Comparison of chromosomal structures isolated under different conditions

Abstract

The biochemical nature of chicken chromosomes isolated using acidic solution (i.e. 50% acetic acid) and using a near neutral solution containing spermine, spermidine, EDTA and EGTA [9], were compared. In both cases, no swelling of the chromosomes was observed. Chromosomes isolated under near neutral conditions contain nucleosome structure, whereas in those isolated under acidic conditions the nucleosome structure is destroyed. This was determined by micrococcal nuclease digestion of the isolated chromosomes followed by gel electrophoresis of the chromosomal DNA. In addition, acidic treatment of the chromosomes resulted in irreversible changes in protein-DNA interactions in chromosomes, as judged by the sedimentation behaviors of the isolated chromosomes in a sucrose gradient containing 2 M NaCl, and by the accessibility of the DNA to restriction endonuclease digestion. The chicken ovalbumin gene in the chromosomes isolated using acidic solutions was highly accessible to digestion by restriction endonucleases, whereas most of the restriction enzyme cleavage sites were protected in the chromosomes isolated under near neutral conditions. These results suggest that acidic treatments of chromosomes, the method used most often for cytogenetic preparations, causes extensive damage to the biochemical nature of chromosome structure; and the solution utilized by Blumenthal et al. [9] is a better solution for chromosome isolation.