Q.17 Two mammalian cell lines with doubling times of 12 h and 36 h were cultured with radioactive thymidine for 8 h. The cells were further cultured without the radioactive thymidine for 72 h. Incorporated radioactivity was measured in equal number of cells in each culture, which revealed that

Thymidine labeling experiments measure DNA synthesis during the S-phase of the cell cycle. In this pulse-chase setup, faster-growing cells incorporate more label relative to slower-growing ones after dilution during the chase period.​

Experiment Overview

Two mammalian cell lines have doubling times of 12 hours (fast) and 36 hours (slow). Cells receive radioactive thymidine for 8 hours (pulse, matching typical S-phase duration), then chase without label for 72 hours. Radioactivity is measured in equal numbers of cells.​

Assume S-phase (Ts) ≈ 8 hours for both, common in mammalian cells. Growth fraction is 1 (all cells cycling).​

Fraction of cells in S-phase (fs) = Ts / Td (doubling time).

• Fast: fs_fast = 8/12 ≈ 0.667

• Slow: fs_slow = 8/36 ≈ 0.222​

Label Incorporation (Pulse)

During the 8-hour pulse, thymidine labels cells in S-phase. Label amount per cell ∝ fs (since equal cells measured).

Fast cells incorporate ~3x more label (0.667 vs. 0.222).​

Label Dilution (Chase)

During 72-hour chase, label dilutes by cell divisions (n = 72 / Td). Each division halves label per cell (DNA semi-conservative replication).​

• Fast: n_fast = 72/12 = 6 divisions; dilution = 2^6 = 64-fold

• Slow: n_slow = 72/36 = 2 divisions; dilution = 2^2 = 4-fold​

Final relative radioactivity per cell:

• Fast: (0.667) / 64 ≈ 0.0104

• Slow: (0.222) / 4 ≈ 0.0555

Slow cells retain ~5.3x more label.​

Option Analysis

(A) Both had same radioactivity: Incorrect. Different fs and dilution rates lead to unequal final label.​

(B) Fast-growing had more: Incorrect. Higher initial incorporation in fast cells outweighed by greater dilution.​

(C) Slow-growing had more: Correct. Less dilution preserves more label despite lower initial uptake.​

(D) Neither had radioactivity: Incorrect. Label persists through semi-conservative replication, though diluted.​

Radioactive thymidine cell labeling experiments are key tools in cell cycle analysis for CSIR NET life sciences preparation. These pulse-chase studies track DNA synthesis by measuring incorporated radioactivity in mammalian cell lines with varying doubling times.

Pulse-Chase Protocol Explained

Cells pulse-labeled with radioactive thymidine for 8 hours incorporate it during S-phase. A 72-hour chase without label allows dilution via divisions. Equal cell counts ensure per-cell comparison.​

Impact of Doubling Time

Fast cells (12h Td) have higher S-phase fraction (~67%), incorporating more label initially. Slow cells (36h Td) incorporate less (~22%). However, chase dilution favors slow growers: 6 vs 2 divisions.​

Slow cells show more radioactivity, answering why growth rate affects labeling outcomes.​