Problems utilizing an enzyme sensitive site assay for photorepair of exogenous DNA with cell-free extracts made from amphibian embryos

Abstract

Attempts were made to assay the ultraviolet-B (UVB) damage repair ability of seven Ontario amphibian species using an enzyme sensitive site restriction-enzyme (ESS) assay. Cell-free protein extracts of amphibian eggs caused the degradation of even high (9 mg/mL) exogenous DNA concentrations. This type of signal loss is characteristic of nuclease digestion. High endogenous concentrations of amphibian nucleases appear to preclude the use of plasmid DNA-ESS assays to determine the UVB damage repair abilities of amphibian eggs. Proper estimation of amphibian ultraviolet damage repair characteristics, using any assay, is reliant upon the generation of cell-free protein extracts created from amphibian embryos covered in protective jelly. The process of releasing the embryo from the glycoprotein-carbohydrate jelly ("dejellying") is achieved by shaking the jelly mass in a solution of 2% L-cysteine. Equivalent exposure to 2% L-cysteine results in a radically different end product with different amphibian taxa. These previously unreported phenomena have important implications for the production, standardization, and reporting of amphibian photorepair data.