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J Biol Chem, Vol. 273, Issue 14, 8360-8368, April 3, 1998
From the Erythropoietin Group, Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom
Hypoxia-inducible expression has been
demonstrated for many groups of mammalian genes, and studies of
transcriptional control have revealed the existence of
hypoxia-responsive elements (HREs) in the cis-acting sequences of
several of these genes. These sequences generally contain one or more
binding sites for a heterodimeric DNA binding complex termed
hypoxia-inducible factor-1 (HIF-1). To analyze this response
further, Chinese hamster ovary cells were stably transfected with
plasmids bearing HREs linked to genes encoding immunoselectable cell
surface markers, and clones that showed reduced or absent
hypoxia-inducible marker expression were selected from a mutagenized
culture of cells. Analysis of these cells revealed several clones with
transacting defects in HRE activation, and in one the defect was
identified as a failure to express the
Selection and Analysis of a Mutant Cell Line Defective in the
Hypoxia-inducible Factor-1
-Subunit (HIF-1)
CHARACTERIZATION OF HIF-1
-DEPENDENT AND -INDEPENDENT
HYPOXIA-INDUCIBLE GENE EXPRESSION
-subunit of HIF-1. Comparison
of hypoxia-inducible gene expression in wild type, HIF-1-defective,
and HIF-1-complemented cells revealed two types of response. For
some genes (e.g. glucose transporter-1), hypoxia-inducible
expression was critically dependent on HIF-1, whereas for other
genes (e.g. heme oxygenase-1) hypoxia-inducible expression
appeared largely independent of the expression of HIF-1. These
experiments show the utility of mutagenesis and selection of mutant
cells in the analysis of mammalian transcriptional responses to hypoxia
and demonstrate the operation of HIF-1-dependent
and HIF-1-independent pathways of hypoxia-inducible gene expression in Chinese hamster ovary cells.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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