Cell-Free Synthesis of Vitellin in the Shrimp Penaeus semisulcatus (de Haan)

Abstract

The vitellogenic ovary of Penaeus semisulcatus contains mRNA specific for vitellin (Vt), low levels of which are found in nonvitellogenic ovaries, and is absent from testes. lmmunoisolation from cell-free translation of poly(A) + RNA using antiserum against purified Vt produced a 35-kDa polypeptide. No differences were found between Vt precipitated from the translation products of the rabbit reticulocyte system and that from the wheat germ extract. The specificity of the immunoprecipitation reaction was demonstrated by the absence of precipitation with nonimmunized rabbit serum and with antibodies prepared against purified hemocyanin or lipoprotein I (LPI), which are crustacean hemolymph proteins. In addition, competition with the radioactively labeled translation product occurred only in the presence of purified Vt, but not BSA or LPI. Vt synthesized in the translation system had a significantly lower molecular weight than that of the purified Vt or that synthesized by ovaries incubated in vitro. The possibility that this difference may be because of the nonglycosylated nature of a cell-free translation product was tested. The removal of oligosaccharides from purified Vt by enzymatic digestion with N-glycosidase F resulted in the appearance of a smaller polypeptide of 36 kDa, which reacted immunologically with Vt antiserum in a Western blot. The size of this fragment is very close to the molecular weight of the translation product.