Cloning, Sequencing and Phylogenetic/Phenetic Classification of an Estrogen Receptor Alpha (α) Subtype of Sheepshead Minnow (\u3ci\u3eCyprinodon variegatus\u3c/i\u3e)

Hypoxia-inducible factor 1α (HIF-1α) is a key transcription factor that controls a variety of cellular and systemic homeostatic responses to hypoxic stress. Expression and function of HIF-1α have not been studied in crustaceans, which experience wide fluctuations of oxygen tensions in their aquatic environment. Here we show that an HIF-1α homolog, gsHIF, is present in the hypoxia-tolerant grass shrimp Palaemonetes pugio. Using RT-PCR and 3′ and 5′RACE, we cloned a full-length gsHIF cDNA (3822 bp) with an open reading frame encoding a 1057 amino acid protein. Similar to vertebrate HIF-1α, gsHIF has one basic helix–loop–helix (bHLH) domain, two PAS domains, an oxygen-dependent degradation domain (ODD) with two proline hydroxylation motifs, and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. In addition to these conserved sequences, gsHIF has a unique 230 amino acid sequence (aa 790–1020) not found in any vertebrate HIF proteins. Phylogenetic analysis indicates that grass shrimp and vertebrate HIFs belong to distinct clades within the HIF protein family. Expression analysis shows that gsHIF is constitutively expressed under normoxic (7.5 ppm DO), moderate (2.5 ppm DO) and severe (1.5 ppm DO) hypoxic conditions. In addition to gsHIF, we cloned a fragment of a second bHLH-PAS transcription factor from the grass shrimp, which had one bHLH and two PAS domains, and an overall 68% amino acid sequence homology with Apis mellifera trachealess protein