Principles of Biochemistry, Volume 3 - A. Lehninger 1985

Molecular Mechanisms of Genetic Information Transfer
More on Genes: Repair, Mutation, Recombination, and Cloning
Expression of cloned genes is enhanced using a promoter

The METABOLISM/31.html">Transcription of many genes and the subsequent Translation of their mRNA to yield the corresponding Proteins require a promoter and an operator, which designate the site where RNA polymerase binds and initiates transcription (Section 29.27); otherwise, there is no guarantee that a foreign Gene integrated into a recipient Cell will be transcribed and translated. To ensure the transcription of an inserted gene, it is frequently necessary to place it within a plasmid or phage k DNA segment immediately downstream of the promoter-operator region. For instance, a gene can be integrated into an E. coli DNA fragment containing the lac Operon, positioned downstream of the operon's promoter-operator region (Section 29-27). If E. coli Cells harboring such recombinant DNA are placed in a medium containing lactose but lacking glucose, the inserted gene will be transcribed and translated along with the segment of the lac operon DNA located between the operator and the inserted gene.

This approach was employed in cloning the gene for Somatostatin, a peptide hormone (consisting of 14 amino acid residues) from the Hypothalamus that regulates the secretion of Insulin, Glucagon, and Growth Hormone. In this case, the somatostatin gene was chemically synthesized and linked to the end of the ß-galactosidase gene. The two linked genes were inserted into an E. coli plasmid, and the resulting recombinant plasmid was introduced into E. coli cells. As a result, these cells synthesized large quantities of a hybrid protein composed of covalently linked ß-galactosidase and somatostatin. Such a molecule, representing a hybrid between an endogenous E. coli protein and a vertebrate protein, is referred to as a chimeric protein. The ß-galactosidase-somatostatin hybrid was cleaved at the peptide bond joining the two proteins, thereby releasing biologically active somatostatin.