Ornithine decarboxylase in <i>Saccharomyces cerevisiae</i>: Chromosomal assignment and genetic mapping of the <i>SPE1</i> gene

Xie, Qiao-wen; Tabor, Celia White; Tabor, Herbert

doi:10.1002/yea.320060602

Cited by 13 publications

(3 citation statements)

References 16 publications

(16 reference statements)

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“…To obtain a yeast strain containing a larger amount of ornithine decarboxylase, the SPE1 gene was inserted into plasmid pVT101U obtained from T. Vernet (9). For this construction, we digested the 2,955-bp fragment from pSPE1U (10) with SstI and HindIII (partial) and inserted the resultant fragment into the multicloning site of plasmid pVT101U. With this plasmid (pVT101U͞SPE1), the transcription of the SPE1 gene is driven by an alcohol dehydrogenase promoter, resulting in a 20-to 200-fold overexpression of ornithine decarboxylase ''constitutively'' (9).…”

Section: Methodsmentioning

confidence: 99%

Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae

Gupta¹,

Hamasaki-Katagiri²,

Tabor³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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As part of our studies on the regulation of polyamine biosynthesis in Saccharomyces cerevisiae, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism. We have found that in S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in in vitro experiments or in the S. cerevisiae genome.T he regulation of ornithine decarboxylase has been studied extensively in a variety of cells and has been shown to involve a very unusual regulatory mechanism. In this system, a specific and unusual protein, called antizyme, is synthesized in response to spermidine͞spermine administration. This protein not only inhibits ornithine decarboxylase but also is important in preparing the ornithine decarboxylase protein for degradation by proteasomes without ubiquitination (reviewed in refs. 1-3). A similar protein has been shown recently to be synthesized in Schizosaccharomyces pombe after the administration of spermidine (4, 5). However, no direct evidence for the presence of an antizyme-like homologue has been demonstrated in Saccharomyces cerevisiae, despite numerous attempts by a number of investigators, nor has an antizyme homologue been found in the sequence of the S. cerevisiae genome (6). In view of this difference, we have been interested in extending our studies on the factors involved in the regulation of ornithine decarboxylase by spermidine in S. cerevisiae, and, in particular, on the factors involved in the degradation of the enzyme, especially because in S. cerevisiae, regulation of the rate of degradation of ornithine decarboxylase seems to be more important than regulation of its biosynthesis (7,8). Materials and MethodsSubcloning of the Yeast SPE1 Gene into a Yeast Constitutive Expression Vector. To obtain a yeast strain containing a larger amount of ornithine decarboxylase, the SPE1 gene was inserted into plasmid pVT101U obtained from T. Vernet (9). For this construction, we digested the 2,955-bp fragment from pSPE1U (10) with SstI and HindIII (partial) and inserted the resultant fragment into the multicloning site of plasmid pVT101U. With this plasmid ...

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Section: Methodsmentioning

confidence: 99%

Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae

Gupta¹,

Hamasaki-Katagiri²,

Tabor³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…We treated pSPE1U (19) water, and resuspended in an equal volume of water. These cells were diluted in H medium (2) containing the necessary supplements to give an initial optical density of 0.01 (see Fig.…”

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confidence: 99%

The presence of an active S-adenosylmethionine decarboxylase gene increases the growth defect observed in Saccharomyces cerevisiae mutants unable to synthesize putrescine, spermidine, and spermine

et al. 1994

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Saccharomyces cerevisiae spe1 delta SPE2 mutants (lacking ornithine decarboxylase) and spe1 delta spe2 delta mutants (lacking both ornithine decarboxylase and S-adenosylmethionine decarboxylase) are equally unable to synthesize putrescine, spermidine, and spermine and require spermidine or spermine for growth in amine-free media. The cessation of growth, however, occurs more rapidly in spe1 delta SPE2 cells than in SPE1 spe2 delta or spe1 delta spe2 delta cells. Since spe1 delta SPE2 cells can synthesize decarboxylated adenosylmethionine (dcAdoMet), these data indicate that dcAdoMet may be toxic to amine-deficient cells.

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“…Briefly, yeast ODC was subcloned from pSPE1U containing the SPE1 gene of Saccharomyces cerevisiae (15) into the modified phagemid pTZ19U (16) into the Nde I- Sma I sites. The resulting plasmid was used to transform E. coli polyamine mutant (HT252).…”

Section: Methodsmentioning

confidence: 99%

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: Purification and characterization of the inhibitory complex

Chattopadhyay

Fernández

Sharma

et al. 2011

Biochemical and Biophysical Research Communications

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Saccharomycescerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC: AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC: AZ complex. The far UV CD spectrum of the full-length antizyme shows that the yeast protein consists of 51% β-sheet, 19% α-helix, and 24% coils. Surface plasmon resonance analyses show that the association constant (K A ) between yeast AZ and yeast ODC is 6× 10 7 (M −1 ). Using purified His-tagged AZ as a binding partner, we have purified the ODC:AZ inhibitory complex. The isolated complex has no ODC activity. The molecular weight of the complex is 90 kDa, which indicates a one to one stoichiometric binding of AZ and ODC in vitro. Comparison of the circular dichroism (CD) spectra of the two individual proteins and of the ODC: AZ complex shows a change in the secondary structure in the complex.

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Ornithine decarboxylase in Saccharomyces cerevisiae: Chromosomal assignment and genetic mapping of the SPE1 gene

Cited by 13 publications

References 16 publications

Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae

Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae

The presence of an active S-adenosylmethionine decarboxylase gene increases the growth defect observed in Saccharomyces cerevisiae mutants unable to synthesize putrescine, spermidine, and spermine

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: Purification and characterization of the inhibitory complex

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