The objective of this proposed project is to synthesize and clone the human insulin gene. The project will involve the following steps: (1) To synthesize two DNA duplexes, one corresponding to the coding region of human insulin A-chain, and the other corresponding to the B-chain. (2) To synthesize two oligodeoxynucleotide duplexes, one containing a start and the other a stop signal for protein synthesis. (3) To join by ligation the DNA coding for the insulin A-chain to the start and stop signals. Similar joining will be carried out for the B-chain. (4) The product from steps 1-3 will be joined to a cloning vehicle, adjacent to a strong promotor. (5) To clone the DNA ligation products from step 4 in E. coli and to identify the clones which contain the insulin gene by hybridization with 32P-labeled single-stranded DNA from step 1. (6) To isolate, purify and characterize the insulin A-chain and B-chain protein products synthesized by E. coli carrying the cloned insulin gene. To produce biologically active human insulin by recombining the A-chain and B-chain isolated from step 7.