IDENTIFICATION OF NEW DYNEIN HEAVY-CHAIN GENES BY RNA-DIRECTED POLYMERASE CHAIN-REACTION

The functional differences among the axonemal dyneins are due, at least in part, to structural differences among the heavy-chain isoforms. Detailed characterization of the various isoforms should reveal the structural bases for functional specialization. To begin the characterization of the dynein heavy-chain family, an RNA-directed polymerase chain reaction (PCR) method was developed in collaboration with Ian Gibbons in his laboratory. Although each inner and outer dynein arm most likely is able to translocate along microtubules, the requirements for the initiation and propagation of bends place different functional demands on different dyneins. The catalytic P-loop has been identified in the complete sequences of several dynein heavy chains and is the absolutely conserved sequence GPAGTGKT which conforms to the GXXXXGKT/S motif found in most ATP-binding proteins. Degenerate oligonucleotide primers were designed to amplify regions of the dynein gene encoding the sequence surrounding the conserved catalytic P-loop. A small amount of mRNA from the cell of interest is reverse transcribed from one of the A primers. The first-strand cDNA is then diluted and a small portion is amplified with the same A primer and an equal concentration of an S primer. © 1995, Academic Press Inc.