HIF-1alpha and -2alpha genes in a hypoxia-sensitive teleost species Megalobrama amblycephala: cDNA cloning, expression and different responses to hypoxia.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF) alpha subunits, i.e. HIF-1alpha, -2alpha and -3alpha. In fish, the molecular constructions, expression characteristics and hypoxic regulation of HIF-alpha subunits are still not well known. In this study, we identified the HIF-1alpha and HIF-2alpha full-length cDNAs in a hypoxia-sensitive fish species Wuchang bream, Megalobrama amblycephala. The whole length of HIF-1alpha cDNA was 3,815bp, consisting of an open reading frame (ORF) encoding 774 amino acid (aa) residues. The HIF-2alpha cDNA totaled 3,121-bp including an 835-aa ORF. The Wuchang bream HIF-1alpha and HIF-2alpha subunits were structurally similar in the DNA-binding and dimerization domains, but differed in the transactivation domain. In adult fish, both HIF-1alpha and HIF-2alpha mRNAs were detected in different tissues under normoxic conditions. HIF-1alpha mRNA was highly expressed in the liver, gill and testis, whereas HIF-2alpha mRNA was abundantly expressed in most of the Wuchang bream tissues. Both HIF-1alpha and HIF-2alpha mRNAs were detected in all stages of embryogenesis and expressed in a ubiquitous pattern. In contrast to HIF-1alpha, the mRNA levels of HIF-2alpha fluctuated in different stages, with higher expression in the zygote, 8-, 28-, 48- and 52-hr post fertilization (hpf) embryos. During hypoxic treatment, the mRNA levels of HIF-2alpha were significantly (p<0.01) up-regulated to 910% in the liver and 320% in the kidney, whereas no significant changes of HIF-1alpha mRNA were observed in the corresponding tissues. These results suggest that the Wuchang bream HIF-1alpha and -2alpha would be involved in different physiological functions under normoxia and hypoxia situations.

Shen RJ ，Jiang XY ，Pu JW ，Zou SM ... -  《-》

Molecular cloning, characterization and expression of two hypoxia-inducible factor alpha subunits, HIF-1alpha and HIF-2alpha, in a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus).

Alteration of gene expression is a crucial component of adaptation by animals to hypoxic conditions and is mediated by specific transcription factors, hypoxia-inducible factors (HIFs), which are composed of alpha and beta subunits. In this study, we report the cloning and characterization of two HIF-alpha subunits, HIF-1alpha and HIF-2alpha, and their expression in various tissues of a hypoxia-tolerant marine teleost, Atlantic croaker (Micropogonias undulatus). The full-length croaker HIF-1alpha (2805 bp) and HIF-2alpha (3205 bp) cDNAs contain open reading frames encoding proteins with 720 and 847 amino acids, respectively, which are highly homologous to the HIF-1alpha and HIF-2alpha proteins of other non-mammalian species. Croaker HIF-1 alpha shares only 43% sequence identity with the croaker HIF-2alpha subunit. However, the basic helix-loop-helix/Per-ARNT-Sim regions appear to be relatively well conserved between the two proteins, with identities of 75-83%. The core oxygen-dependent degradation domain regions in croaker HIFs are well conserved, suggesting a similar mechanism of HIF degradation to that in other vertebrate species. Northern blot analysis showed that croaker HIF-1alpha and HIF-2alpha mRNAs (transcript sizes 3.0-3.8 kb) are highly expressed in the brain, heart, liver, and gonads under hypoxic conditions, whereas muscle tissues show lower levels of expression. Short-term (1.7 mg/L dissolved oxygen, DO for 3 days to 1 week) and long-term (1.7, 2.7 and 3.7 mg/L DO for 3 weeks) hypoxia exposure caused significant increases in HIF-1alpha and HIF-2alpha mRNA expression in croaker ovaries compared to mRNA levels in fish held in normoxic conditions (DO: 6.5 mg/L). However, HIF transcript levels in hypoxia-exposed fish had returned to control values 24 h after the DO in the tanks was restored to normoxic levels. The results suggest that the upregulation of both HIF-1alpha and HIF-2alpha subunits at the transcriptional level is an important component of adaptation of croaker to chronic hypoxia and HIF-alphas are potentially useful molecular indicators of

Rahman MS ，Thomas P 《GENE》

Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih).

Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver of Wuchang bream (Megalobrama amblycephala Y.) using RT-PCR and rapid amplification of cDNA ends (RACE). They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical isoelectric points of 5.25 and 5.30, respectively. Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region. Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD). Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates, while Ma-HSP70 shared more than 85.0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86.5% identity. Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region. The two proteins also contained many protein kinase C phosphorylation sites, N-myristoylation sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins were alpha-helix and random coil. Fluorescent real-time quantitative RT-PCR was used to study the effects of heat shock (34 degrees C), crowding stress (100g L(-1)) and challenge with bacteria Aeromonas hydrophila on the mRNA expression of the two HSP70s in Wuchang bream liver. The results indicated that, during 24 h stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased at first and then decreased under heat stress, and appeared to increase continuously under crowding stress. After bacterial challenge, the mRNA levels of both Ma-HSC70 and Ma-HSP70 increased at first and then decreased. The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream.

Ming J ，Xie J ，Xu P ，Liu W ，Ge X ，Liu B ，He Y ，Cheng Y ，Zhou Q ，Pan L ... -  《-》

Cloning of hif-1alpha and hif-2alpha and mRNA expression pattern during development in zebrafish.

Hypoxia-inducible factors (HIFs) regulate gene expression in response to hypoxia and in vertebrates they are known to participate in several developmental processes, including angiogenesis, vasculogenesis, heart and central nervous system development. Over the last decade, major progress in unraveling the molecular mechanisms that mediate regulation of HIF proteins by oxygen tension has been reported, but our knowledge on their developmental regulation during embryogenesis in model organisms is limited. Expression of hif-1alpha and hif-2alpha genes has been characterized during normal mouse development and they were found to be expressed from stages E7.5, later in E9.5 and E15.5 in several different tissues such as the brain, heart and blood vessels. However, there is no detailed temporal information on their expression at other embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe the cloning and detailed expression pattern of zebrafish hif-1alpha and hif-2alpha genes. Sequence analysis revealed that zebrafish Hif proteins are highly homologous to other vertebrate orthologues. Zebrafish hif-1alpha and hif-2alpha are both expressed throughout development in discrete territories in a dynamic pattern. Interestingly, in the notochord the expression of hif-1alpha is switched off, while hif-2alpha transcription is turned on, signifying that the two genes might have partially overlapping, although non-redundant functions in development. This is the first time that a detailed comparison of the expression of hif-1alpha and hif-2alpha is directly assessed in a vertebrate model system throughout development.

Rojas DA ，Perez-Munizaga DA ，Centanin L ，Antonelli M ，Wappner P ，Allende ML ，Reyes AE ... -  《GENE EXPRESSION PATTERNS》

Cloning of hypoxia-inducible factor 1alpha cDNA from chick embryonic ventricular myocytes.

Hypoxia-inducible factor 1 (HIF-1) is composed of HIF-1alpha and arylhydrocarbon nuclear receptor translocator (ARNT), which belong to the basic-helix-loop-helix-Per/ARNT/Sim (bHLH-PAS) family of transcription factors. HIF plays key roles in oxygen homeostasis and embryonic cardiovascular development. In this study, we have cloned cDNAs encoding the chick HIF-1alpha from cultured embryonic ventricular myocytes (CEVM) and then examined its expression in various embryonic tissues. The deduced amino acid sequence of the chick HIF-1alpha cDNA showed 79% identity with that of the human HIF-1alpha cDNA. In contrast, sequence homology between the chick HIF-1alpha and endothelial PAS protein 1 (EPAS1), another member of the bHLH-PAS proteins, was only low (49%). HIF-1alpha mRNA was expressed abundantly in CEVM, but scarcely in the liver, which was quite different from expression pattern of EPAS1 mRNA. These data suggest that HIF-1alpha may be involved in embryonic cardiovascular development. HIF-1alpha and EPAS1 may play distinct roles during developmental stages.

Takahashi T ，Sugishita Y ，Nojiri T ，Shimizu T ，Yao A ，Kinugawa K ，Harada K ，Nagai R ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》