IMAGE-ANALYSIS OF MURINE CHROMATIN FIBER STRUCTURE AFTER IN-SITU DIGESTION WITH RESTRICTION ENDONUCLEASES

OBJECTIVE: To determine and quantify the differences produced in chrom atin structure after selective in situ DNA digestion with restriction endonucleases (RE). STUDY DESIGN: Chromatin fiber structure from a mur ine cell line teas analyzed under light and electron microscopy before and after in situ digestion with AluI, HinfI and HaeIII using digital image analysis (DIA). The proposed DIA-biased method entails the gene ration of a binary image and a skeleton characteristic of the chromati n fiber. Morphologic features (surface, perimeter, shape, number of tr iple points and Euler number) of the chromatin structure can then be q uantified from the digitized images. The results of these experiments weve compared with those obtained from direct digestion of naked DNA u sing the same endonucleases in an attempt to cor relate the distributi on of restriction sites, according to the DNA fragment size obtained, with the extent of chromatin disorganization after RE in situ digestio n. RESULTS: Homologous chromatin fiber regions are differentially affe cted by the action of each XE, although they are indistinguishable und er light microscopy. CONCLUSION: The proposed analytical routine const itutes a valuable tool for uncovering subtle alterations in the struct ure of chromatin fiber that has been modified tinder different experim ental conditions.