Pierisin-6 Gene Case Study
Identification and characterization of Pierisin gene in P. napi
The strategy was followed to amplify the Pierisin-6 gene was given in the supplementary Fig. 1. The mRNA of pierisin-6 gene was purified from the total RNA of fifth instar larvae of P. napi, the conserved region was amplified using gene specific primers (supplementary table-1), cloned into pGEM®-T vector and sequenced. Further, 3’ and 5’ untranslated regions of pierisin-6 gene were identified by 3’/5’ RACE-PCR. The amplified cDNA sequence consisted of 2946 bp which contains a complete coding region (cds) at nucleotide positions 232 to 2784, 5’ and 3’ untranslated region (UTR) with poly (A) sequences. The complete mRNA of Pierisin-6 from P. napi shared 95.3% and 97.4% of sequence similarity with Pierisin-5 from P. Canidia [5] in nucleotide and amino acids …show more content…
As shown in Fig. 5, the results point out a significant increased in internucleosomal DNA fragmentation of HeLa and HepG2 cells. The DNA isolated from cells treated with Pierisin-6 (2 and 25 ng/ml, respectively) concentration were incubated for 48 h and subjected to agarose gel electrophoresis. DNA ladder characteristic of apoptosis were observed in both cell lines (Fig. 5). No DNA fragmentation occurred in the untreated cells.
3.9. Caspase-3 expression in apoptotic cells
The molecular effectors that activate a series of protease that leads to programmed cell death by intrinsic and extrinsic pathways are known as caspases. Caspase-3 dependent apoptotic cell death was induced by Pierisin-6 in human cancer cell lines were . The HeLa and HepG2 cells were exposed to Pierisin-6 protein (2 and 25 ng/ml, respectively) for different time intervals (6, 2, 24, 36, 48 and 72 h) and the activation of Caspases-3 were detected. Caspase-3 activity increased significantly after Pierisin-6 treatment when compared with untreated cells (Fig. 6).
3.10. DNA content and cell cycle
The strategy was followed to amplify the Pierisin-6 gene was given in the supplementary Fig. 1. The mRNA of pierisin-6 gene was purified from the total RNA of fifth instar larvae of P. napi, the conserved region was amplified using gene specific primers (supplementary table-1), cloned into pGEM®-T vector and sequenced. Further, 3’ and 5’ untranslated regions of pierisin-6 gene were identified by 3’/5’ RACE-PCR. The amplified cDNA sequence consisted of 2946 bp which contains a complete coding region (cds) at nucleotide positions 232 to 2784, 5’ and 3’ untranslated region (UTR) with poly (A) sequences. The complete mRNA of Pierisin-6 from P. napi shared 95.3% and 97.4% of sequence similarity with Pierisin-5 from P. Canidia [5] in nucleotide and amino acids …show more content…
As shown in Fig. 5, the results point out a significant increased in internucleosomal DNA fragmentation of HeLa and HepG2 cells. The DNA isolated from cells treated with Pierisin-6 (2 and 25 ng/ml, respectively) concentration were incubated for 48 h and subjected to agarose gel electrophoresis. DNA ladder characteristic of apoptosis were observed in both cell lines (Fig. 5). No DNA fragmentation occurred in the untreated cells.
3.9. Caspase-3 expression in apoptotic cells
The molecular effectors that activate a series of protease that leads to programmed cell death by intrinsic and extrinsic pathways are known as caspases. Caspase-3 dependent apoptotic cell death was induced by Pierisin-6 in human cancer cell lines were . The HeLa and HepG2 cells were exposed to Pierisin-6 protein (2 and 25 ng/ml, respectively) for different time intervals (6, 2, 24, 36, 48 and 72 h) and the activation of Caspases-3 were detected. Caspase-3 activity increased significantly after Pierisin-6 treatment when compared with untreated cells (Fig. 6).
3.10. DNA content and cell cycle