Abstract
We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they may not possess the extent of secondary modification evident with the nuclear histones. This purified rDNA chromatin also possesses RNA polymerase I activity, and many other nonhistone proteins, including two very abundant proteins (26 and 38 kDa) that may be either ribonucleoproteins or nucleolar matrix proteins. Micrococcal nuclease digestion of the metrizamide-purified rDNA chromatin produces particles containing 145-base pair DNA fragments identical in length to those in total chromatin and which contain both transcribed and nontranscribed rDNA sequences. Some smaller fragments (30, 70, and 110 base pairs) are also seen, but their sequence content is not known. These particles sediment uniformly at 11 S in sucrose gradients containing 15 mM NaCl, and at 4-11 S in gradients containing 0.35 M NaCl. Particles enriched in gene or nontranscribed spacer sequences are not resolved in these sucrose gradients or in metrizamide gradients. Our findings suggest that the rDNA chromatin fraction we have identified contains transcriptionally active genes and that an organized, particle-containing structure exists in active rDNA chromatin.
Highlights
Follow this and additional works at: https://digitalcommons.odu.edu/medicaldiagnostics_fac_pubs Part of the Genetics Commons, Molecular Biology Commons, Molecular Genetics Commons, and the Structural Biology Commons
Characterization of Proteins Associated with Purified rDNA Chromatin-We first characterized the proteins associated positions of two abundant nonhistone proteinassociated with rDNA chromatin; the asterisks mark the positioonfsthe four core histones
Both nonhistones arefound in therDNA chromatin region and RNP region of the gel; the top nonhistone protein trails throughout the with rDNA chromatin using a two-dimensional gel electro- gel
Summary
Dept. of Microbiology, University of Virginia inactive chromatin, the model does not propose that rDNA chromatin is depletedof histones, as suggestedby some of the studies mentioned above. Of Microbiology, University of Virginia inactive chromatin, the model does not propose that rDNA chromatin is depletedof histones, as suggestedby some of the studies mentioned above. Other studies argue that ribosomal RNA genes possess typical nucleosome-likefeatures and a nearly full complement of histones. I have alsobeen detected in the200-base pair DNA fragments in micrococcal nuclease-derived chromatin subunits from X. laeuis (Reeves and Jones, 1976; Reeves, 1976, 1977). Thesperocedures have led to assaysof RNA polymerase activity andnuclease digestion studies using the purified chromatin fraction.since thePhysarum rDNA sequence is palindromic, contain-. Possible to compare directly the structures of active “gene” ciated with agarose gel-purified rDNA chromatin. Matin was purified from nuclear chromatin and RNPsby electrophoresis in a 0.4% Sea/Plaque agarose gel as described by Amero et al
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