APOPTOTIC DNA FRAGMENTATION IS DETECTED BY A SEMIQUANTITATIVE LIGATION-MEDIATED PCR OF BLUNT DNA ENDS

Apoptosis is a form of programmed cell death (PCD) characterized by mo rphological changes and stereotypical DNA degradation described as a n ucleosomal 'ladder'. However, nucleosomal ladders have only been clear ly demonstrated in vertebrate tissues when large numbers of cells die in synchrony. Their absence may be explained by asynchronous death und er physiological conditions, or by distinct molecular mechanisms. In t his study, nucleosomal ladders were revealed by a ligation-mediated po lymerase chain reaction (LMPCR), that amplifies DNA fragments with blu nt, 5' phosphorylated ends. Numerous tissues from different organisms were examined which demonstrated that nucleosomal ladders (a) accompan y physiological cell death in mammalian tissues where previously DNA f ragmentation has not been detected; (b) are produced during invertebra te cell death; (c) are invariably generated via the production of blun t, 5' phosphorylated double strand breaks. These results suggest that PCD in multicellular organisms consistently involves apoptotic mechani sms and that the endonuclease activity is evolutionarily conserved.