Molecular cloning and characterization of ornithine decarboxylase cDNA of the nematode <i>Panagrellus redivivus</i>

Besser, H von; Niemann, George; Domdey, B; Walter, Rolf D.

doi:10.1042/bj3080635

Cited by 10 publications

(10 citation statements)

References 36 publications

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“…Genomic DNA was isolated from a mixed-stage population of worms, essentially by the method of Jowett [18], cleaved with restriction enzymes (Boehringer Mannheim) and sizefractionated in a 0.9 % agarose gel. The DNA was blotted on to nylon membranes (GeneScreen Plus ; New England Nuclear, Bad Homburg, Germany) and probed with a 0.9 kb genomic P. redi i us PCR fragment generated as previously described [17]. Standard hybridization conditions for GeneScreen Plus membranes [5iSSC, 1 % (w\v) SDS, 50 % formamide, 5i Denhardt's solution, 100 µg of tRNA] were performed overnight at 42 mC in the presence of at least 100 ng of $#P-labelled DNA at 5i10& c.p.m.…”

Section: Dna Purification Restriction Analyses Southern Blot Isolamentioning

confidence: 99%

“…Sequence-specific primers based on the cDNA sequence of P. redi i us ODC (P-ODC 18 ; [17]) were used to amplify the complete coding region of the ODC cDNA. The 5h oligonucleotide possessed a BglII restriction site and the 3h antisense oligonucleotide possessed an EcoRI restriction site.…”

Section: Production and Characterization Of Recombinant P Redivivus mentioning

confidence: 99%

“…To establish a model organism for nematodes, we investigated the polyamine biosynthesis of the free-living nematode Panagrellus redi i us, which can be maintained under axenic culture conditions. The ODC cDNA of P. redi i us has been isolated previously [17]. Here we report the structure of the ODC gene and the biochemical properties of the recombinant ODC expressed in Escherichia coli.…”

Section: Introductionmentioning

confidence: 97%

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Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Niemann

Besser

Walter

1996

Biochemical Journal

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A southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5' non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni(2+)-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mumol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for L-ornithine was determined as 44 microM. The Ki values for putrescine, alpha-diffluoromethylornithine, alpha-hydrazino-ornithine and alpha-methylornithine were calculated as 51, 34, 0.34 and 42 microM respectively.

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Section: Dna Purification Restriction Analyses Southern Blot Isolamentioning

confidence: 99%

Section: Production and Characterization Of Recombinant P Redivivus mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

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Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Niemann

Besser

Walter

1996

Biochemical Journal

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“…ODCs from C. fasciculata and L. donovani (19) consist of 721 and 707 aa, respectively, whereas the enzyme from mammals (36), yeast (37), T. brucei (16,29), Neurospora crassa (38), Xenopus laevis (39), and the nematode Panarellus redividus (40) contains between 423 and 484 aa. Comparison of the amino acid sequence for ODC from C. fasciculata with those of other eukaryotic species reveals several conserved regions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cloning of a trypanosomatid gene coding for an ornithine decarboxylase that is metabolically unstable even though it lacks the C-terminal degradation domain

Svensson

Ceriani²,

Wallström³

et al. 1997

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

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Mammalian ornithine decarboxylase (ODC) is among the most labile of cellular proteins, with a half-life of usually less than an hour. Like other short-lived proteins ODC is degraded by the 26S proteasome. Its degradation is not triggered by ubiquitination, but is stimulated by the binding of an inducible protein, antizyme. Truncations and mutations in the C terminus of mammalian ODC have been shown to prevent the rapid turnover of the enzyme, demonstrating the presence of a degradation signal in this region. Moreover, ODCs from the trypanosomatid parasites Trypanosoma brucei and Leishmania donovani, which lack this Cterminal domain, are metabolically stable, and recombination of T. brucei ODC with the C terminus of mammalian ODC confers a short half-life to the fusion protein when expressed in mammalian cells. In the present study we have cloned and sequenced the ODC gene from the trypanosomatid Crithidia fasciculata. To our knowledge, this is the first protozoan shown to have an ODC with a rapid turnover. The sequence analysis revealed a high homology between C. fasciculata ODC and L. donovani ODC, despite the difference in stability. We demonstrate that C. fasciculata ODC has a very rapid turnover even when expressed in mammalian cells. Moreover, ODC from C. fasciculata is shown to lack the C-terminal degradation domain of mammalian ODC. Our findings indicate that C. fasciculata ODC contains unique signals, targeting the enzyme for rapid degradation not only in the parasite but also in mammalian cells.

