© 1983 Oxford University Press
research-article |
Isolation and preliminary characterization of u.v.-sensitive mutants from the human cell line EUE
Istituto di Mutagenesi e Differenziamento C.N.R., via Svezia 10, 56100 Pisa, Italy
1To whom all correspondence should be addressed
Five u.v. light-sensitive clones were isolated in the EUE cell line by means of a modified form of the original 5-bromode-oxyuridine (BUdR)-light method worked out by Puck and Kao for the isolation of nutritional mutants. A cell population was mutagenized with ethylmethanesulfonate. After the expression time, cells were u.v.-irradiated and incubated with BUdR to label excision patches in repair proficient cells. A subsequent irradiation with ‘black’ light caused DNA strand breakage in BUdR-substituted cells. During BUdR treatment, hydroxyurea and a fluorochrome (Hoechst 33258) were added to possibly enhance the analogue incorporation into DNA and to increase the photolability of BUdR containing sequences, respectively. Out of 192 colonies selected with this method, 38 were isolated and tested for their u.v.-sensitivity. Five of them showed significant, reproducible differences with respect to the parental line. As a partial characterization, the five u.v.-sensitive clones were assayed for unscheduled [3H]thymidine incorporation after exposure to u.v. light, by means of liquid scintillation spectrometry and autoradio-graphy. In all clones, DNA repair synthesis was significantly decreased with respect to the parental line.