Peroxisomes research - Article Example

1. Introduction Peroxisomes are multi-functional organelles, playing major role in cellular metabolism. Zellweger syndrome is one of the several peroxisome-associated disorders are identified in man, some of which are lethal. In yeasts, peroxisomes are generally involved in the primary metabolism of specific carbon and/or nitrogen sources used for growth. Since peroxisomes lack DNA, all peroxisomal proteins, both soluble and membrane-bound, are post-translationally imported into the organelle (De Duve, 1983). PEX3 gene corresponds for peroxisomes and it encodes a 52 kDa protein essential for peroxisome biogenesis and maintenance. The peroxisome also functions to relieve cells of oxidative stress via proteins such as catalase (Cta1 gene) that are present in the peroxisomal matrix. Oxidative stress can be monitored by the accumulation of H2O2. The present contribution relates in determining the role of iPEX3A and iPEX3B in peroxisomal function and the potential role of these proteins in cell. 2. Materials and Methods: 1. Organisms and media. Yeast (S. cerevisae); Yeast Potato Dextrose media (YPD); Low Fluorescent Media(YNB). pLUC plasmid 2. Reagents OxyBurst Green H2 DCFDA; Hydrogen peroxide Dimethyl sulphoxide (DMSO) Phosphate buffer saline (PBS), 1X Kanamycin 3. Genomic DNA Isolation kit and Luciferase assay system kit (Promega). 4. PCR 5. Inverted fluorescent microscope 6. Miscellaneous Microfuge tubes(1.5 ml or 2 ml) PCR tubes Micropipettes Micropipette tips Luminometer Sterile Petri plates Incubator (30C) Centrifuge Methods: DNA Isolation: The DNA isolation of S. cerevisae was performed form the DNA isolation kit as per the supplier's instructions. Detection of H2O2 Yeast strains were grown in 5 ml YPD for 18h at 30C. The cells were spin down and were washed twice with 1ml low fluorescent media (YNB). The final pellet was resuspended with 500 ls low fluorescent YNB. To each sample 10 ls OxyBurst Green H2DCFDA, SE was added. The dye was dissolved in DMSO to make a 10mg/ml stock. The cells were incubated with dye for 30 mins at 30C. These samples were washed 3 times with low fluorescent media (YNB). 1000 cells were plated in each well of a multi-well microscope slide. The cells were counted using inverted-fluorescent microscope. The excitation and emission filters were used for green fluorescence. Luciferase measurement The genomic DNA was amplified using PCR 500 bp upstream region of Act1 Cta1, iPex3A and iPex3B open reading frames. The PCR fragments were inserted upstream of Luciferase reporter gene in pLUC plasmid. The promoter-pLUC plasmids were transformed into wild-type yeast via the PEG - LiAC transformation method. The Transformants were plated on YPD plates with kanamycin. Each colony of the transformant was grown in 5ml YPD for 18 hours at 30C. The cells were spin down in a centrifuge. The cells were resuspended in 1mL low fluorescence media (YNB). This sample was divided into two parts. To one sample 100uls of 0.003% hydrogen peroxide was added. The tubes were incubated at 28C for 30 minutes. The cells were spin down. The cells were washed 3 times with 1ml 1XPBS. The final pellet was resuspended in 500 ls of 1X PBS. The cells were separated into 10,000 cell aliquots. One ml lysis reagent to the cells and incubated for 5 mins. The luciferase assay reagent was added to lysed cells. The intensity of light emitted from sample was measured with a luminometer. Statistical analysis: Each sample were measured six times and standard deviation was determined. Further, t-test were carried out to signify the difference in the values. Results: To determine the role of iPEX3A and iPAX3B in peroxisomal function set of experiments were carried out. In first sets of experiments accumulation of H2O2 were determined in different mutant having single mutation in cat1, ipex3A and ipex3B. Table 1 displays the results of H2O2 treatment on these three mutants along with wild type (positive control) cells. There was significant increase (p Read More