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Figures
- Fig. 1.
Sequence and partial alignment of ferritin heavy chain from L. littorea. (A) Nucleotide (upper) and deduced amino acid (lower) sequences of the cDNA encoding the C-terminal end of ferritin heavy chain from the L. littorea library. The nucleotide sequence has the GenBank accession number AY090096. (B) Alignment of the C-terminal amino acid sequence of ferritin heavy chain from L. littorea with sequences from Lymnaea (pond snail; accession number P42577), chiton (BAA21810), starfish (AAB60883), salmon (P49946), and human (P02794). Identical residues are indicated by (–); stops indicate gaps.
- Fig. 2.
(A) Expression of ferritin heavy chain (HC) transcripts in hepatopancreas of L. littorea over a time course of anoxia exposure (12–120 h) and aerobic recovery (1 h after 120 h anoxia) as shown by northern blots (top). Scanned intensity of anoxic samples was normalized against the ethidium bromide (EtBr) stained gel (middle) and the 18S ribosomal RNA band (bottom) and then plotted relative to the intensity of the control band. (B) Histograms show means ± s.e.m. for N=3 independent blots using RNA extracted from different snails. aSignificantly different from the normoxic control as determined by the Dunnett's test, P<0.01. bSignificantly different from 1 h aerobic recovery, P<0.05. Con, control; R, recovery.
- Fig. 3.
Nuclear run-off assays examining transcriptional regulation of ferritin heavy chain. (A) Assays were performed using nuclei prepared from the hepatopancreas of normoxic and 48 h anoxic L. littorea. Transcripts labeled with [32P]-UTP were hybridized to nylon membranes carrying immobilized inserts coding for the clones indicated. Ribosomal protein L26 was included as a positive control (Larade et al., 2001). (B) Histograms show the ratio of transcript levels in anoxic versus control samples, means ± s.e.m. for N=3 independent trials, each consisting of nuclei isolated from the hepatopancreas of five snails.
- Fig. 4.
Ferritin heavy chain translation status measured using polysome profiles of L. littorea hepatopancreas extracts prepared using post-mitochondrial supernatants centrifuged on 15%–30% continuous sucrose density gradients. Fractions were collected with high sucrose (30%) in fraction 1, decreasing to 15% in fraction 15. Bar graphs show absorbance at 254 nm, representing the relative amount of total RNA in each fraction. P, polysomes; M, monosomes. Line graphs show ferritin heavy chain transcript levels as determined from northern blots of total RNA isolated from each fraction. (A) normoxia; (B) 72 h anoxia; (C) 6 h aerobic recovery following 72 h anoxia.
- Fig. 5.
Ferritin heavy chain protein levels during anoxia and aerobic recovery. (A) Western blots of hepatopancreas protein samples (20 μg) from normoxic, 24 h anoxic, 72 h anoxic and 1 h recovered (after 72 h anoxia) snails probed with an anti-ferritin antibody (Andersen et al., 1995). (B) Histograms show means± s.e.m. for N=3 samples. aSignificantly different from the control value as determined by the Dunnett's t-test, P<0.01.
- Fig. 6.
Expression of ferritin heavy chain transcripts in hepatopancreas exposed to various conditions in vitro. Total RNA was isolated from the explanted hepatopancreas incubated under each condition and resolved on a 1.5% formaldehyde gel, blotted onto nitrocellulose and hybridized at 45°C to 32P-labeled probes produced from the ferritin heavy chain cDNA clone. Levels of mRNA transcripts are illustrated during normoxia (Con) and stress or second messenger exposure for paired hepatopancreas samples. The scanned intensity of experimental samples listed in the table is relative to the corresponding normoxic control. Values are means ± s.e.m. for N=3 independent hepatopancreas samples. aSignificantly different from control values as determined by the Student's t-test, P<0.01.
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