An effective strategy for cloning the mitochondrial citrate carrier: identification, characterization and tissue distribution in silver eel

Abstract

A general and effective strategy to identify cDNA encoding for mitochondrial silver eel citrate carrier (CIC) has been developed. In particular using prim-ers directed towards the highly conserved signature motif of the ortholog citrate cDNA sequences, the full-length silver eel cDNA from liver was obtained. This is 1193 bp in length with 5' and 3' untranslated regions of 90 and 149 bp, respectively. The open rea-ding frame encodes a mature protein of 297 amino acids, preceded by a presequence of 20 amino acids. Additionally the mature CIC overexpressed in Es-cherichia coli and reconstituted into phospholipids vesicles showed the same substrate specificity of the native protein previously characterized in silver eel. The tissue distribution of silver eel CIC mRNAs was investigated and transcripts were detected at high levels in swim bladder whereas a weaker signal was found in brain, gill, intestine and liver.