Steroid hormones, in conjunction with cytoplasmic receptor molecules, have pronounced effects on cell differentiation and gene action. The chicken oviduct is a particularly valuable model system for studying the control of gene activity because it is one of the few eukaryotic systems where one can show that a well characterized effector acts directly on the genome to modulate the synthesis (transcription) of a specific messenger RNA (ovalbumin mRNA) which can be studied both functionally and chemically. The kinetics of mRNA induction will be explored in depth; but the major goal will be to understand the mechanism by which steroids affect gene expression. The basic approach to this problem will be to fractionate the oviduct genome (chromatin) by affinity chromatography. Chromatin-bound steroid receptors will be linked to insolubilized steroid, followed by enzymatic shearing of the chromatin to variable sized pieces and isolation of the receptor binding sites and adjacent chromatin. This will allow the following key questions to be asked: (1) What is the distance between the steroid receptor binding sites and the structural gene for ovalbumin? (2) Are the steroid receptors clustered? If so, how many? (3) Is the DNA in the receptor binding site unique or reiterated? (4) Are the synergistic effects observed with hormonal combinations due to contiguous receptor binding sites? (5) Are there unique chromatin proteins in the receptor binding sites and do they change during development and/or gene activation? (6) Is the receptor region of the chromatin enriched in RNA polymerase, i.e. "open"? The eventual goal will be to add steroid receptors along with other factors to effect specific gene transcription in an isolated system. It is anticipated that partially purified chromatin containing the responsive genes will hasten this approach. The answers to these questions should provide a clearer picture of the organization of the eukaryotic genome as well as insight into the regulatory mechanisms involved in gene activation which is at the crux of the problem of cell differentiation in general as well as certain types of genetic disorders.
NIH ExPorter
NIH ExPorter