Molecular cloning and characterization of a novel mRNA present in the squid giant axon

• Published in: Journal of neuroscience research 1997-07, Vol.49 (2), p.144-153
• Main Authors: Chun, Jong-Tai, Gioio, Anthony E., Crispino, Marianna, Eyman, Maria, Giuditta, Antonio, Kaplan, Barry B.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; axon; Axons - chemistry; Base Sequence; cDNA clone; Cloning, Molecular; Decapodiformes; DNA, Complementary - genetics; Genetic Code; Loligo; Marine; Molecular Sequence Data; RNA, Messenger - isolation & purification; squid
• ISSN: 0360-4012; 1097-4547
• DOI: 10.1002/(SICI)1097-4547(19970715)49:2<144::AID-JNR3>3.0.CO;2-9

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT‐PCR analysis indicate that there are 1.8 × 106 molecules of pA6 mRNA (≈0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system 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 protein contains a single transmembrane domain located in the middle of the molecule and a phosphate‐binding loop situated near the N terminus. The C‐terminal region of the protein contains two potential phosphorylation 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 channel protein. J. Neurosci. Res. 49:144–153, 1997. © 1997 Wiley‐Liss, Inc.