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Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica
In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp)...
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2006
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464398/ https://www.ncbi.nlm.nih.gov/pubmed/16733544 http://dx.doi.org/10.1371/journal.ppat.0020048 |
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author | Bracha, Rivka Nuchamowitz, Yael Anbar, Michael Mirelman, David |
author_facet | Bracha, Rivka Nuchamowitz, Yael Anbar, Michael Mirelman, David |
author_sort | Bracha, Rivka |
collection | PubMed |
description | In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development. |
format | Text |
id | pubmed-1464398 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-14643982006-05-26 Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica Bracha, Rivka Nuchamowitz, Yael Anbar, Michael Mirelman, David PLoS Pathog Research Article In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development. Public Library of Science 2006-05 2006-05-26 /pmc/articles/PMC1464398/ /pubmed/16733544 http://dx.doi.org/10.1371/journal.ppat.0020048 Text en © 2006 Bracha et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Bracha, Rivka Nuchamowitz, Yael Anbar, Michael Mirelman, David Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica |
title | Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica
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title_full | Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica
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title_fullStr | Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica
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title_full_unstemmed | Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica
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title_short | Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica
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title_sort | transcriptional silencing of multiple genes in trophozoites of entamoeba histolytica |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464398/ https://www.ncbi.nlm.nih.gov/pubmed/16733544 http://dx.doi.org/10.1371/journal.ppat.0020048 |
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