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. CONCLUSION: 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

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, -1

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, "</p><p>BMC Molecular Biology 2006;7():15-15.</p><p>Published online 20 Apr 2006</p><p>PMCID:PMC1473195.</p><p></p>man HIF-1α; fHIF2α, HIF-2α; hHIF2α, human HIF-2α; hHIF3α, human HIF-3α; gcHIF4α, grass carp HIF-4α. The number on the left of each row denotes the amino acid position. Identical amino acids shared by most of the HIF-α s are shaded in black while similar amino acids are shaded in grey. Dashes (--) indicate gaps inserted for improved alignment. Domains that are typical characteristic of HIF-α proteins are marked on the alignment according to the amino acid positions in human HIF-1α. The two conserved proline residues within the ODD domain are indicated by open arrows (⇩). The closed arrow () indicates the asparagine residue (Asn-803) in C-TAD which controls HIF-1 binding to CBP/p300

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, -0

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, "</p><p>BMC Molecular Biology 2006;7():15-15.</p><p>Published online 20 Apr 2006</p><p>PMCID:PMC1473195.</p><p></p>ions delineating the different domains are indicated and include: bHLH, basic helix-loop-helix domain; PASA/B, Per-ARNT-Sim A/B domains; PAC, domain C-terminal to PAS motifs; ODD, oxygen dependent degradation domain; N-TAD, N-terminal transactivation domain; and C-TAD, C-terminal transactivation domain

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, -2

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp, "</p><p>BMC Molecular Biology 2006;7():15-15.</p><p>Published online 20 Apr 2006</p><p>PMCID:PMC1473195.</p><p></p>quences by maximum parsimony (PROTPARS) using the PHYLIP package version 3.57 c [25]. The bootstrap support (SEQBOOT program, PHYLIP package) for each branch (1000 replications) is shown. The standing of the abbreviations and the GenBank/EMBL/Swissprot accession number of the bHLH-PAS-A/B sequences used are provided in