Peroxisomes research Article Example | Topics and Well Written Essays - 1250 words

Peroxisomes research - Article Example 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 int ensity 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