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Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropod Littorina littorea

Tamara E. English^* and Kenneth B. Storey

Institute of Biochemistry and Department of Biology, College of Natural Sciences, Carleton University, 1125 Colonel By Drive, Ottawa Ontario, Canada K1S 5B6

View larger version (68K): [in a new window]   Fig. 1. cDNA sequence and translated amino acid sequence of L. littorea metallothionein, LLMET. Underlined nucleotides show the start codon, the stop codon (asterisk) and the polyadenylation signal (AATAAA), respectively. The encoded amino acid sequence is shown in bold. Note the high percentage of cysteine residues, most of which are arranged in a repeating Cys-X-Cys pattern.

View larger version (36K): [in a new window]   Fig. 2. (A) Translated amino acid sequence of clone LLMET (Periwinkle, encoding the protein Ll-MT) aligned with amino acid sequences of metallothioneins (MT) from other mollusc species: land snail Helix pomatia cadmium (Cd) MT (midgut-derived, GenBank accession number P33187) and copper (Cu) MT (accession number P55947); blue mussel Mytilus edulis 20-Ib (accession number S47576) and oyster Crassostrea virginica (accession number S17156). Conserved amino acids are highlighted in black. Gray highlights indicate residues that are shared between Ll-MT and at least two other sequences in the figure. The boxed residues at the C terminus comprise a highly conserved motif, Cys-X-Cys-X(3)-Cys-Thr-Gly-X(3)-Cys-X-Cys-X(3)-Cys-X-Cys-Lys, which categorizes Ll-MT into the mollusc family (family 2) of the MT classification system. (–) indicates an inserted gap. (B) Homology tree of MTs from selected invertebrate species. The percentage identities between the groups are indicated to the right of the forks. Sequences were aligned and the homology tree generated using the protein alignment matrix (PAM; Dayhoff et al., 1978) as employed in DNAman. The sequences were obtained from GenBank; accession numbers for species not listed in A are: lobster Homarus americanus (accession number A37039), mud crab Scylla serrata (accession number P02805), sea urchin Strongylocentrotus purpuratus (accession number P04734) and nematode Caenorhabditis elegans (accession number A34905).

View larger version (51K): [in a new window]   Fig. 3. Northern blot hybridization of clone LLMET against total RNA (15 µg per lane) isolated from foot muscle (A) or hepatopancreas (B) of L. littorea showing changes in transcript levels over a time course of freezing or anoxia stress. Samples were collected from control (C, untreated), stressed (1, 12 or 24 h of anoxia or freezing exposure) and 24 h recovered (after 6 days of stress exposure) snails. Representative northern blots (top) are shown with the corresponding ethidium bromide-stained ribosomal RNA bands (middle) from the formaldehyde gel. Histograms (bottom) show normalized band intensities. Values are means ± S.E.M. for N=3 independent trials using RNA isolated from different individuals. Measured pixel densities for each band were first normalized against their corresponding rRNA band and then mean band intensity was calculated for each treatment. Finally, experimental values were plotted relative to the corresponding control value that was set to 1. Asterisks indicate values that differ significantly from controls, Student's t-test (P<0.05).

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