Abstract
A method involving short pulses of 5‐bromodeoxyuridine (brUdRib) followed by irraidation with 313 nm light was used to locate the time of replication of certain genes during the cell cycle of two cell lines, AF8 and AL106. AF8, a temperature‐sensitive mutant of BHK21/13 cells, grows at 33°C but not at 39.5°C. AL106, a hybrid clone of tsAF8 and SV‐40 transformed Lesch‐Nyhan fibroblasts (LNSV), which retains all hamster chromosomes and one human chromosome (No. 3), has the ability to grow at 39.5°C. AF8 and AL106 cells synchronized at the G~1~‐S boundary were released from their block and pulsed with brUdRib for 2‐hour periods during the S phase. The cells were subsequently irradiated with 313 nm light. Colony‐forming efficiency and revertants frequency were studied. Incorporation of brUdRib during the early S phase (0–4 hours from the begining of S), decreased the colony‐forming efficiency of AL106 cells both at 33°C and 39.5°C, and also of AF8 cells at 33°C. No AF8 colonies grew at the nonpermissive temperature regardless of the treatment. Thus the time of replication of genes responsible for colony‐forming ability was the same in tsAF8 at the permissive temperature and in AL106 at both temperatures. The time of replication of the genes responsible for the ts function in AF8 cells was located by determining the revertants frequency in synchronized AF8 cells pulsed with brUdRib and irradiated during 1‐ to 2‐hour periods of the S phase. Back‐mutants were scored by counting the number of clones capable of growing at 39.5°C (nonpermissive for AF8 cells). The highest frequency of induced back‐mutations occurred in synchronized AF8 cells pulsed with brUdRib (and irradiated) between two to four hours from the begining of the S phase. Exposure to brUdRib during other periods of the S phase or during G~1~ had no effect on the reversion rate.
This method can be used to locate the time of replication (in S) of ts genes in other temperature‐sensitive mutants or of other specific genes in other conditional mutants.