First published online May 30, 2008
Journal of Experimental Biology 211, 1964-1968 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.017368
The cloning of eel osmotic stress transcription factor and the regulation of its expression in primary gill cell culture
W. K. F. Tse,
S. C. Chow and
C. K. C. Wong*
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong
Kong
View larger version (33K):
[in this window]
[in a new window]
|
Fig. 1. The cloning of eel Ostf cDNA. (A) Strategy for cloning the eel Ostf
sequence. (B) DNA sequence of the 615 bp eel Ostf cDNA and the deduced amino
acid sequence. (C) Comparison of amino acid sequences of the cloned eel Ostf
and tilapia Ostf1 (GenBank accession no. AY679524). The mismatch or deleted
residues are underlined. The conserved TSC-22 domain is bold.
|
|
View larger version (9K):
[in this window]
[in a new window]
|
Fig. 2. The expression levels of Ostf mRNA in PercollTM-gradient-isolated gill
pavement cells (PVCs) and chloride cells (CCs). Gill epithelia were dissected
and digested; the cell suspension was centrifuged in PercollTM gradient
solution. The respective gill cell type was isolated, and total RNA was
extracted and reverse transcribed for real-time PCR assay. (A) Comparison of
Ostf expression levels in isolated freshwater PVCs and CCs.
*P<0.05 compared with the PVC. (B) Change in Ostf mRNA
expression levels in the gill PVCs and CCs isolated from fish at day 0.25, 1,
3 and 7 of seawater acclimation. A significant induction of Ostf mRNA in gill
CCs after 0.25 days of seawater acclimation was measured.
*P<0.05 compared with freshwater CCs. A small but
significant increase of Ostf mRNA in isolated gill PVCs after 0.25 days of
seawater acclimation was noted.  P<0.05 compared
with the isolated freshwater PVCs. The results were obtained from four
independent experiments.
|
|
View larger version (7K):
[in this window]
[in a new window]
|
Fig. 3. Induction of Ostf mRNA expression levels in primary gill cell culture.
Primary gill cells were grown overnight in isotonic medium (320 mOsmol
l–1) and were then subjected to hypertonic treatments and/or
actinomycin D (Act D) treatment. After 6 h (0.25 days) of treatment, total RNA
was extracted and reverse transcribed for real-time PCR assay. (A)
Hypertonicity was achieved by the addition of either (1) membrane-nonpermeable
solute (NaCl) or (2) membrane-permeable solute (urea) to the medium, making
its osmolarity 500 mOsmol l–1. A significant induction of
Ostf mRNA expression was observed in the hypertonic condition prepared by the
addition of NaCl. (B) Effect of Act D on hypertonicity-induced Ostf mRNA
expression. Hypertonicity and Act D cotreatment abolished the induced Ostf
transcript levels. *P<0.05 compared with the control
cells. The results were obtained from five independent experiments.
|
|
View larger version (9K):
[in this window]
[in a new window]
|
Fig. 4. Effects of organic osmolytes or colchicine on Ostf mRNA expression levels
in primary gill cell culture. Primary gill cells were grown overnight in the
isotonic medium (320 mOsmol l–1) and were then subjected to
hypertonic treatment (NaCl, 500 mOsmol l–1) with the addition
of (A) either 5 mmol l–1 of inositol, betaine or taurine or
(B) 100 µmol l–1 colchicine. After 6 h of treatment, total
RNA was extracted and was reverse transcribed for real-time PCR assay. Note
that the cotreatment of the hypertonicity-exposed cells with the organic
osmolytes or colchicine abolished the induced Ostf mRNA expression.
*P<0.05 compared with the control cells. The results
were obtained from five independent experiments.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2008
