Cloning and characterization of sarco/endoplasmic reticulum Ca2+-atpase (SERCA) from crayfish axial, muscle

The discontinuous pattern of muscle growth during the moulting cycle of a f reshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Re ticulum Ca2+-ATPase (SERCA), We describe the cloning, sequencing and charac terization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abd ominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) f ollowed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp ope n reading frame coding for 1002 amino acid residues with a molecular mass o f 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme cont ains all the conserved domains found in 'P'-type ATPases, and the hydropath y profile suggests a transmembrane organization typical of other SERCAs, It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% i dentity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitc h muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle iso form SERCA2 and 67% identity with SERCA3, Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conser ved and that most differences were in the NH2 terminus, the central loop re gion and the COOH terminus. Northern analysis of total RNA from crayfish ti ssues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SE RCA was most abundant in muscle (axial abdominal, cardiac and stomach), whe re it is involved in Ca2+ resequestration during relaxation, and in eggs, w here it may be implicated in early embryogenesis. The level of SERCA mRNA e xpression in axial abdominal muscle varied during the moulting cycle as det ermined by slot-blot analysis. SERCA expression was greatest during intermo ult and decreased to approximately 50% of this level during pre- and postmo ult. Patterns of gene expression for SERCA and other sarcomeric proteins du ring the crustacean moulting cycle may be regulated by ecdysteroids and/or mechanical stimulation.