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

Abstract

Ornithine decarboxylase (ODC), which initiates the biosynthesis of the polyamines putrescine, spermidine, and spermine, is encoded by the spe-l 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 SI 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-l 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.Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to form putrescine, the first step in polyamine biosynthesis leading to spermidine and spermine formation (Fig. 1). In most organisms, ODC is a dimer of 52,000-to 55,000-Da subunits, the activity of which is highly regulated in response to growth stimuli and to the polyamines themselves (31,50,62,63). The amplitude, the speed, and the unusual mechanisms of regulation of the enzyme suggest to many researchers that ODC and the polyamines have a special relationship to growth. Accordingly, many molecular studies of this enzyme in organisms ranging from bacteria to humans have appeared in the last few years (3,5,22,28,37).The most conspicuous features of ODC regulation in mammals in response to polyamines are the control of ODC synthesis without a change in ODC mRNA levels and the polyamine-induced increase in the turnover rate of the enzyme (31). In the fungus Neurospora crassa, the regulatory responses of ODC at the level of enzyme protein are the same as in many mammalian systems (3,16). In this study, we describe the sequence of the cloned OD...

Ornithine Decarboxylase of Stagonospora (Septoria) nodorum Is Required for Virulence toward Wheat

“…ODC Gene Isolation-Degenerate oligonucleotides designed against conserved amino acids within the ODC proteins of human (32), yeast (16), N. crassa (17), and Datura otramanium (33) were used as PCR primers to amplify an internal fragment of the S. nodorum ODC gene. A product of 609 bp was obtained using S. nodorum DNA as a template and this was cloned into pBluescript SK Ϫ (Stratagene) and sequenced to confirm its identity.…”

“…The ability to disrupt chromosomal gene loci in haploid fungi is well established and allows a direct test of gene function in growth and pathogenicity. ODC genes have been isolated and disrupted in yeast (16), Neurospora crassa (17), and Ustilago maydis (18). Yeast and N. crassa are non-pathogens and disruption in U. maydis prevents formation of the diploid state, which is a pre-requisite for pathogenicity.…”

Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA

“…The predicted amino acid sequence of the plant ODC was aligned with ADC of oat [21], ADC of tomato [36], ADC of E. coli [38], ODC of yeast [51], ODC of Neurospora crassa [52], ODC of human [53], ODC of mouse [54], ODC of Xenopus [55], ODC of Trypanosoma brucei [56], ODC of Drosophila [57], ODC of Leishmania donovanii [58], DapDC of Pseudomonas aeruginosa [59] and the product of the tabA gene of Pseudomonas syringae cv. tabaci [41].…”

Selective inhibition of cytosine‐DNA methylases by polyamines

“…However, clear-cut examples that polyamines recognize selective sites in the DNA are rare. The most striking specific interactions of polyamines with nucleic acids occur during transcriptional and translational regulation of their own synthesis [23,24]. Additional examples of a certain degree of specific interaction between polyamines and DNA is the observation that some tricationic polyamines bind selectively to GC sequences [25], and a report describing the [28].…”