Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis.
Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well-characterized DNA-protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same length. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an "equivalent sphere" (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3-9%T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.
Orbán
L
L
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-0001.
Garner
M M
MM
Wheeler
D
D
Tietz
D
D
Chrambach
A
A
D000818:Animals; D002645:Chickens; D004247:DNA / Q000097:blood / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000097:blood / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004912:Erythrocytes / Q000648:ultrastructure; D008433:Mathematics; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry* / Q000648:ultrastructure; D010316:Particle Size
D004268:DNA-Binding Proteins; D009707:Nucleosomes; D004247:DNA