Design of degenerate oligonucleotide primers for cloning of G-protein a subunits

Heterotrimeric G proteins couple the seven-transmembrane domain receptors to various intracellular effectors, such as adenylyl cyclase, phospholipase C-β, and ion channels. Discrete signal transduction pathways are mediated through G proteins distinguished by the particular α, β, and γ subunit composition. The G-protein α-subunit genes are encoded by a large and evolutionarily conserved multigene family, as are the β and γ subunits. Sequence alignment of α-subunit genes from fungi, plants, and animals reveals several highly conserved amino acid motifs that are thought to contribute to guanosine-5'-triphosphate (GTP) binding. Degenerate polymerase chain reaction (PCR) primers which target these motifs have been used to amplify G-protein α subunits selectively from mice, Drosophila, Caenorhabditis elegans, Dictyostelium, Arabidopsis, and Neurospora. The PCR products can be easily cloned, screened, and sequenced over a precise region to identify rapidly novel genes. The PCR cloning technique is a powerful means of revealing possible novel G-protein α subunits that play central roles in the biological processes of a variety of organisms.