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Modulation of Ovalbumin Synthesis by Estradiol-17ß and Actinomycin D as Studied in Explants of Chick Oviduct in Culture

From the ¹ From the Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305

A secondary administration of estradiol-17ß to chicks augments the incorporation of ³H-uridine and ³H-lysine into oviduct magnum RNA and protein, respectively. The incorporation of isotopes into ovalbumin, the major protein synthesized by this tissue, is enhanced to a greater extent than into total protein. There is a concomitant organization of monosomes into polysomes during the first 9 hours of secondary stimulation. Actinomycin D, at a dose of 5 mg per kg, blocks the estrogen-mediated increase in isotope incorporation into RNA, total protein, and ovalbumin. At a slightly lower dose of actinomycin D, 2 mg per kg, the induction of protein synthesis, including ovalbumin, is almost normal, although the incorporation of ³H-uridine into bulk RNA is inhibited to nearly the same extent as at the higher dose. Isotope incorporation into ribosomal RNA appears to be selectively inhibited. This result agrees with earlier findings which suggested that rRNA synthesis is not required for the induction of ovalbumin synthesis during early secondary stimulation.

Although we have tried several hormonal combinations and chick sera, we have been unable to induce ovalbumin synthesis in explants of the oviduct magnum in tissue culture. Once ovalbumin synthesis has been induced in vivo, however, ovalbumin continues to be synthesized in tissue culture. The electrophoretic pattern on sodium dodecyl sulfate acrylamide gels of soluble proteins synthesized in vivo and in culture are the same. Thus, the tissue culture technique can be used confidently to measure the synthesis of specific proteins. Protein synthesis was investigated in both short (20 min) and long term (30 hour) cultures. Kinetic studies of protein synthesis during short incubations suggest that ovalbumin is synthesized and released from polysomes at the same rate as the average oviduct protein. Insulin is necessary in long term cultures to maintain the incorporation of isotopes into protein; it has no differential effect on ovalbumin synthesis. During a 30-hour culture period, the rate of ovalbumin synthesis declines relative to total soluble protein synthesis, with a half-life of about 14 hours. The addition of actinomycin D inhibits ³H-uridine incorporation into RNA by 95%, but has little effect on ovalbumin synthesis; however, the synthesis of nonsecretory proteins is inhibited (half-life = 7 hours). The secretory proteins, ovalbumin and conalbumin, become a larger proportion of the proteins synthesized after actinomycin D treatment. We conclude that ovalbumin messenger RNA (or some factor which regulates ovalbumin synthesis) is not synthesized in explants in tissue culture, but it has a longer intrinsic half-life than bulk mRNA (or factors).

The details of the immunoprecipitation methods employed to detect and measure ovalbumin synthesis are presented. The sodium dodecyl sulfate acrylamide gel electrophoretic technique used to verify the specific precipitation of ovalbumin has general applicability for proving the specificity of immunoprecipitation of antigens from radioactive homogenates.

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				O. Sharma Induction of ovalbumin synthesis in immature chicks by actinomycin D and thioacetamide Science, October 6, 1978; 202(4363): 68 - 69. [Abstract] [PDF]