Open AccessAUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS
Author(s) -
Nelson Janet S. R.,
Schiffer Lewis M.
Publication year - 1973
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.1973.tb01593.x
Subject(s) - dna polymerase , microbiology and biotechnology , polymerase , thymidine , dna , biology , primer (cosmetics) , dna polymerase i , biochemistry , chemistry , polymerase chain reaction , reverse transcriptase , organic chemistry , gene
A technique has been developed allowing the autoradiographic detection of incorporation of 3 H‐thymidine‐5′‐triphosphate ( 3 H‐TTP) into nuclear DNA of smears of Sarcoma‐180 (S‐180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg ++ , and Ficoll, with the cell's own DNA acting as primer. The incorporation of 3 H‐TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg ++ and produces a product which is DNase sensitive and RNase resistant. DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing 3 H‐TTP labeling index in autoradiographs of aging tumors. The 3 H‐TTP labeling index has also been shown to exceed but parallel the in vivo 3 H‐thymidine ( 3 H‐TdR) pulse labeling index for all tumor ages examined. In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.