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Characterization of apoptosis-like endonuclease activity in avian thymocytes
1Department of Poultry Science, University of Georgia, Athens 30602-2772.
Abstract:
In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.
Insights
Avian thymocytes possess internucleosomal DNA cleavage activity during apoptosis. This endonuclease activity, similar to mammalian counterparts, degrades DNA and is inhibited by EDTA and zinc ions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis involves DNA fragmentation.
- Internucleosomal DNA cleavage is a hallmark of apoptosis.
- Investigating this process in avian systems is crucial.
Purpose of the Study:
- To investigate internucleosomal DNA cleavage activity in avian thymocytes during apoptosis.
- To characterize the nuclease activity responsible for DNA degradation.
Main Methods:
- Incubation of avian thymocyte nuclear proteins with chicken red blood cell (cRBC) nuclei.
- Analysis of DNA degradation using agarose gel electrophoresis and fluorescence-activated flow cytometry.
- Assessing nuclease activity on radiolabeled DNA.
Main Results:
- Avian thymocyte nuclear extract exhibited endonuclease activity degrading cRBC chromatin at internucleosomal sites.
- Flow cytometry confirmed DNA loss in cRBC nuclei treated with thymocyte proteins.
- The nuclease activity was inhibited by EDTA, zinc ions, and aurintricarboxylic acid.
Conclusions:
- Avian thymocytes possess an endonuclease activity during apoptosis that cleaves DNA at internucleosomal sites.
- This avian nuclease activity shares characteristics, including cofactor requirements and inhibitor susceptibility, with its mammalian counterpart.
- The findings suggest a conserved mechanism of DNA fragmentation during apoptosis across species.