Examining the cell cycle regulator, p21, in the regeneration competent newt species, Notophthalmus viridescens
Nottrodt, Rachel E.
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Adult newts are capable of completely regenerating lost structures including limb, tail, and spinal cord tissues after injury. A unique mammal capable of similar epimorphic regeneration is the Murphy Roths Large (MRL) mouse with the ability to fully close ear hole punctures. Examination of these mice revealed that they do not express the protein p21, a cyclin dependent kinase (CDK) inhibitor, which controls cell cycle progression from G1 to S phase by monitoring the cell for DNA damage. Knockout p21 mice were also able to fully close ear hole punctures validating the role of p21 in this phenomenon. Based on these findings, my research seeks to examine p21 during newt tail and caudal spinal cord regeneration. Since a lack of p21 allows for regeneration in these mice, otherwise considered regeneration-incompetent, it is of interest to study p21 in the regeneration-competent newt, Notophthalmus viridescens. Using qPCR, I demonstrated that transcripts of a p21-like gene are significantly down-regulated at 14 and 21 days post amputation. Initial Western blots indicated temporal changes in p21 expression across regeneration time points, but at a molecular weight smaller than the predicted 21kDa. The discrepancy in molecular weight led to mass spectrometry protein identification analysis that detected a p16-interacting protein in newt tail that shares 87.5% identity with the Xenopus p16- interacting protein. p16 is also a CDK inhibitor, acting at the G1-S transition to specifically inhibit cyclin D and CDK4/6. Continued survey for p21 protein expression was unable to detect p21 within newt tissues tested. This outcome suggests either the tested antibodies are unable to cross-react with the newt p21 protein, or that it is expressed at very low levels in the newt making it virtually undetectable, or the newt lacks p21 protein like the MRL mouse. 5’-3’ RACE was unable to determine the newt-specific p21 sequence, but the newt Histone H3a gene was amplified and found to contain some sequence similarity to p21 in other species. This work represents the first attempt to study p21 gene and protein expression in regenerating newt caudal tail and spinal cord tissue. The novel finding of p16-interacting protein suggests the potential for alternative cell cycle regulation methods, via p16, previously thought to be lost in the newt.