G-BAND EXPRESSION AND MEGABASE FRAGMENTATIONS IN APOPTOSIS

Apoptosis seems characterized by a cascade of megabase to 200-bp fragm entations and by a commitment to perish at the initial level, How that could be achieved seems unclear, Preferential cleavage of transcripti onally active chromatin by apoptotic nuclease activity has long been s uggested. We show here the manifestation of self-inflicted G-banding p atterns in mitotic chromosomes, or G-band expression, occurring concur rently with a pattern of megabase fragmentations in two apoptotic syst ems that we have established in human Chang liver cells using (a) stau rosporine and (b) vanadyl(4) prepulsing, We further show that rare-cut ting NotI and MluI restriction endonucleases with C-G dinucleotide seq uence specificity had produced similar G-bandings and megabase fragmen tations cascading down to the 200-bp ladder fragmentation that were al so associated with the expression of characteristic apoptotic morpholo gies by the digested cells. CpG-specific methylation using the methyla se SssI abolished the DNA fragmentation cascade, G-banding, and apopto tic expressions induced by NotI and MluI, implicating endonuclease cle avage of active chromatin, where CpG islands are concentrated, as the initiating event, Reproducing the G-bandings and megabase fragmentatio ns by directly applying NotI and MluI endonucleases to fixed chromosom es and extracted genomic DNA, respectively, further confirmed the noti on of endonucleolytic cleavage of active chromatin as the causation. N uclease digested light G-band regions of chromosomes appeared to be th e chromosome sites providing the megabase fragments. Transcriptionally active genes of the genome are known to be preferentially cleaved by nuclease activity and are established as being concentrated in the lig ht G-bandings that correspond to R-bandings, which are also known to b e the sites of more frequent cytogenetic breakpoints. Manifestation of self-inflicted G-banding patterns (G-banding expression) in apoptosis would then imply cleavage of the transcriptionally active genes in ev ery light G-band site of every chromosome in the genome, This must be suicidal, (C) 1998 Academic Press.