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Isolation of UV-sensitive variants of human FL cells by a viral suicide method
Abstract:
A new method (viral suicide method) for the isolation of UV-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with UV-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their UV sensitivity. Nineteen of 238 clones examined were sensitive to UV irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their UV sensitivity for 4 months in culture. UV sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (D0) are 2.9, 3.7, and 7.8 J/m2 for UVS-1, UVS-2, and the parental FL cells, respectively- They are no more sensitive than FL cells to X-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability.
Insights
A novel viral suicide method effectively isolates UV-sensitive human cell mutants. Two stable mutants, UVS-1 and UVS-2, were identified, exhibiting increased UV sensitivity and altered host cell reactivation capabilities.
Area of Science:
- Cell Biology
- Radiation Biology
- Virology
Background:
- UV sensitivity is crucial for understanding DNA repair mechanisms.
- Human FL cells are a common model for studying cellular responses to radiation.
- Host cell reactivation (HCR) is a key DNA repair pathway.
Purpose of the Study:
- To develop a new method for isolating UV-sensitive human cell mutants.
- To characterize the UV sensitivity and DNA repair capacity of isolated mutants.
Main Methods:
- The viral suicide method involved infecting mutagenized human FL cells with UV-irradiated Herpes simplex virus.
- Surviving colonies were screened for UV sensitivity and defects in host cell reactivation (HCR).
- UV sensitivity was quantified by extrapolation number (n) and mean lethal dose (D0).
Main Results:
- Nineteen UV-sensitive clones were isolated from 238 initial colonies.
- Two stable UV-sensitive mutants, UVS-1 and UVS-2, were identified and maintained for 4 months.
- UVS-1 and UVS-2 showed increased UV sensitivity (lower D0 values) compared to parental FL cells.
- UVS-2 exhibited reduced HCR ability, while UVS-1 had similar HCR ability to FL cells.
- Mutants were not more sensitive to X-irradiation than parental cells.
Conclusions:
- The viral suicide method is effective for isolating UV-sensitive mutants.
- UVS-1 and UVS-2 represent valuable tools for studying UV-induced DNA damage and repair pathways.
- Differential HCR abilities in the mutants provide insights into specific repair mechanisms.