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Isolation of Chinese hamster ovary cells with reduced poly(ADP-ribose) polymerase activity
R A MacLaren1, M V Witmer, E Richardson
1Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104.
Abstract:
The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.
Insights
Researchers developed a method to isolate poly(ADP-ribose) polymerase-deficient Chinese hamster ovary cells. These mutants show increased sensitivity to DNA ethylating agents, aiding the study of this enzyme
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- The precise biological functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, cell-cycle regulation, and cellular differentiation remain incompletely understood.
- Identifying cells with deficiencies in PARP activity is crucial for elucidating its specific roles within cellular processes.
Purpose of the Study:
- To develop and implement a method for isolating Chinese hamster ovary (CHO) cells exhibiting reduced poly(ADP-ribose) synthesis.
- To characterize the functional consequences of diminished PARP activity in these isolated cell mutants.
Main Methods:
- A novel screening method was employed to directly assess poly(ADP-ribose) polymerase activity in individual colonies of CHO cells.
- Colonies with significantly decreased poly(ADP-ribose) production were identified using nylon replica screening and subsequently isolated for further analysis.
Main Results:
- A series of CHO cell mutants with 50% or less of the normal poly(ADP-ribose) polymerase activity were successfully isolated.
- These PARP-deficient mutants exhibited normal cell generation times.
- The isolated mutants demonstrated a 20% increased sensitivity to DNA ethylating agents compared to the parental CHO cell line.
- Conversely, the mutants displayed normal sensitivity to gamma-ray irradiation.
Conclusions:
- The developed screening method is effective for isolating poly(ADP-ribose) polymerase-deficient CHO cell mutants.
- The findings suggest a role for poly(ADP-ribose) polymerase in cellular defense against DNA ethylating agents.
- The study provides valuable cellular models for further investigation into the specific biological functions of PARP.