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Journal of Experimental Biology, Vol 203, Issue 22 3411-3423, Copyright © 2000 by Company of Biologists

JOURNAL ARTICLES

Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase

Z Zhang, D Chen and MG Wheatly
Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA. michele.wheatly@wright.edu

The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed 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 open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues 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. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of this level during pre- and postmoult. Patterns of gene expression for SERCA and other sarcomeric proteins during the crustacean moulting cycle may be regulated by ecdysteroids and/or mechanical stimulation.

This article has been cited by other articles:

				M. G. Wheatly and Y. Gao Molecular biology of ion motive proteins in comparative models J. Exp. Biol., September 1, 2004; 207(19): 3253 - 3263. [Abstract] [Full Text] [PDF]

				D. Chen, Z. Zhang, M. G. Wheatly, and Y. Gao Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from crayfish J. Exp. Biol., September 1, 2002; 205(17): 2677 - 2686. [Abstract] [Full Text] [PDF]

				M Wheatly, Z Zhang, J Weil, J Rogers, and L Stiner Novel subcellular and molecular tools to study Ca(2+) transport mechanisms during the elusive moulting stages of crustaceans: flow cytometry and polyclonal antibodies J. Exp. Biol., January 3, 2001; 204(5): 959 - 966. [Abstract] [PDF]