Endogenous DNases as a Tool for Isolation of Nuclear Matrix: Critical Parameters of Nucleolysis

The degree of nucleolysis is of critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences, as demonstrated using an example of inactive (c-fos, c-myc, andC) and active (p53, albumin, and28S rRNA) genes in resting hepatocytes. The optimal degree of nucleolysis is characterized by degradation of loop domains of chromatin, with the relatively uniform molecular weight distribution of DNA being preserved. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched with or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix