TY - JOUR
T1 - Comparison of chromosomal structures isolated under different conditions
AU - Kuo, M. Tien
N1 - Funding Information:
The authorw ould like to thank ProfessorP . Chambon, Strasbourg,F rance, for allowing us to use the ovalbumin acne clones. MS Mildred Lai and MS Bhanumati Iyer ior their excellent technicala ssistance.T his study was supported in part by grants G-831 from the Robert A. Welch FoundationsC D-2S from the ACS, Inc.. and (X-28573-01 from the NIH.
PY - 1982/3
Y1 - 1982/3
N2 - 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.
AB - 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.
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U2 - 10.1016/0014-4827(82)90109-4
DO - 10.1016/0014-4827(82)90109-4
M3 - Article
C2 - 7067738
AN - SCOPUS:0020326603
SN - 0014-4827
VL - 138
SP - 221
EP - 229
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 1
ER -