Abstract
BackgroundCaudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (β-actin).ResultsIn this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation.ConclusionOur results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.
Highlights
Caudata species such as salamanders are affected by environmental changes, which can drastically reduce their population
We aimed to (1) investigate the effects of ionizing radiations (X-rays, and γ-rays), and nonionizing radiation (ultraviolet (UV) light) on H. lichenatus, focusing on changes in gene expression; (2) clone and identify the coding sequences of β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from H. lichenatus, which can be used as endogenous controls, and that of p53, which can be used as a marker of environmental change; and (3) confirm that these cloned open reading frames can be translated to proteins in transfected cells
We showed that hyp53 expression suppressed the proliferation of cultured cells and that hyp53 mRNA responded to radiation, similar to that observed in mammals
Summary
Caudata species such as salamanders are affected by environmental changes, which can drastically reduce their population. The expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (β-actin). Amphibians require both water and land for survival; they are affected by environmental changes, especially those caused by ionizing radiation [1, 2]. Unlike in mammal species such as humans, molecular analyses, such as gene expression analysis, in Caudata after exposure to ionizing radiation are rarely conducted.
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