Introduction
This research investigates the construction of cDNA from sucrose gradient-fractionated mRNA derived from SVT2 for p53 specific sequences. The paper investigates an increase in the p53 levels to help derive the relationship between the protein and the transformation of cells.
Objective of the Study
The presence of the protein in low amounts in unaltered growth cells and its short half-life suggests that p53 plays a regulatory role during the growth of cells. Comprehensions of the mechanisms during cell transformation that increase p53 levels will help contrive the relationship between p53 and cell alteration. This research will fill a research gap by using SVT2 cells to prepare a cDNA clone specific to the p53 protein to determine factors affecting cell transformation.
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Observations/Findings by Other Authors
Previous authors have concluded that the differential expression of p53 in altered and unaltered cells results from several mechanisms. The simian virus 40 (SV40) alteration increases the half-life of p53 cellular antigen, resulting in significant accumulation of the protein without disrupting the translatable p53 mRNA levels. They have also suggested that the changes in translatable mRNA levels justify the difference in the p53 levels of differentiated and undifferentiated F9 teratocarcinoma cells. Similarly, protein stabilization is likely to cause an accumulation in the cells altered by the Epstein-Barr virus and the adenovirus. One study has also claimed that transcriptional activation occurs during the induction of lymphocytes.
Results
Screening of the cDNA revealed a substantial enrichment of the protein after the preparation of polysomes from SVTZ cells and their immunoprecipitation with a monoclonal antibody against p53.When a second cDNA probe with total cytoplasmic mRNA was carried out, the single colony reacted with the immunoselected probe but failed to react with the total cytoplasmic probe.
Further identification of the DNA showed that the DNA collected from various cDNA clones did not bind any p53 mRNA. However, the pp53-208 DNA selected a significant amount of the messenger that indicates a p53 mRNA-derived insert. The excision of the insert from pp53-208 DNA demonstrated the hybridization of two extra clones. The two clones strongly bonded to p53 mRNA while several other clones failed to select any detectable amounts of this mRNA. Hybridization of pp53-208 to mouse genomic DNA showed only a single band that suggested that a cloned DNA is compatible to a single mouse gene.
Materials and Methods
The construction of the cDNA library was carried out by preparing and fractionating RNA from SVT2 cells. The cDNA library was later screened by hybridizing them to colony blots. The cells were selected for testing after being grown in 250ml of M9 medium and their amplification in cytidine at 1mg/ml. Genomic DNA analysis is carried out by digesting EcoRI, transferring it to nitrocellulose sheets, and hybridizing it with an insert.
How the Study advances the Present Subject
The report covers an aspect of the subject that had not been studied previously. The authors have adequately integrated the SVT2 cells, which have not been used previously in altering cDNA. It advances this subject by investigating alternative factors that lead to increased p53 levels during cell transformation.
Conclusion
The increased amounts of P53 detected during cell alteration appear to be directly related to the alterations in mRNA levels. The changes detected in the gene expression of p53 and the factors that affect the changes will help examine how p53 and cell transformation relate also allows for further screening of different cells and tissues to produce more p53 mRNA.
Critical analysis
The report is conclusive as it gives a detailed explanation of the subject and the findings from the experiments. The experiments carried out to fulfill the author’s objective and provide alternatives to the previous studies. The results are presented in figures and tables that enable the reader to analyze the results correctly. The impact of SVT2 is demonstrated in the research through the various tests carried out. The author’s conclusion is justified due to the alternative tests they carried out to ensure they have the right results. However, further research is needed to determine alternative mechanisms that support the increase in p53 after cell transformation.
References
Oren, M., & Levine, A. (1983). Molecular cloning of a cDNA specific for the murine p53 cellular tumor antigen. Proceedings of the National Academy of Sciences of the United States of America, 80 (1), 56-59. Retrieved May 15, 2021, from http://www.jstor.org/stable/13319
