bairwa, N. K..

7 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↗

bioRxiv2019

Genetic Interaction between RLM1 and F-box Motif Encoding gene SAF1 Contributes to Stress Response in Saccharomyces cerevisiae

Stress response is mediated by transcription of stress responsive genes. F-box motif protein Saf1 involves in SCF-E3 ligase mediated degradation of the adenine deaminase, Aah1 upon nutrient stress. Four transcription regulators, BUR6, MED6, SPT10, SUA7, have been reported for SAF1 gene in genome database of Saccharomyces cerevisiae. Here in this study an in-silco analysis of gene expression and transcription factor databases was carried out to understand the regulation of SAF1 gene expression during stress for hypothesis generation and experimental analysis. The GEO profile database analysis showed increased expression of SAF1 gene when treated with clioquinol, pterostilbene, gentamicin, hypoxia, genotoxic, desiccation, and heat stress, in WT cells. SAF1 gene expression in stress conditions correlated positively whereas AAH1 expression negatively with RLM1 transcription factor, which was not reported earlier. Based on analysis of expression profile and regulatory association of SAF1 and RLM1, we hypothesized that inactivation of both the genes may contribute to stress tolerance. The experimental analysis with the double mutant, saf1{Delta}rlm1{Delta} for cellular growth response to stress causing agents, showed tolerance to calcofluor white, SDS, and hydrogen peroxide. On the contrary, saf1{Delta}rlm1{Delta} showed sensitivity to MMS, HU, DMSO, Nocodazole, Benomyl stress. Based on in-silico and experimental data we suggest that SAF1 and RLM1 both interact genetically in differential response to genotoxic and general stressors.

genetics↗

bioRxiv2019

Absence of Replication fork associated factor CTF4 and F-box motif Encoding Gene SAF1 leads to reduction in Cell Size and Stress Tolerance Phenotype in S. cerevisiae

Chromosome transmission fidelity factor, Ctf4 in S. cerevisiae associates with replication fork and helps in the sister chromatid cohesion. At the replication fork, Ctf4 links DNA helicase with the DNA polymerase. The absence of Ctf4 invokes replication checkpoint in the cells. The Saf1 of S.cerevisiae interacts with Skp1 of SCF-E3 ligase though F box-motif and ubiquitinates the adenine deaminase Aah1 during phase transition due to nutrient stress. The genetic interaction between the CTF4 and SAF1 has not been studied. Here we report genetic interaction between CTF4 and SAF1 which impacts the growth fitness and response to stress. The single and double gene deletions of SAF1 and CTF4 were constructed in the BY4741 genetic background. The strains were tested for growth on rich media and media containing stress causing agents. The saf1{Delta}ctf4{Delta} cells with reduced cell size showed the fastest growth phenotype on YPD medium when compared with the saf1{Delta}, ctf4{Delta}, and WT. The saf1{Delta}ctf4{Delta} cells also showed the tolerance to MMS, NaCl, Glycerol, SDS, Calcofluor white, H2O2, DMSO, Benomyl, and Nocodazole when compared with the saf1{Delta}, ctf4{Delta}, and WT cells. However, saf1{Delta}ctf4{Delta} cells showed the sensitivity to HU when compared with WT and saf1{Delta}. Based on these observations we suggest that SAF1 and CTF4 interact genetically to regulate the cell size, growth and stress response.

genetics↗

bioRxiv2019

The absence of F-box motif Encoding Gene SAF1 and Chromatin Associated factor CTF8 together contributes to MMS Resistant and HU Sensitive phenotype in S. cerevisiae

The Replication factor-C compex which related to cohesion, constitutes, three subunits called Ctf18, Ctf8 and Dcc1. These three subunit complex assist the loading of PCNA onto the chromosome. None of the replication factor C components are essential for cell viability. The null mutant of the CTF8 in S.cerevisiae shows the chromosome instability and high frequency of chromosome loss. The SAF1 gene product of S. cerevisiae involved in the degradation of adenine deaminase factor Aah1p by SCF-E3 ligase, which itself is the part of E3 ligase. The ubiquitin marked degradation of Aah1p occurs during nutrient stress which lead to cell enter into the quiescent state. The N-terminus of Saf1p interacts with the Skp1 of SCF-E3 ligase and at C-terminus recruits with Aah1p. Here we have investigated about the binary genetic interaction between the SAF1 and CTF8 genes. The strains containing single and double gene deletions of SAF1 and CTF8 were constructed in the BY4741 genetic background. Further the mutant strains were evaluated for growth fitness, genome stability and response to genotoxic stress caused by hydroxyurea (HU) and methyl methane sulfonate (MMS). The saf1{Delta}ctf8{Delta} strain showed the increased growth phenotype in comparison to saf1{Delta}, ctf8{Delta}, and WT strain on YPD medium. However saf1{Delta}ctf8{Delta} strain when grown in the presence MMS showed resistance and HU sensitive phenotype when compared with saf1{Delta}, ctf8{Delta}. The frequency of Ty1 retro-transposition was also elevated in saf1{Delta}ctf8{Delta} in comparison to either saf1{Delta} or ctf8{Delta}. The number of cells showing the two or multi-nuclei phenotype was also increased in saf1{Delta}ctf8{Delta} cells when compared with the either saf1{Delta} or ctf8{Delta}. Based on these observations, we report that the absence of both the gene SAF1 and CTF8 together leads to MMS resistance, HU sensitivity, and genome instability. This report warrants the investigation of mechanisms of differential growth phenotype due to loss of SAF1 and CTF8 together in presence of genotoxic stress in future.

genetics↗

bioRxiv2019

Loss of F-box Motif Encoding Gene SAF1 and RRM3 Together Leads to Synthetic Growth Defect and Sensitivity to HU, MMS in S.cerevisiae

Unearthing of novel genetic interaction which leads to synthetic growth defects due to inactivation of genes are needed for applications in precision medicine. The genetic interactions among the molecular players involving different biological pathways need to be investigated. The SAF1 gene of S.cerevisiae encodes for a protein product which contain N-terminal F-box motif and C-terminal RCC1 domain. The F-box motif interacts with Skp1subunit of the SCF-E3 ligase and C-terminus with Aah1 (adenine deaminase) for ubiquitination and subsequent degradation by 26S proteasome during phase transition from proliferation state to quiescence phase due to nutrient limitation stress. The replication fork associated protein Rrm3 of S.cerevisiae belongs to Pif1 family helicase and function in removal of the non-histone proteins during replication fork movement. Here we have investigated the genetic interaction among both the genes (SAF1 and RRM3) and their role in growth fitness and genome stability. The single and double gene knockout strains of SAF1and RRM3 genes was constructed in BY4741 genetic background and checked for the growth fitness in presence of genotoxic stress causing agents such as hydroxyurea and methyl methanesulfonate. The strains were also evaluated for nuclear migration defect by DAPI staining and for HIS3AI marked Ty1 retro-transposition. The saf1{Delta}rrm3{Delta} showed the extremely slow growth phenotype in rich medium and sensitivity to genotoxic agents such as HU and MMS in comparison to single gene mutant (saf1{Delta}, rrm3{Delta}) and WT cells. The saf1{Delta}rrm3{Delta} also showed the defects in nuclear migration as evident by multi-nuclei phenotype. The saf1{Delta}rrm3{Delta} also showed the elevated frequency of Ty1 retro-transposition in JC2326 background in comparison to either saf1{Delta} or rrm3{Delta}. Based on these observations we report that thatSAF1 and RRM3 functions in parallel pathway for growth fitness and stability of the genome.

genetics↗