THE SYNTHESIS, ISOLATION, AMPLIFICATION AND TRANSCRIPTION OF THE OVALBUMIN GENE

ABSTRACT RNA was transcribed from chick oviduct chromatin and the concentration of ovalbumin mRNA sequences in the RNA transcripts was measured by hybridization with a radioactive complementary DNA probe synthesized against purified ovalbumin mRNA using viral reverse transcriptase. Ovalbumin mRNA sequences were more abundant in RNAs transcribed from chromatin of estrogen-stimulated chick oviduct than that of chicks withdrawn from hormone. Reconstitution experiments indicated that the nonhistone protein fraction of the estrogen-stimulated chromatin was responsible for maintaining expression of the ovalbumin gene in vitro . In order to study the specific interactions between these regulatory proteins and the ovalbumin gene, we attempted to synthesize, isolate and amplify this gene. Using reverse transcriptase and the complete ovalbumin complementary DNA (cDNA ov ) as a template, a double-stranded DNA (the synthetic ovalbumin gene) was synthesized in vitro . This double-stranded synthetic ovalbumin gene has been successfully incorporated into bacterial plasmids and amplified. By an alternative method designed to obtain DNA sequences adjacent to the coding portion of the ovalbumin gene, we have also purified the natural ovalbumin gene from total chick DNA by affinity chromatography. Using separate columns in which purified ovalbumin mRNA (mRNA ov ) and CDNAOV were covalently linked to phospho-cellulose, the respective complementary ovalbumin DNA strands were enriched about 10, 000-fold from total chick DNA sheared mechanically to a mean length of four to five thousand base pairs. RNA transcribed from these partially purified DNA preparations contained ovalbumin sequences in a much greater concentration than RNA synthesized from total chick DNA.