Isolation of a protein fraction that binds preferentially to chicken middle repetitive DNA

We have fractionated oviduct tissue extracts by using a combination of ion-exchange and DNA-Sephadex chromatography. By comparing the electrophoretic patterns of proteins eluted from competing specific and nonspecific DNA columns, we isolated a fraction which bound with specificity to columns containing the chicken middle repetitive sequence "CR1". This fraction showed a clear preference for binding to separate, cloned CR1 fragments derived from either the 5' or the 3' transition region of the ovalbumin gene domain when examined by using nitrocellulose filter binding assays. To localize the protein binding site, a CR1 clone was digested with various restriction enzymes, and the resulting fragments were examined for preferential protein binding. Results suggest that the binding site lies within a 39-nucleotide sequence which is highly conserved among different CR1 elements. This finding represents the first isolation of a protein which demonstrates a preference for binding to a middle repetitive sequence and suggests that this interaction may have a biological role. The DNA column competition adsorption method should have general application to the isolation of other gene-regulating proteins possessing DNA sequence preference.     

The presence of ovalbumin mRNA coding sequences in multiple restriction fragments of chicken DNA.

Chicken DNA has been digested with restriction enzymes and the size distribution of the DNA fragments containing ovalbumin specific sequences has been examined after separation of the fragments on agarose gels and transfer to nitrocellulose sheets. Hybridisation with terminally 32P-labelled ovalbumin mRNA fragments or with RNA populations transcribed from the DNA of a hybrid plasmid containing ovalbumin sequences was used to locate the DNA fragments coding for ovalbumin. Digestion with enzymes which do not cut within the portion of the ovalbumin gene synthesised from ovalbumin messenger RNA in vitro has shown the presence of several defined fragments carrying ovalbumin specific sequences. Possible explanations of these observations are discussed.