Abstract
The purpose of these experiments was to obtain proinsulin mRNA from catfish pancreatic islets and synthesize its cDNA. Poly(A)-rich mRNA was electrophoresed on preparative agarose-urea gels. One RNA fraction was obtained which translated predominantly preproinsulin. This mRNA was estimated to be approx. 210 000 M r (650 nucleotides) when electrophoresed under denaturing conditions. [ 3H]Proinsulin cDNA was hybridized to excess RNA to monitor purification of mRNA from total islet RNA. Greater than 94% of proinsulin messenger contained poly(A) sequences. [ 3H]Proinsulin cDNA hybridized to its template mRNA with a Rot 1 2 of 4.4 × 10 −3 mole · sec/1. The overall purification was 80-fold by this type of analysis. Thermal denaturation studies indicated a high degree of fidelity of hybrid formation between [ 3H]proinsulin cDNA and proinsulin mRNA. Proinsulin comprised 20% of total islet protein when synthesis was measured in vivo (Albert and Permutt, 1979) and 12–20% when total islet mRNA was translated in a cell-free system. Using the [ 3H]proinsulin cDNA probe it was estimated that proinsulin mRNA accounted for approx. 15% of total islet mRNA.
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