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Cell growth requires the uptake of water, driven by an osmotic gradient across the plasma membrane. When the external osmolarity increases, many eukaryotic cells are capable of osmoregulation by increasing their internal osmolarity (1). The molecular mechanisms used by eukaryotic cells to sense changes in external osmolarity and transduce that information into an osmoregulatory response are poorly understood. The yeast Saccharomyces cerevisiae responds to increases in external osmolarity by increasing glycerol synthesis and decreasing glycerol permeability, thereby accumulating cytoplasmic glycerol up to molar concentrations (2, 3).
We isolated osmoregulation-defective mutants of yeast (4) by first screening mutagenized cells for the failure to grow on high-osmolarity medium [YEPD (1% yeast extract, 2% bactopeptone, 2% dextrose) supplemented with 0.9 M NaCl or 1.5 M sorbitol]. Mutants that grew well on YEPD but not on high-osmolarity medium (Osm sup S ) were then assayed for cellular glycerol accumulation 1 hour after the addition of 0.4 M NaCl to the medium (3). Osm sup S mutants with a reduction in the glycerol response were all recessive and fell into one of four complementation groups, identifying four HOG (high osmolarity glycerol response) genes, HOG1 to HOG4. Of this collection of mutants, we further analyzed two mutants, hog1-1 and hog4-1. The reduced glycerol response and Osm sup S of hog1-1 and hog4-1 cosegregated 2:2 in tetrads resulting from a backcross to wild type and are thus the result of a single mutation.
Genomic DNA fragments were cloned (5) that complemented the Osm sup S phenotype of hog1-1 and hog4-1, respectively. To locate HOG1 and HOG4 on each genomic clone, we generated subclones and tested for complementation of the Osm sup S phenotype of the respective hog mutant (6). The chromosomal locus of each clone was marked with a selectable marker and shown to be tightly linked to the original hog mutation (7), demonstrating that HOG1 and HOG4 (or closely linked genes) had been cloned.
The nucleotide sequence of the hog1-1--complementing DNA (8) revealed that HOG1 (GenBank accession number L06279) is a member of the MAP (mitogen-activated protein) kinase family (9). The HOG1 sequence contains a single, large open reading frame of 1.2 kb encoding a 416-amino acid protein with a molecular size of 47 kD. Northern (RNA) blot hybridization with the...