The Journal of Cell Biology, Vol 93, 278-284, Copyright © 1982 by The Rockefeller University Press
Analysis of DNA attached to the chromosome scaffold
MT Kuo
Two different methods have been described to investigate whether any
specific DNA sequences are intimately associated with the metaphase
chromosome scaffold. The chromosome scaffold, prepared by dehistonization
of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many
looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor
Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from
dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI
digestion followed by removal of the looped DNA by sucrose gradient
sedimentation. Alternatively, the scaffold DNA was prepared from
micrococcal nuclease- digested intact chromosomes using sucrose gradients
containing 2M NaCl. Solution hybridization of the radioactively labeled
scaffold DNA with a large excess of total nuclear DNA revealed that, in
either case, the scaffold DNA is not a unique sequence class of genomic
DNA. Southern- blotting hybridization also showed that the scaffold DNA
prepared from EcoRI-digested dehistonized chromosomes was not enriched (or
depleted) in the ovalbumin gene sequences. The possibility of a dynamic
interaction of protein and DNA in the chromosome scaffold and the
possibility that the scaffold is a preparative artifact are discussed.
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