Cloning and characterization of a novel calmodulin‐like gene (CaM) from crayfish Procambarus clarkii

Abstract

Calmodulin (CaM), the Ca2+ sensor in living cells, is essential for biological functions mediated by Ca2+dependent mechanisms. This study describes the cloning and characterization of a novel CaM from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcCaM). We also examined tissue specific distribution and expression of pcCaM mRNA as a function of molting by real-time PCR. The complete sequence of pcCaM consists of 739 bp and codes for 149 amino acid residues (molecular mass of 16.8 kDa). The deduced pcCaM protein sequence matched most closely with published CaM sequences from human CaM (96.5%) to pearl-oyster (97.9%) and killfish (98.5%). The real-time PCR analysis confirmed that pcCaM mRNA was ubiquitously expressed in all tissues tested (gill, hepatopancreas, antennal gland, axial abdominal and cardiac muscle); however it was most abundant in muscles. The real-time PCR analysis also revealed that pcCaM mRNA expression was upregulated in pre/postmolt stages as compared with intermolt expression in both epithelial and in non-epithelial tissues. The expression of pcCaM paralleled our previous findings in the trends for plasma membrane calcium ATPase (PMCA) during molting of crayfish, suggesting that their cellular function in regulating intracellular Ca2+ is linked. PMCA is thought to be regulated by calmodulin. Colocalization study of PMCA and CaM protein in tail muscle with confocal microscopy techniques confirmed the correlation of these two proteins in cellular calcium metabolism in crayfish. (Supported by Grant IBN 0445202 to MGW, YG and CMG).