Isolation and characterization of a Saccharomyces cerevisiae mutant with a disrupted gene for the IP subunit of succinate dehydrogenase.
A partial cDNA clone corresponding to the iron-sulfur protein of succinate dehydrogenase (EC 1.3.99.1) has been isolated by an application of the polymerase chain reaction (Gould, S. J., Subramani, S., and Scheffler, I. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1934-1938). We used this clone for targeted gene disruption to isolate clones of Saccharomyces cerevisiae which were totally defective in this enzyme. The disruption of the gene was verified by Southern analysis. A Northern analysis revealed the existence of a new transcript which includes the 5' end of the mRNA for the IP subunit, but extends into the vector sequence disrupting the gene. A Western analysis suggests that the large flavoprotein subunit (FP) is still made in the mutant and imported into the mitochondria, but at significantly lower levels.
Lombardo
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Department of Biology, University of California, San Diego 92093.
Scheffler
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D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D008928:Mitochondria / Q000201:enzymology; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D013385:Succinate Dehydrogenase / Q000235:genetics*
D004271:DNA, Fungal; D012331:RNA, Fungal; D013385:Succinate Dehydrogenase