Ornithine decarboxylase gene of Neurospora crassa: isolation, sequence, and polyamine-mediated regulation of its mRNA.

Ornithine decarboxylase (ODC), which initiates the biosynthesis of the polyamines putrescine, spermidine, and spermine, is encoded by the spe-1 gene of the fungus Neurospora crassa. This gene and its cDNA have been cloned and sequenced. The gene has a single 70-nucleotide intron in the coding sequence. The cDNA, comprising the entire coding region, recognizes a single 2.4-kb mRNA in Northern (RNA) blots. The mRNA transcript, defined by S1 mapping, has an extremely long, 535-base leader without strong secondary-structure features or an upstream reading frame. The translational start of the protein is ambiguous: a Met-Val-Met sequence precedes the Pro known to be the N terminus of the ODC polypeptide. The polypeptide encoded by the N. crassa spe-1 gene (484 amino acids) has 46% amino acid identity with that of Saccharomyces cerevisiae (466 amino acids) and 42% with that of mouse (461 amino acids). Alignment of the longer N. crassa sequence with S. cerevisiae and mouse sequences creates gaps in different sites in the S. cerevisiae and mouse sequences, suggesting that N. crassa ODC is closer to an ancestral form of the enzyme than that of either yeast or mouse ODC. N. crassa ODC, which turns over rapidly in vivo in the presence of polyamines, has two PEST sequences, found in most ODCs and other proteins with rapid turnover. In striking contrast to other eucaryotic organisms, the variation in the rate of ODC synthesis in response to polyamines in N. crassa is largely correlated with proportional changes in the abundance of ODC mRNA. Spermidine is the main effector of repression, while putrescine has a weaker effect. However, putrescine accumulation appears to increase the amount of active ODC that is made from a given amount of ODC mRNA, possibly by improving its translatability. Conversely, prolonged starvation for both putrescine and spermidine leads to the differentially impaired translation of ODC mRNA.

Polyamine metabolism and growth of neurospora strains lacking Cis-acting control sites in the ornithine decarboxylase gene.

Ornithine decarboxylase (ODC) initiates the synthesis of polyamines (putrescine, spermidine, and spermine) and is highly regulated. We wished to know the importance of the control of ODC synthesis to the rates of growth and polyamine synthesis in the fungus, Neurospora crassa. We identified two control sites of the spe-1 gene, encoding ODC. One was an upstream activation region (UAR) and the other was the DNA encoding the long ODC mRNA leader, which governs polyamine-mediated repression of enzyme synthesis. Transformants receiving copies of spe-1 sequences lacking the UAR compensated for the deficiency by derepression or enzyme stabilization; polyamine synthesis was almost normal. A transformant lacking the spe-1 mRNA leader DNA constitutively expressed ODC mRNA and ODC activity, and synthesized excessive putrescine, especially when provided exogenous ornithine. This transformant grew normally and had only mildly elevated pools of spermidine, the major polyamine of this organism. We conclude that ODC activity normally limits polyamine synthesis, and ornithine becomes limiting in the ODC-constitutive strain. In this strain, however, spermidine synthesis remains rigorously limited by another step of the pathway, as yet unidentified. Thus the control of ODC activity in Neurospora is not vital to growth in laboratory culture, and synthesis of toxic levels of spermidine is limited by other mechanisms.