Homogeneous rate of degradation of nuclear DNA during apoptosis.

DNA fragmentation during apoptosis is characterized by endonucleolytic cleavage of chromosomal DNA into an oligonucleosomal ladder. To determine if actively transcribed genes are more susceptible to cleavage during apoptosis than non-transcribed genes, the rate of fragmentation of differentially expressed genes was measured in B-lymphocyte hybridoma cells. Five genes were studied based on their transcriptional activity and/or nuclear localization, and mitochondrial DNA was assayed as a negative control for apoptotic fragmentation. Apoptosis was induced in the hybridoma cells by ultraviolet light, and DNA was prepared at multiple time points after ultraviolet irradiation. Degradation into an oligonucleosomal ladder appeared as early as 2 h after treatment, showing that fragmentation is rapidly activated in hybridoma cells. The DNA was then digested with restriction enzymes, separated by gel electrophoresis and hybridized with the gene-specific probes for Southern blot analyses. Loss of gene-specific signals was measured by quantitation of autoradiographs. The results show all of the nuclear genes were degraded at the same rate regardless of their transcriptional status or nuclear localization. The data suggest that once the cell activates its destruction program, nuclear DNA is rapidly degraded in a homogeneous manner.