Purification and initial characterization of primate satellite chromatin.
Nucleoprotein hybridization, a method for the purification of specific DNA sequences as chromatin, was employed to fractionate primate centromeric alpha satellite chromatin as a first step in the identification and analysis of novel centromere-enriched proteins. In order to optimize the amount of material available for further study, cultured African green monkey cells were employed because satellite DNA represents approximately 25% of the genome. Two chromatin preparations were compared for the yield and total protein content of purified material. Regardless of the preparation, alpha satellite sequences were enriched to near purity. Since intact satellite chromatin is relatively refractile to the enzymatic digestion steps in the method, the total amount of solubilized material available for purification is rather low. In contrast, nuclei treated with acidic washes to extract histone H1 provided solubilized material enriched in satellite sequences. In addition, this material is more efficiently utilized in an affinity chromatography step. However, the extraction of many non-histones at low pH resulted in very low yields of protein in the purified fraction. Two-dimensional gel comparisons of proteins associated with H1-containing satellite chromatin after iodination of total chromatin proteins revealed a number of polypeptides enriched to varying degrees in the purified fraction. The electrophoretic mobilities of a few enriched polypeptides corresponded to previously identified heterochromatin-associated proteins while many others appear to be novel. The work presented validates nucleoprotein hybridization as a purification method for highly repeated sequences as chromatin in analytical amounts. The fact that a number of the enriched proteins are visible in stained gels of bulk chromatin proteins suggests that further biochemical analysis can be carried out on these polypeptides directly.
Jasinskas
A
A
Department of Molecular Biology and Biochemistry, BioSci II, University of California, Irvine, 92697-3900, USA.
Hamkalo
B A
BA
D000818:Animals; D002503:Centromere / Q000737:chemistry*; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000737:chemistry* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004276:DNA, Satellite / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006570:Heterochromatin; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D008836:Micrococcal Nuclease; D009707:Nucleosomes; D014709:Vero Cells
D002843:Chromatin; D004276:DNA, Satellite; D006570:Heterochromatin; D006657:Histones; D009707:Nucleosomes; D008836:Micrococcal Nuclease