Cloning Neuropeptide Tyrosine cDNA

Abstract

The nervous system is composed of a diverse population of cells selectively expressing different genes which ultimately determine the type of neurotransmitters and neuropeptides produced and released. Neuropeptide tyrosine (NPY) is one of the most abundant and widely expressed peptides in the mammalian nervous system (1). It was originally isolated by virtue of a chemical assay that detects the presence of a carboxyl-terminal amide in proteins (2); a hallmark of potential hormonal function. Its widespread expression in the central and peripheral nervous systems, in addition to the carboxyl-terminal amide, suggested that NPY could be a critical neurotransmitter in a variety of neuronal processes. We were therefore interested in obtaining the cDNA for human NPY in order to study NPY gene expression and screen for the NPY gene (3,4). The only available information was the amino acid sequence of porcine NPY and the amino acid sequences of two related peptides, pancreatic polypeptide and peptide YY (5,6). These experiments were performed in the early 1980s before the advent of genome sequencing and polymerase chain reaction (PCR) technology. Today, experiments to obtain cDNAs for proteins for which only amino acid sequence is available are designed very differently and include computer searches of nucleotide databases and/or various PCR strategies. The experiments described in this chapter represent the classical way by which cDNAs were cloned from corresponding protein sequence.