REES, B.B.*; SCHULTE, P.M.: Is intron 2 of the Fundulus lactate dehydrogenase B gene a hypoxia responsive element?

Previous studies have shown that liver lactate dehydrogenase B (LDH-B) activity increases during hypoxic exposure of F. heteroclitus. Several glycolytic genes in mammals are known to be upregulated during hypoxia via the binding of hypoxia-inducible factor (HIF-1) to a conserved sequence motif, the hypoxia responsive element (HRE). The F. heteroclitus Ldh-B gene contains a putative HRE in the second intron, which might be involved in the up-regulation of this gene in response to hypoxia. We used two complementary approaches to evaluate this hypothesis. First, we reasoned that if this DNA element were functionally important it would be conserved among multiple species. Therefore, we sequenced intron 2 of Ldh-B from 13 species in the genus Fundulus. The 3′ region of the intron was highly conserved and contained 2 putative HREs and a sequence similar to the mammalian cyclic AMP responsive element (CRE). The number and spacing of these elements is virtually identical to the arrangement of elements in the hypoxia responsive promoter of mammalian Ldh-A. In addition, we assessed the capacity of this intron to confer hypoxia-inducibility upon reporter gene expression using in vivo transient transfection. Although the addition of intron 2 upstream of the minimal Ldh-B promoter led to a marginal increase in reporter gene expression during normoxia, intron 2 failed to enhance gene expression under hypoxia. In this experiment, however, there was no increase in LDH-B enzyme activity, suggesting that the fish may have responded behaviorally or physiologically to environmental hypoxia in such a way as to minimize tissue hypoxia. We are currently exploring the operation of Ldh-B intron 2 as an HRE in cell culture. Supported by National Science Foundation grant IBN 9723050.