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Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.

Pubmed ID: 1986231

Authors

  • Minard KI
  • McAlister-Henn L

Journal

Molecular and cellular biology

Publication Data

January 14, 1991

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM-33218

Mesh Terms

  • Amino Acid Sequence
  • Base Sequence
  • Blotting, Southern
  • Cloning, Molecular
  • Codon
  • DNA Mutational Analysis
  • Fungal Proteins
  • Gene Expression Regulation, Fungal
  • Genes, Fungal
  • Isoenzymes
  • Malate Dehydrogenase
  • Molecular Sequence Data
  • Molecular Weight
  • Restriction Mapping
  • Saccharomyces cerevisiae