Estrogen administration to immature chicks causes selective increase in transcription of mRNAs of egg white proteins in the oviduct. In addition, estrogen confers stability to the induced mRNAs. Rapid withdrawal of estrogen from the hormone stimulated chicks results in accelerated degradation of induced mRNAs as well as rRNAs, resulting in the cessation of egg white protein synthesis. The mechanisms which regulate mRNA stability are unknown. The goal of the proposed project is to investigate the mechanism by which mRNAs for egg white proteins and rRNA are degraded during estrogen withdrawal, specifically the role of 2'-5' linked oligo (adenylic acid) (2-5A) activated endonuclease. This is because the enzyme which synthesizes 2-5A (2-5A synthetase) is known to be regulated by a variety of inducers beside interferon, including estrogens. Additional control of 2-5A synthetase-endonuclease system can be achieved through degradation of 2-5A by phosphodiesterase. We would also investigate whether rapid degradation of induced mRNAs and rRNA in oviduct during acute hormone withdrawal is unique to estrogen. Does withdrawal from nonestrogenic hormones, progesterone or glucocorticoids (administration of these hormones induces ovalbumin synthesis when given as secondary stimulant) render RNAs unstable? 2-5A dependent endonuclease will be assayed by degradation of rRNA into characteristic and discrete products. Competition radiobinding assay complemented with ability of 2-5A to activate an endoribonuclease and inhibit protein synthesis in cell-free extracts will be used to detect 2-5A synthesis. 2-5A will be characterized by HPLC. mRNAs for ovalbumin, cellular mRNAs (Beta-actin, Alpha-tubulin) and rRNA will be analyzed by dot blot and Northern blot hybridization techniques using specific cloned DNA probes. 2-5A synthetase and the enzyme which degrades 2-5A will be quantitated.