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“…Parasitic nematodes appear to lack ODC and are mainly dependent on polyamine uptake and interconversion for their supply of polyamines (Walter 1988). It is remarkable that the cDNA for ODC has been cloned from the free-living nematode Panagrellus redivivus (Besser et al 1995), which resembles parasitic nematodes in overall structure and development, to study polyamines at the biochemical and molecular level. The sequence of the isolated odc gene shows 32% amino acid identity to odc genes from invertebrates and lacks the PEST sequence.…”

Section: Spermidine Metabolism In Nematodesmentioning

confidence: 99%

Spermidine metabolism in parasitic protozoa - a comparison to the situation in prokaryotes, viruses, plants and fungi

Kaiser¹,

Gottwald²,

Wiersch³

et al. 2003

FOLIA PARASIT

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Abstract. Targeting polyamines of parasitic protozoa in chemotherapy has attracted attention because polyamines might reveal novel drug targets for antiparasite therapies (Müller et al. 2001). The biological function of the triamine spermidine in parasitic protozoa has not been studied in great detail although the results obtained mainly imply three different functions, i.e., cell proliferation, cell differentiation, and biosynthesis of macromolecules. Sequence information from the malaria genome project databases and inhibitor studies provide evidence that the current status of spermidine research has to be extended since enzymes of spermidine metabolism are present in the parasite (Kaiser et al. 2001). Isolation and characterisation of these enzymes, i.e., deoxyhypusine synthase (EC 1.1.1.249) (DHS) and homospermidine synthase (EC 2.5.1.44) (HSS) might lead to valuable new targets in drug therapy. Currently research on spermidine metabolism is based on the deposition of the deoxyhypusine synthase nucleic acid sequence in GenBank while the activity of homospermidine synthase was deduced from inhibitor studies. Spermidine biosynthesis is catalyzed by spermidine synthase (EC 2.5.1.16) which transfers an aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine. Spermidine is also an important precursor in the biosynthesis of the unusual amino acid hypusine and the uncommon triamine homospermidine in eukaryotes, in particular in pyrrolizidine alkaloid-producing plants (Ober and Hartmann 2000). Hypusine is formed by a two-step enzymatic mechanism starting with the transfer of an aminobutyl moiety from spermidine to the ε-amino group of one of the lysine residues in the precursor protein of eukaryotic initiation factor eIF5A by DHS (Lee and Park 2000). The second step of hypusinylation is completed by deoxyhypusine hydroxylase (EC 1.14.9929) (Abbruzzese et al. 1985). Homospermidine formation in eukaryotes parallels deoxyhypusine formation in the way that in an NAD + -dependent reaction an aminobutyl moiety is transferred from spermidine. In the case of homospermidine synthase, however the acceptor is putrescine. Thus the triamine homospermidine consists of two symmetric aminobutyl moieties while there is one aminobutyl and one aminopropyl moiety present in spermidine. Here, we review the metabolism of the triamine spermidine with particular focus on the biosynthesis of hypusine and homospermidine in parasitic protozoa, i.e., Plasmodium, Trypanosoma and Leishmania, compared to that in prokaryotes i.e., Escherichia coli, a phytopathogenic virus and pyrrolizidine alkaloid-producing plants (Asteraceae) and fungi.

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Molecular cloning and characterization of ornithine decarboxylase cDNA of the nematode Panagrellus redivivus

Cited by 10 publications

References 36 publications

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

Cloning of a trypanosomatid gene coding for an ornithine decarboxylase that is metabolically unstable even though it lacks the C-terminal degradation domain

Spermidine metabolism in parasitic protozoa - a comparison to the situation in prokaryotes, viruses, plants and fungi

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