A novel hypoxia-response element in the lactate dehydrogenase-B gene of the killifish Fundulus heteroclitus.
Previous studies have suggested that the lactate dehydrogenase-B gene (Ldh-B) of the Atlantic killifish, Fundulus heteroclitus, is a hypoxia-responsive gene. Here, we demonstrate that the F. heteroclitus Ldh-B promoter confers hypoxia-dependence upon reporter gene expression in transiently transfected mammalian (Hep3B) and fish (RTG-2 and RTH-149) cells in culture. Mutation and deletion analyses identified a putative hypoxia-response element (HRE) between 109 and 90 nucleotides upstream of the major start site. This HRE is characterized by the sequence 5'-GATGTG-3' spaced by 8 nucleotides from a perfect inverted repeat, and both sites are necessary for hypoxic induction of reporter gene expression in mammalian and fish cells. This HRE differs from the canonical sequence at one nucleotide position that is invariant among HREs from a wide range of hypoxia-sensitive genes. In fish cells, maximal induction of reporter gene expression driven by this HRE occurred at the lowest oxygen level tested (0.5%), took 48 h to 96 h, and was independent of glucose concentration (between 5.6 and 25 mM). Under all conditions tested, hypoxic induction of gene expression was lower in RTH-149 cells than in RTG-2, suggesting a potential defect in hypoxia signaling in RTH-149 cells. These results demonstrate that the F. heteroclitus Ldh-B promoter contains a novel HRE that is capable of driving reporter gene expression in a sequence-specific and oxygen-, time-, and cell line-dependent manner.
Rees
Bernard B
BB
Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA. brees@uno.edu
Figueroa
Yanira G
YG
Wiese
Thomas E
TE
Beckman
Barbara S
BS
Schulte
Patricia M
PM
D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D023781:Fundulidae / Q000235:genetics*; D017930:Genes, Reporter / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*
D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; C409667:lactate dehydrogenase 1; D008156:Luciferases; D005947:Glucose