Cloning and expression analysis of two distinct HIF-alpha isoforms--gcHIF-1alpha and gcHIF-4alpha--from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus.
BACKGROUND
Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.


RESULTS
We have cloned and characterized two distinct HIF-alpha cDNAs--gcHIF-1alpha and gcHIF-4alpha--from the hypoxia-tolerant grass carp. The deduced gcHIF-1alpha protein is highly similar to the HIF-1alphas (57-68%) from various vertebrate species, while gcHIF-4alpha is a novel isoform, and shows an equivalent degree of amino acid identity (41-47%) to the HIF-1alpha, HIF-2alpha and HIF-3alpha proteins so far described. Parsimony analysis indicated that gcHIF-4alpha is most closely related to the HIF-3alpha proteins. Northern blot analysis showed that mRNA levels of gcHIF-1alpha and gcHIF-4alpha differ substantially under normoxic and hypoxic conditions, while Western blot studies demonstrated that the endogenous protein levels for both gcHIF-1alpha and gcHIF-4alpha are similarly responsive to hypoxia. Our findings suggest that both gcHIF-1alpha and gcHIF-4alpha are differentially regulated at the transcriptional and translational levels. HRE-luciferase reporter assays show that both proteins function as transcription activators and play distinct roles in modulating the hypoxic response in grass carp.


CONCLUSIONS
There are at least two distinct HIF-alpha isoforms--gcHIF-1alpha and gcHIF-4alpha--in the hypoxia-tolerant grass carp, which are differentially expressed and regulated in different fish organs in response to hypoxic stress. Overall, the results suggest that unique molecular mechanisms operate through these two HIF-alpha isoforms, which underpin the hypoxic response in the hypoxia-tolerant grass carp.
Law
Sheran H W
SH
Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, People's Republic of China. sheranlaw@yahoo.com
Wu
Rudolf S S
RS
Ng
Patrick K S
PK
Yu
Richard M K
RM
Kong
Richard Y C
RY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D002347:Carps / Q000145:classification / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D023061:Gene Order; D000860:Hypoxia / Q000378:metabolism / Q000662:veterinary; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D016415:Sequence Alignment / Q000662:veterinary
D017931:DNA Primers; D018076:DNA, Complementary; D051795:Hypoxia-Inducible Factor 1, alpha Subunit