MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL MESSENGER-RNA PRESENT IN THE SQUID GIANT-AXON

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to t his mRNA population has facilitated the identification of several of t he constituent mRNAs which encode several cytoskeletal and motor prote ins as well as enolase, a glycolytic enzyme, In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cD NA library. The pA6 mRNA is relatively small (550 nucleotides in lengt h) and is expressed in both nervous tissue and skeletal muscle. The ax onal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative TT-PCR anal ysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (simila r to 0.45 pg) in the analyzed segment of the giant axon and suggest th at the level of pA6 mRNA in the axonal domain of the giant fiber syste m might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative prot ein contains a single transmembrane domain located in the middle of th e molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphoryl ation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channe l protein. (C) 1997 Wiley-Liss, Inc.