ISOLATION, CHARACTERIZATION, AND DISRUPTION OF THE YEAST GENE ENCODING CYTOSOLIC NADP-SPECIFIC ISOCITRATE DEHYDROGENASE

The cytosolic isozyme of NADP-specific isocitrate dehydrogenase (IDP2) was purified from a Saccharomyces cerevisiae mutant containing a chro mosomal disruption in the gene encoding the mitochondrial isozyme (IDP 1). IDP2 was shown to be a homodimer with a subunit molecular weight o f approximately 45 000 and an isoelectric point of 5.5. Amino acid seq uences were obtained for tryptic peptides of IDP2 and used to plan pol ymerase chain reactions. A resulting 400 bp DNA fragment was used as a hybridization probe to isolate the IDP2 gene from a yeast genomic DNA library. The complete nucleotide sequence of the IDP2 coding region w as determined and translated into a 412-residue amino acid sequence. I DP2 and IDP1 were found to be identical in 71% of the aligned residue positions. The identity of the IDP2 gene was confirmed by genomic repl acement with a disrupted IDP2 coding region. Haploid yeast strains lac king either or both IDP2 and IDP1 were constructed by genetic crosses of mutant strains containing disruptions in chromosomal IDP2 and IDP1 loci. No dramatic differences in growth rates with common carbon sourc es could be attributed to these disruptions.