Distribution of Tightly Bound Proteins in the Chicken Ovalbumin Gene Region

A method for investigating the distribution of the DNA-tightly bound proteins (TBP) in the chicken ovalbumin gene regions is described. The TBP are operationally defined as the proteins that remain bound to the DNA in the presence of 2 M NaCl. Nuclei from chicken erythrocytes, livers, and oviducts were lysed in 2 M NaCl and sheared, and the lysed chromatin was chromatographed through a Sepharose 4B column to separate protein and DNA which contains the TBP. The DNA fraction, after digestion with restriction endonuclease EcoRI, was passed through a GF/C glass fiber filter. The filter retains the DNA-TBP but not the protein-free DNA. In all cases, only about 0.5% of the total genomic DNA was retained on the filter. The ovalbumin gene sequences in the DNA-TBP were analyzed by the Southern blot. It was found that the copy number of the ovalbumin gene in the DNA-TBP isolated from erythrocyte or liver nuclei is not significantly different from that in the total unfractionated nuclear DNA. Liquid hybridization of nick-translation-labeled DNA-TBP with a large excess of total chicken nuclear DNA also demonstrated that the DNA is not a specific subset of the genome. In the nuclei from laying hen oviducts in which the ovalbumin gene but not the globin is actively expressed, approximately 3-fold enrichment of the ovalbumin gene was found in the DNA-TBP. The enrichment could have been due to a contamination of transcriptional complexes during the purification of the DNA-TBP, since no depletion in the globin gene sequences was found in the same sample. These results suggest a random distribution of the TBP in that genome, due possibly to a transient interaction between DNA and TBP. Precautions for evaluation of the results dealing with the TBP published in the literature are also discussed.