Translation of mRNA for 3,4-dihydroxyphenylalanine decarboxylase isolated from epidermis tissue of Calliphora vicina R. -D. in a heterologous system. Dependence of mRNA concentration on the insect steroid hormone ecdysone

Abstract

RNA was isolated from the epidermis of Calliphora vicina larvae by phenol--chloroform extraction. The RN A sedimenting in sucrose gradients between 5 and 18 S was submitted to chromatography on oligo(dT)-cellulose columns. The fraction binding to the oligo(dT) is able to stimulate protein synthesis in a system consisting of mouse liverribosomal subunits, pH-5 factors from rat liver and initiation factors from rabbit reticulocytes. Optimal Mg2+ concentration for the translation of insect mRNA is 3.5 mM, that of K+ 76 MM. Initiation factors prepared from epidermis of Calliphora larvae are less efficient in the translation of insect mRNA than initiation factors isolated from reticulocytes. The pH-5 fraction from epidermis inhibits protein synthesis independent of the source of the mRNA fraction used. One of the proteins synthesized in the reconstituted system under the direction of insect mRNA has been identified as 3,4-dihydroxyphenylalanine (DOPA) decarboxylase by immunoprecipitation with specific antiserum against DOPA decarboxylase and comigration in dodecylsulphate-acrylamide electrophoresis with pure DOPA decarboxylase. Both mRNA from white prepupae and from 6--7-days-old larvae contain sequences coding for DOPA decarboxylase. However, white prepupae contains 3--4 times more DOPA decarboxylase-mRNA than 6--7-days-old larvae. The content of DOPA decarboxylase mRNA is proportional to the amount of active DOPA decarboxylase molecules present in the animals from which the mRNA was isolated.