CLONING AND SEQUENCING OF AN INSULIN-LIKE RNA FROM HUMAN BRAIN

Abstract

Publisher Summary This chapter discusses the cloning and sequencing of an insulin-like RNA from human brain. In a study described in the chapter, a cloned sequence encoding human insulin was used to identify RNAs homologous to insulin in a human fetal brain. These sequences do not encode insulin but rather a family of sequences related to insulin. To further characterize these sequences, double-stranded DNA was synthesized using total fetal brain RNA as template ; this was inserted into the Pst site of pBR322 using the oligo d(G)–d(C) joining procedure. A cDNA clone was used encoding the β peptide of insulin to screen the colonies that were obtained. From 300 transformants, 14 colonies that hybridized to the human insulin probe under nonstringent hybridization conditions were identified. The region homologous to insulin was localized by restriction enzyme digestion and blot hybridization. The inserted DNA was sequenced by the chemical method. The clone represented the 31 portion of RNA of 4000 bases. The repeat of (TG) was found in the intron of the Gγ-globin and the Aγ-globin. To characterize the RNA transcripts corresponding to the sequences in this clone, restriction fragments of pAG-B6 were used to probe RNA from fetal brain. This clone corresponds to a 4000-base RNA transcript that hybridizes to the insulin probe. This sequence is present in brain RNA and not in liver RNA.