title: Isolation of catfish proinsulin messenger RNA and synthesis of its complementary DNA.
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 Mr (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 x 10(-3) mole x sec/l. 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.
mesh_terms: D000818:Animals; D004587:Electrophoresis, Agar Gel; D005399:Fishes; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000302:isolation & purification*; D011384:Proinsulin / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000302:isolation & purification*
chemical_list: D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D011384:Proinsulin