Cloning, expression and characterization of the ornithine decarboxylase gene from Dictyostelium discoideum

Abstract

Ornithine decarboxylase (ODC) is a rate limiting enzyme in polyamine synthesis that decarboxylates ornithine to form the diamine putrescine. We report here the isolation, expression and characterization of a homolog of ODC from Dictyostelium discoideum. DdODC is conserved and shows sequence and structural homology with that from human. Both ODC transcript and protein are expressed at all stages of development and show high expression in prestalk/stalk cells. It is cytosolic and predominantly perinuclear in localization. Both overexpression of DdODC and putrescine treatment resulted in inhibition of cell proliferation. KEY WORDS: D. discoideum, ODC expression, putrescinePolyamines are ubiquitously present from bacteria to mammals Tabor 1985, Janne 2004) and regulate cell growth, proliferation, differentiation and cell death. The fast proliferating prokaryotes mostly contain putrescine and spermidine (Tabor and Tabor 1976) while the slow proliferating eukaryotes predominantly have spermidine and spermine (Fillingame 1975). A decrease in intracellular polyamines occurs during aging (Minois et al., 2011) but it still remains unclear whether this is a cause or consequence of aging. Addition of exogenous spermidine can extend lifespan in various animal models through epigenetic modifications, induction of autophagy and suppression of necrosis (Eisenberg et al., 2009).Ornithine decarboxylase (ODC, EC 4.1.1.17) catalyses the first and the rate limiting step of polyamine biosynthesis where Lornithine is converted to putrescine (Heller et al., 1976). It is strictly regulated during development (Heby and Persson 1990) and is involved in synthesis of spermidine and spermine. Decarboxylation of ornithine by ODC is the only pathway for de novo synthesis of polyamines in mammals and higher eukaryotes. The ability to strictly regulate cellular polyamine levels within a very narrow range is critical since extreme levels of polyamines (high or low) result in damaging effects, both on life of the cell and organism as a whole (Pegg and McCann 1982).This study was undertaken to understand the role of putrescine, which is the most abundant polyamine found in Dictyostelium discoideum (Saran 1998 Abbreviations used in this paper: DdODC, Dictyostelium discoideum ODC; ODC, ornithine decarboxylase.grow and divide mitotically but upon starvation and in response to the chemoattractant, cAMP, they enter the developmental phase to ultimately form a fruiting body consisting of dead vacuolated stalk cells and viable spores. Since ODC is responsible for the formation of putrescine we characterized this enzyme. Harris and North (1982) did not find any significant ODC activity during development but Campagne and Lowik (1985) have reported high activity at the slug stage. Our earlier report (Saran 1998) has shown high putrescine levels during slug stage. This is the first report on molecular cloning, expression and characterization of a putative ODC-like sequence from D. discoideum. We found it to be structurally conserved and predomina...

Identification and Cloning of S-Adenosylmethionine Decarboxylase (Samdc) Gene From Dictyostelium Discoideum.

“…Growth and development of Dictyostelium discoideum:-D. discoideum Ax2 (axenic strain) cells were grown in HL5 (axenic) medium at 22°C under shaken conditions (200 rpm) 1 . Nearly after 40 hours of growth, the cells reached a log phase (~3-5x10 6 cells/mL) after which they were collected and plated for development.…”

“…Development was also carried after treatment with 20 mM putrescine 1 . 10 or 20 μL drops of cells at a density of 1x10 7 cells/mL were spotted on non-nutrient agar plates 1 .…”

“…10 or 20 μL drops of cells at a density of 1x10 7 cells/mL were spotted on non-nutrient agar plates 1 . Construction of the overexpressing strain [act15/samdc-eyfp]/Ax2:-samdc (DDB_DDB0237590) gene was PCR amplified from Ax2 genomic DNA.…”

“…To get a deeper insight into the role of spermidine we have in the present communication identified and expressed the S-adenosylmethionine decarboxylase (samdc) gene, which is involved in the biosynthesis of the polyamines, spermidine and spermine. We also measured the levels of samdc mRNA during development in wild type cells, odc overexpressing cells and after putrescine treatment, which we have earlier reported to increase the spermidine levels 1 .…”

“…We have exploited the model organism, Dictyostelium discoideum to understand the role of various polyamines during growth and development. In our earlier publication 1 , we have identified and expressed ornithine decarboxylase (odc) gene from D. discoideum and found putrescine to be more important for growth but not development. On the other hand, we also found the importance of spermidine during development but not during growth.…”