Hypoxia-inducible factor 1alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau.

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak, which has been living at 3000-5000 m altitude for at least two millions of years, was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1alpha ORFs (open reading frames) encoding for two isoforms of HIF-1alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1alpha mRNA and protein are tissue specific expression. Its HIF-1alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1alpha protein may play an important role in the adaptation to hypoxia environment.

Wang DP ，Li HG ，Li YJ ，Guo SC ，Yang J ，Qi DL ，Jin C ，Zhao XQ ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

cDNA cloning, gene organization and variant specific expression of HIF-1 alpha in high altitude yak (Bos grunniens).

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic-helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1alpha is the oxygen-regulated subunit of HIF-1, which regulates the transcription of genes involved in oxygen homeostasis in response to hypoxia. Yak (Bos grunniens), a mammal native to high altitude (HA) region ( approximately 3500-5500 m), has successfully adapted over many generations to the chronic hypoxia of HA. In the present work, cDNA encoding HIF-1alpha has been cloned from the blood of yak. Tissue specific expression of the mRNA was analyzed in blood, heart, lung, liver and kidney by RT-PCR with primers from three different regions of cDNA. The HIF-1alpha expression was liver and blood specific. The HIF-1alpha mRNA contains 823 bp long 3'UTR that is AU-rich and contains ten AUUUA pentamers and two overlapping copies of the nonamer UUAUUUAUUUAUU. Three potential microRNAs, hsa-miR-107/mmu-miR-107/rno-miR-107, hsa-miR-18b and hsa-miR-135a/mmu-miR-135a/rno-miR-135a, targeting 3'UTR of yak HIF-1alpha, were identified by using target prediction software. The CDS encodes for 823 residues of amino acids and showed 99%, 95%, 92%, 90% and 90% similarity to domestic cattle, human, plateau pika, mouse and rat HIF-1alpha, respectively. HIF-1alpha cDNA, cloned and sequenced in the present work has revealed the evolutionary conservation through multiple sequence alignment. Liver and blood specific stability of HIF-1alpha mRNA appears miR-107 regulated.

Dolt KS ，Mishra MK ，Karar J ，Baig MA ，Ahmed Z ，Pasha MA ... -  《GENE》

Cloning of hypoxia-inducible factor 1alpha cDNA from a high hypoxia tolerant mammal-plateau pika (Ochotona curzoniae).

Hypoxia-inducible factor 1 is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species living only at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Furthermore, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa.

Zhao TB ，Ning HX ，Zhu SS ，Sun P ，Xu SX ，Chang ZJ ，Zhao XQ ... -  《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

[Genetic cloning and expression of hypoxia inducible factor 1 alpha in high altitude hypoxic adaptation species Tibetan antelope (Pantholops hodgsonii)].

In order to investigate the role of the hypoxia inducible factor 1 alpha (HIF-1α) in the adaptation mechanism to high altitude hypoxia, the cloning of the HIF-1α gene cDNA of Tibetan antelope (Pantholops hodgsonii), using RT-PCR and RACE, was applied, and the comparative analysis of the tissue-specific expressions of HIF-1α among Tibetan antelope, Tibetan sheep and plain sheep was performed using real-time PCR and Western blot. The sequence analysis indicated that the cDNA sequences acquired by cloning from the HIF-1α gene of Tibetan antelope comprised a 2 471-bp open reading frame (ORF) and a 1 911-bp 3'UTR. The similarity between its coding sequence, predicted amino acid sequence and HIF-1α of other mammals exceeded 87%, in which the similarity with cow was up to more than 98%, which showed that this sequence was the cDNA of HIF-1α of Tibetan antelope. The results of real-time PCR and Western blot showed that expressions of HIF-1α mRNA and protein appeared in Tibetan antelope's lung, cardiac muscle and skeletal muscle, with the highest expression in lung. HIF-1α mRNA and protein had obvious differential expression in these tissues. Further research showed that Tibetan antelope and Tibetan sheep possessed higher expressions of HIF-1α protein in the three tissues above-mentioned compared with plain sheep, and the expressions of HIF-1α mRNA and protein in Tibetan antelope's lung, cardiac muscle and skeletal muscle were higher than those of Tibetan sheep. It illustrates that the hypoxic HIF-1α-specific expression is one of the molecular bases of high altitude hypoxia adaptation in Tibetan antelope.

Liu F ，Wuren T ，Ma L ，Yang YZ ，Ge RL ... -  《-》

Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P < 0.05), but the HIF-2α gene is significantly different in the heart, spleen, and kidney (P < 0.05). Furthermore, the methylation levels in the 5' flanking regulatory regions of HIF-1α and HIF-2α in yak kidney were significantly decreased than cattle counterparts (P < 0.05). Identifying these genes and the comparison of different expressions facilitates the understanding of the biological high-altitude hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

Xiong X ，Fu M ，Lan D ，Li J ，Zi X ，Zhong J ... -  《-》