Purification and Characterization of Proinsulin mRNA from Rat B‐Cell Tumor

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Abstract
Proinsulin mRNA was purified from rat B‐cell tumor induced by streptozotocin and nicotinamide. The purification was achieved through the use of oligo(dT)‐cellulose chromatography and sucrose gradient centrifugation. As judged by translational activity in a cell‐free protein‐synthesizing system of wheat germ, proinsulin mRNA was purified 362‐fold from total nucleic acid of the B‐cell tumor. Purified proinsulin mRNA migrated as a single symmetrical peak on sucrose gradient centrifugation and as a single band on polyacrylamide gel electrophoresis in the presence of formamide. The translation product of purified proinsulin mRNA in the cell‐free protein‐synthesizing system was found to be only preproinsulin, as analyzed by dodecylsulfate/polyacrylamide gel electrophoresis and immunoprecipitation.The molecular weight of proinsulin mRNA, determined by polyacrylamide gel electrophoresis in the presence of formamide, was approximately 200000 (equivalent to approximately 570 nucleotides). Almost all of the proinsulin mRNA was bound to oligo(dT)‐cellulose, and the translation of proinsulin mRNA in a cell‐free protein‐synthesizing system of wheat germ was completely inhibited by 7‐methylguanosine 5′‐phosphate. These results indicated that the nucleotide sequences in proinsulin mRNA consisted of 58% translatable sequences and 42 % untranslatable sequences, presumably containing a capped structure, a 7‐methylguanosine residue, at the 5′ terminus and a poly(A) sequence at the 3′ terminus.The translation product of purified proinsulin mRNA, preproinsulin, was found to consist of two types of preproinsulin I (57%) and preproinsulin II (43%). This result indicated that the isolated proinsulin mRNA consisted of two types of proinsulin I mRNA and proinsulin II mRNA in approximately equal amounts.Complementary DNA was synthesized with proinsulin mRNA as a template. From the experiment of hybridization kinetics under conditions of excess RNA, the r0t1/2, of proinsulin mRNA was found to be 2.1 × 10−4 mol 1−1 s. This result also confirmed that proinsulin mRNA had been isolated in a high degree of purity.