Year-end Sale % OFF 
Abstract
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.
Highlights
Epigenetic gene silencing is a heritable change in gene expression that occurs without a change in nucleotide sequence
The down-regulation of the SAPLIP 1-encoding gene was first noted by Dr Upi Singh (Stanford University, United States), following hybridizations of genomic microarrays with cDNA produced from mRNA of G3 trophozoites as compared to that of HM-1:IMSS trophozoites
The transcription levels of various SAPLIPs as well as other housekeeping genes was examined by RT-PCR, and as shown in Figure 1A the transcripts of Entamoeba histolytica amoebapore A gene (Ehap-a), Ehap-b, and SAPLIP 1 were downregulated in the silenced G3 trophozoites, while others, such as Ehap-c, SAPLIP 5, and SAPLIP 14 showed transcription levels simliar to those in the parent strain HM-1:IMSS
Summary
Epigenetic gene silencing is a heritable change in gene expression that occurs without a change in nucleotide sequence. TGS has been shown to occur in plants following transfection with plasmids containing a transgene promoter region without the transcribed sequences [6]. Epigenetic silencing of the Entamoeba histolytica amoebapore A gene (Ehap-a) occurred following transfection of trophozoites of virulent strain HM-1:IMSS with a hybrid plasmid containing a 59 upstream region (473 bp) of the Ehap-a gene [12]. Sequence analysis of the 473 bp upstream segment revealed that in addition to the promoter region of the Ehap-a gene, it included 140 bp of an adjacent, short interspersed nuclear element (SINE1) that is transcribed in the opposite orientation and contained a unique thymidine-rich stretch of 48 bp. E. histolytica has been shown to harbor non-long terminal repeats that are either long interspersed (LINE) or SINEs [13,14,15]. Other studies have shown that the transcriptional activity of some non-LTR transposable elements is sensitive to the presence of homologous transgenes and that the transcriptional activation of retrotransposons can alter the expression of adjacent genes in wheat as well as in mice [18,19,20]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
