Stable episomal transfection and gene expression in Entamoeba dispar

Moshitch-Moshkovitch, S; Stolarsky, Tamara; Mirelman, David; Alon, Rinat N.

doi:10.1016/s0166-6851(96)02766-1

Cited by 15 publications

(8 citation statements)

References 16 publications

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“…Similar amounts of actin mRNA were detected in all the samples (data not shown). As previously shown (Moshitch‐Moshkovitch et al ., 1996), the small amounts of Crithidia fasciculata cells, which are usually added to the E. dispar cultures, were not transfected under these conditions.…”

Section: Resultssupporting

confidence: 81%

Regulation of expression of ribosomal protein L‐21 genes of Entamoeba histolytica and E. dispar is at the post‐transcriptional level

Moshitch-Moshkovitch¹,

Petter²,

Levitan³

et al. 1998

Molecular Microbiology

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SummaryTwo genes, EhgLE3 and Ehg34, encoding the ribosomal protein L21 (rp-L21) were identified and characterized from Entamoeba histolytica. Their coding regions are highly conserved, but their flanking regions differ significantly. Analogous genes (EdgLE3 and Edg34) were characterized in E. dispar. The two rp-L21 copies are transcribed at similar levels in the two parasites. However, their relative binding to the polyribosomal complex during active translation is different. In E. histolytica, binding of EhgLE3 transcripts to the polyribosomes is significantly higher in comparison with that of Ehg34 transcripts, whereas in E. dispar the binding pattern is inverse. The importance of each of the rp-L21 flanking regions to gene translation was investigated by constructing hybrid plasmids containing the CAT reporter gene flanked by rp-L21 flanking regions. The plasmids were stably transfected into E. histolytica and E. dispar, and CAT mRNA and enzymatic activity levels were determined. All plasmids promoted transcription of CAT. Yet, in E. histolytica, high levels of CAT activity were observed only when gLE3 upstream regions flanked CAT. In contrast, in E. dispar, high levels of CAT activity were observed when g34 upstream regions flanked CAT. The downstream regions showed no significant effect on CAT translation.

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Section: Resultssupporting

confidence: 81%

Regulation of expression of ribosomal protein L‐21 genes of Entamoeba histolytica and E. dispar is at the post‐transcriptional level

Moshitch-Moshkovitch¹,

Petter²,

Levitan³

et al. 1998

Molecular Microbiology

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“…To name but a few, the signal for the initiation of the invasion process, the mechanism of elimination of the mucus barrier, the role of EDG, and the in vivo participation of the different molecules in this multifactorial process have not been demonstrated. The recent advances in amebic transfection technology (56,98,104,106,122,168) will undoubtedly shed new light on the involvement of specific molecules in the pathogenic mechanism. The uncertainty of the ploidy of E. histolytica has hindered the production of gene knockout clones like those produced for the study of the pathogenicity of Toxoplasma gondii and Leishmania spp.…”

Section: Discussionmentioning

confidence: 99%

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

Espinosa‐Cantellano¹,

Martı́nez-Palomo²

2000

Clinical Microbiology Reviews

191

115

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“…The pSA21 plasmid construct containing the sequence of interest was then digested with BamHI and SacI, which releases a cassette containing the sequence of interest flanked by 5Ј and 3Ј actin regulatory sequences. This cassette was then transferred to the pEhAct-Neo (38) vector, after removal of its cassette by digestion with BamHI and SacI.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR product obtained was digested with SacII and SalI and ligated to the 3Ј Ehactin flanking region that was obtained by digestion of the pSA21 (29) plasmid with SalI and BamHI. This cassette containing the 5Ј upstream segment of the Ehap-a gene, the full-length EhLimA sequence, and the 3Ј Ehactin flanking region was cloned into the pEhAct-Neo (38) vector digested at SacII and BamHI. The resulting plasmid was transfected into the G3 strain, and transfectants were selected as previously described (7).…”

Section: Methodsmentioning

confidence: 99%

EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane inEntamoeba histolytica

2007

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The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.Many cellular functions are dependent on specific proteinprotein interactions. These protein-protein interactions are governed by a variety of protein binding domains one of which is the LIM domain. Since first described nearly 2 decades ago (21, 28, 57), LIM domain-containing proteins have been identified in a wide range of eukaryotes, including protozoa (16,30,45), plants (5,20,40), and yeast (Saccharomyces cerevisiae) (39), stressing the evolutionary importance of these proteins. In the parasitic protozoan Entamoeba histolytica, a few studies have revealed the presence of such proteins (17, 36), although to date, no LIM domain-containing proteins have been characterized in this organism. The name LIM is derived from the three developmentally regulated transcription factors, LIN-11 of Caenorhabditis elegans (21), Isl1 of rat (28), and MEC-3 of C. elegans (57) in which the LIM domain was initially identified. The LIM domain is a cysteine-rich motif consisting of two zinc finger-like modules and displaying the consensus sequence CX 2 CX 16-23 HX 2 CX 2 CX 2 CX 16-21 CX 2 (C/H/D/) (31). This domain is found in a variety of different proteins with diverse functions, including transcription factors and cytoskeleton-associated proteins, and may be found in association with other functional domains, such as homeodomains, protein kinase domains, or other protein binding domains (...

show abstract

Stable episomal transfection and gene expression in Entamoeba dispar

Cited by 15 publications

References 16 publications

Regulation of expression of ribosomal protein L‐21 genes of Entamoeba histolytica and E. dispar is at the post‐transcriptional level

Regulation of expression of ribosomal protein L‐21 genes of Entamoeba histolytica and E. dispar is at the post‐transcriptional level

Pathogenesis of Intestinal Amebiasis: From Molecules to Disease

EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane inEntamoeba histolytica

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