Abstract
To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme. Four mutants, PE4, PE7, PE69, and PE85, were isolated. They were able to grow slowly at 25 degrees C on minimal medium but required putrescine or spermidine supplementation for growth at 35 degrees C. The mutants did not complement with one another or with ODC-less spe-1 mutants isolated in earlier studies. In all of the mutants isolated to date, the mutations map at the spe-1 locus on linkage group V. Strains carrying mutations PE4, PE7, and PE85 displayed a small amount of residual ODC activity in extracts. None of them had a temperature-sensitive enzyme. The enzyme of the PE85 mutant had a 25-fold higher Km for ornithine (5mM) than did the enzyme of wild-type or the PE4 mutant (ca. 0.2 mM). The enzyme of this mutant was more stable to heat than was the wild-type enzyme. These characteristics were normal in the mutant carrying allele PE4. The mutant carrying PE85 was able to grow well at 25 degrees C and weakly at 35 degrees C with ornithine supplementation. This mutant and three ODC-less mutants isolated previously displayed a polypeptide corresponding to ODC in Western immunoblots with antibody raised to purified wild-type ODC. We conclude that spe-1 is the structural gene for the ODC.
Highlights
To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme
ODC in Neurospora crassa is regulated over a large range of activity [20], as it is in most organisms
Our results clearly indicate that the spe-l locus is the structural gene for ODC
Summary
To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme. The enzyme of this mutant was more stable to heat than was the wild-type enzyme These characteristics were normal in the mutant carrying allele PE4. The mutant carrying PE85 was able to grow well at 25°C and weakly at 35°C with ornithine supplementation This mutant and three ODC-less mutants isolated previously displayed a polypeptide corresponding to ODC in Western immunoblots with antibody raised to purified wild-type ODC. It was not known whether this mutant and two others isolated previously [18] were structural gene mutants. We attempted here to define the structural gene for ODC by isolating mutants with kinetically altered enzyme and by defining immunologically the cross-reacting material in ODC-less mutants
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