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Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) from crayfish

Dongdong Chen, Zhiping Zhang, Michele G. Wheatly^* and Yongping Gao

Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA

View larger version (107K): [in a new window]   Fig. 1. (A) The complete nucleotide and deduced amino acid sequence of crayfish cardiac muscle SERCA cDNA (accession number AF025848). Nucleotides and amino acids are numbered to the right of the sequence. The phosphorylation site, fluorescein isothiocyanate (FITC) site and the 5'-p-fluorosulfonylbenzoyladenosine (FSBA)/-[4-(N-2-chloroethyl-N-methylamino)]benzylamide ATP (CIRATP) binding site are underlined and labeled. The start codon and the stop codon are indicated in bold letters. The slash in the sequence at nucleotide 2980 is the site at which the heart muscle sequence diverges from the axial abdominal muscle sequence (Zhang et al., 2000; accession no. AF025849). (B) The nucleotide sequence of crayfish axial abdominal muscle SERCA (Zhang et al., 2000) beginning from nucleotide 2980.

View larger version (29K): [in a new window]   Fig. 2. Comparison of the C-terminal amino acid sequences of SERCAs. The amino acid sequence of SERCA C termini shown are for (A) crayfish axial abdominal muscle (Zhang et al., 2000; accession no. AAB82291); (B) crayfish heart muscle (present study; accession no. AAB82290); (C) the 4.5 kb Artemia protein (Palmero and Sastre, 1989; accession no. P35316); (D) the 5.2 kb Artemia protein (Escalante and Sastre, 1993; accession no. CAA51262); (E) rat SERCA2a (Brandl et al., 1986; accession no. P11508); (F) rat SERCA2b (Lytton et al., 1989; accession no. P11057). The slash mark denotes the point at which the amino acid sequence of each set of proteins diverges.

View larger version (55K): [in a new window]   Fig. 3. Hydropathy plot of (A) crayfish cardiac muscle SERCA (present study; accession no. AAB82291) in comparison with (B) crayfish axial muscle (Zhang et al., 2000; accession no. AAB8229), (C) Artemia SERCA 5.2 kb isoform (Escalante and Sastre, 1993; accession no. CAA51262), (D) Artemia SERCA 4.5 kb isoform (Palmero and Sastre, 1989; accession no. P35316), (E) rat SERCA2b (Lytton et al., 1989; accession no. P11057) and (F) rat SERCA2a (Brandl et al., 1986; accession no. P11508). Hydrophobicity values were determined by the method of Kyte and Doolittle (1982) using a window of 12 residues (MacDNASIS, Rainbow Technologies). Putative transmembrane domains are numbered based on the model of Brandl et al. (1986). The arrows indicate the extra potential transmembrane domain.

View larger version (69K): [in a new window]   Fig. 4. Northern blot analysis of the distribution of the two crayfish SERCA isoforms (from heart and axial muscle) in intermoult tissues. Messenger RNAs from eggs, heart muscle and axial abdominal muscle were hybridized with the following probes: (A) the 426 bp fragment corresponding to crayfish nucleotides 2086-2546 in both heart and axial muscle SERCA; (B) the 723 bp XbaI—poly(A) tail fragment of the putative heart SERCA; (C) the 515 bp XhoI—poly(A) tail fragment of the putative muscle SERCA. The positions of marker nucleotides are shown at the left.

View larger version (72K): [in a new window]   Fig. 5. Northern blot analysis of crayfish heart SERCA mRNA in heart muscle during different stages of the moulting cycle. (Top) Heart muscle total RNA (5 µg) from 18 crayfish at different moult stages was used: intermoult (N=6), late premoult (N=6) and postmoult (1-2 days, N=6). The membrane was hybridized to a heart-specific SERCA probe (723 bp) and exposed to X-ray film for 24h. (Bottom) 18S RNA concentration on the corresponding formaldehyde/agarose gel before being transferred to the membrane to serve as control (visualized by Ethidium Bromide staining).

View larger version (52K): [in a new window]   Fig. 6. Southern blot analysis of crayfish SERCA gene copy number. 15 µg of genomic DNA were digested with HindIII or EcoRI, separated on a 0.8 % agarose gel, transfered to a nylon membrane, and hybridized with a probe that is from nucleotides 2785-3019 of the crayfish tail muscle SERCA.