Cell-free protein synthesis in lysates of Drosophila melanogaster cells.
A procedure is described for preparing cell-free protein synthesizing lysates from Drosophila melanogaster tissue culture cells and embryos. Preparation of translationally active lysates from tissue culture cells is dependent on the presence of rat liver supernatant during cell lysis to inhibit ribonuclease activity. After micrococcal nuclease treatment of the lysate, protein synthesis is dependent on the addition of exogenous messenger RNA. The fidelity of translation is very high. The conditions for optimal translation have been determined. In addition, the effects on translation of a variety of supplements, including rat liver supernatant, have been analyzed. The products of translation by the Drosophila lysate have been compared with those of wheat germ extracts and of micrococcal nuclease treated rabbit reticulocyte lysates. Translation in vitro of bovine parathyroid hormone messenger RNA yielded two products tentatively identified as preproparathyroid hormone and proparathyroid hormone, as well as an unidentified third product. This result suggests that insect enzymes can accurately process mammalian precursor proteins.
D000818:Animals; D002460:Cell Line; D004331:Drosophila melanogaster / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D010281:Parathyroid Hormone / Q000096:biosynthesis; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D010281:Parathyroid Hormone; D011498:Protein Precursors; D012333:RNA, Messenger