Isolation and characterization of mutations in the HXK2 gene of Saccharomyces cerevisiae.
Several hundred new mutations in the gene (HXK2) encoding hexokinase II of Saccharomyces cerevisiae were isolated, and a subset of them was mapped, resulting in a fine-structure genetic map. Among the mutations that were sequenced, 35 were independent missense mutations. The mutations were obtained by mutagenesis of cloned HXK2 DNA carried on a low-copy-number plasmid vector and screened for a number of different phenotypes in yeast strains bearing chromosomal hxk1 and hxk2 null mutations. Some of these mutants were characterized both in vivo and in vitro; they displayed a wide spectrum of residual hexokinase activities, as indicated by three assays: in vitro enzyme activity, ability to grow on glucose and fructose, and ability to repress invertase production when growing on glucose. Of those that failed to support growth on fructose, only a small minority made normal-size, stable, and inactive protein. Analysis of the amino acid changes in these mutants in light of the crystallographically determined three-dimensional structure of hexokinase II suggests important roles in structure or catalysis for six amino acid residues, only two of which are near the active site.
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Biology Department, Massachusetts Institute of Technology, Cambridge 02139.
Bloom
L M
LM
Zhu
Z M
ZM
Walsh
C T
CT
Botstein
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D000596:Amino Acids / Q000032:analysis; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005838:Genotype; D006593:Hexokinase / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D000596:Amino Acids; D007527:Isoenzymes; D006593:Hexokinase