All attempts to identify ornithine decarboxylase in the human pathogen Trypanosoma cruzi have failed. The parasites have instead been assumed to depend on putrescine uptake and S-adenosylmethionine decarboxylase (AdoMetDC) for their synthesis of the polyamines spermidine and spermine. We have now identified the gene encoding AdoMetDC in T. cruzi by PCR cloning, with degenerate primers corresponding to conserved amino acid sequences in AdoMetDC proteins of other trypanosomatids. The amplified DNA fragment was used as a probe to isolate the complete AdoMetDC gene from a T. cruzi genomic library. The AdoMetDC gene was located on chromosomes with a size of approx. 1.4 Mbp, and contained a coding region of 1110 bp, specifying a sequence of 370 amino acid residues. The protein showed a sequence identity of only 25% with human AdoMetDC, the major differences being additional amino acids present in the terminal regions of the T. cruzi enzyme. As expected, a higher sequence identity (68-72%) was found in comparison with trypanosomatid AdoMetDCs. When the coding region was expressed in Escherichia coli, the recombinant protein underwent autocatalytic cleavage, generating a 33-34 kDa alpha subunit and a 9 kDa beta subunit. The encoded protein catalysed the decarboxylation of AdoMet (Km 0.21 mM) and was stimulated by putrescine but inhibited by the polyamines, weakly by spermidine and strongly by spermine. Methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of human AdoMetDC, was a poor inhibitor of the T. cruzi enzyme. This differential sensitivity to MGBG suggests that the two enzymes are sufficiently different to warrant the search for compounds that might interfere with the progression of Chagas' disease by selectively inhibiting T. cruzi AdoMetDC.
The degradation of ornithine decarboxylase (ODC) is mediated by antizyme, a protein regulated by the end-products of ODC activity, the polyamines. High levels of polyamines induce a ϩ1 ribosomal frameshift in the translation of the rat antizyme message leading to the expression of a full-length protein.We have studied whether the regulation of antizyme expression occurs only at the level of translation or whether polyamine levels also affect the transcription of the antizyme gene. Thus, we have cloned and sequenced the mouse homologues of the rat ODC-antizyme gene and cDNA. Northern blot analysis shows that although high concentrations of polyamines do not affect the steady-state levels of antizyme message in L1210 leukemia cells, polyamine depletion using 2-(difluoromethyl)ornithine [Orn(F 2 Me)] leads to a marked decrease in mRNA levels. Results of transient transfections of luciferase-reporter-gene constructs driven by antizyme promoter fragments in untreated and Orn(F 2Me)-treated Balb/C 3T3 cells indicate that the transcription of the antizyme gene is altered upon polyamine depletion. The amount of antizyme protein on Western blots was also altered by polyamine depletion and addition, and the polysomal distribution of antizyme message suggests a general translational increase of the message when polyamine concentrations are high. These results indicate a role for polyamines in the transcriptional and translational regulation of ornithine decarboxylase antizyme.Keywords : polyamine ; ornithine decarboxylase; antizyme; translational frameshifting; transcription.Polyamines are polycations, which bind and interact with protein necessary for making the interaction and the degradation of ODC have been identified, but it is uncertain whether antinumerous molecules within a cell. They are present in all living zyme is degraded at the same time as ODC or whether it is cells, are essential for growth, and play important roles in differrecycled [12Ϫ17]. Recently another factor in the regulation of entiation of eukaryotic cells [1,2]. Cells acquire polyamines by ODC has been described, when the antizyme-inhibitor cDNA uptake from the exterior and/or synthesize them de novo. Orniwas cloned and sequenced [18]. This protein binds to antizyme, thine decarboxylase (ODC) is the key regulatory enzyme and with a much higher affinity than that of ODC [19]. The regulaone of the rate-limiting enzymes in the polyamine-biosynthesis tion of antizyme inhibitor has so far not been investigated. The pathway. It catalyses the conversion of ornithine into putrescine, finding that antizyme plays a role in the regulation of polyamine a diamine that is the precursor of the polyamines spermidine and uptake by an unknown mechanism adds further complexity to spermine [3]. ODC activity is induced by growth factors mostly the tightly regulated polyamine homeostasis [20, 21]. through increased transcription of the ODC gene [4]. ODC isThe level of antizyme mRNA in a cell is usually high even necessary for cell-cycle progression and over expression...
A genomic clone for a mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene was isolated from a cosmid library. Surprisingly, the gene proved to be intronless. With the exception of three base substitutions (changing 2 amino acids in the deduced protein), the 1002-nucleotide sequence of the open reading frame was identical to that of mouse AdoMetDC cDNA. Moreover, the gene contained a poly(dA) tract at the 3' end and was flanked by 13-base pair direct repeats. Our findings suggest that this gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the genome. By polymerase chain reaction, we positively identified the intronless gene in the mouse genome, and, by primer extension analysis, we proved the gene to be functional. Thus, its transcripts were found in many cell lines and tissues of the mouse and were particularly abundant in the liver. When the open reading frame of the intronless gene was expressed in Escherichia coli HT551, a strain with no AdoMetDC activity, it was found to encode a 38-kDa protein, corresponding to AdoMetDC proenzyme. Although the change of methionine 70 to isoleucine was close to the cleavage site at serine 68, this protein underwent proenzyme processing, generating a 31-kDa alpha subunit and an 8-kDa beta subunit. Importantly, the protein encoded by the intronless gene was functional, i.e. it catalyzed the decarboxylation of S-adenosylmethionine, and its specific activity was comparable with that of recombinant human AdoMetDC purified according to the same procedure.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of polyamines. We have previously identified a mouse AdoMetDC gene that exhibits the hallmarks of a retroposon; that is, it has no introns, is flanked by direct repeats, and has a poly(dA) tract at its 3'-end. This gene, termed Amd-2, is not a processed pseudogene; rather, it is transcribed in a variety of mouse tissues and encodes a functional enzyme. In the current report, we present the sequence of a 6.7-kb genomic segment of the Amd-2 locus. Several sequences of interest, including an intercisternal A particle (IAP) element, a transposon-related sequence, and several expressed sequence tags (ESTs), were found within or near Amd-2. We also show, through analysis of an interspecific backcross, that Amd-2 is located on Chr 12, tightly linked to the gene (Odc) that encodes ornithine decarboxylase, another key enzyme in polyamine synthesis. Finally, we show that Amd-2 is present among several divergent species of the genus Mus. Thus, the integration event that generated Amd-2 may have occurred early during Mus evolution.
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