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 deter mine if actively transcribed genes are more susceptible to cleavage du ring apoptosis than non-transcribed genes, the rate of fragmentation o f differentially expressed genes was measured in B-lymphocyte hybridom a cells. Five genes were studied based on their transcriptional activi ty and/or nuclear localization, and mitochondrial DNA was assayed as a negative control for apoptotic fragmentation. Apoptosis was induced i n the hybridoma cells by ultraviolet light, and DNA was prepared at mu ltiple time points after ultraviolet irradiation, Degradation into an oligonucleosomal ladder appeared as early as 2 h after treatment, show ing that fragmentation is rapidly activated in hybridoma cells, The DN A was then digested with restriction enzymes, separated by gel electro phoresis and hybridized with the gene-specific probes for Southern blo t analyses. Loss of gene-specific signals was measured by quantitation of autoradiographs, The results show all of the nuclear genes were de graded at the same rate regardless of their transcriptional status or nuclear localization. The data suggest that once the cell activates it s destruction program, nuclear DNA is rapidly degraded in a homogeneou s manner.