First published online March 9, 2004
Journal of Experimental Biology 207, 1353-1360 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00872
Accumulation and translation of ferritin heavy chain transcripts following anoxia exposure in a marine invertebrate
Kevin Larade* and
Kenneth B. Storey
Institute of Biochemistry and Department of Biology, Carleton
University, 1125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6
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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.
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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.
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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.
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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.
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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.
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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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© The Company of Biologists Ltd 2004
