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.
Two cloned derivatives of the Kc cell line of Drosophila were shown to produce DOPA decarboxylase following administration of the steroid moulting hormone 20-hydroxyecdysone. In the continuous presence of the hormone at a concentration of 2 X 10(-7) M, DOPA decarboxylase activity first appeared between 48 and 72 h. Because of this lag, the tissue culture system promises to serve as a useful model for those in vivo situations where increases in the hormone titre precede increases in DOPA decarboxylase activity. In clone 7C4, after maximal enzyme activity was achieved at 144 h, the enzyme activity per cell decreased as the cells resumed division following the hormone-induced division arrest. In clone 7E10, cell division never resumed in the presence of 20-hydroxyecdysone and DOPA decarboxylase activity per cell increased continuously from the time it first appeared. When line 7E10 was exposed to a 6-h pulse of the steroid, enzyme activity appeared about 18 h earlier than in the presence of continuous hormone and, further, the cells were released from division arrest. Enzyme activity per cell then declined from an early 96-h maximum. The enzyme produced by the cell lines was immunologically distinct from the enzyme produced in vivo and ion-exchange column chromatography resolved the enzyme from cells and intact organisms into two species.(ABSTRACT TRUNCATED AT 250 WORDS)
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