Cloning, genomic organization, and osmotic response of the aldose reductase gene.
Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We have cloned and characterized the rabbit AR gene to determine how it is regulated by hyperosmolality. The length of the gene, not including 5' or 3' flanking regions, is approximately 14.7 kilobases (kb) organized into 10 exons and 9 introns. The transcription start site is 36 base pairs upstream of the initiator methionine codon. A 5-kb fragment containing approximately 3.5 kb of 5' flanking region was isolated. The 3.5-kb sequence was examined for basal promoter activity and hyperosmotic response in luciferase reporter gene constructs. A 235-base-pair fragment (base pairs -208 to +27) was able to drive the downstream reporter gene in transfected PAP-HT25 cells under isoosmotic conditions (300 mosmol/kg of H2O). When this fragment plus the remaining upstream sequence (from approximately base pair -3429 to base pair +27) was used, cells in hyperosmotic medium (500 mosmol/kg of H2O) showed about 40-fold induction of luciferase expression compared with cells in isoosmotic medium. The upstream fragment (from approximately base pair -3429 to base pair -192) also conferred osmotic response to a heterologous promoter (B19). This finding evidences putative osmotic response element(s) (OREs) within a specific DNA fragment in a eukaryotic genome. Identification and characterization of OREs within this fragment and their associated trans-acting factors should reveal the molecular mechanisms of gene regulation in osmotic stress.
Ferraris
J D
JD
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Williams
C K
CK
Martin
B M
BM
Burg
M B
MB
García-Pérez
A
A
D000449:Aldehyde Reductase / Q000096:biosynthesis* / Q000235:genetics* / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D006706:Homeostasis; D007438:Introns; D007679:Kidney Medulla / Q000201:enzymology / Q000502:physiology*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011817:Rabbits; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013154:Spleen / Q000201:enzymology; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D000449:Aldehyde Reductase