Eukaryotic translation initiation factor 5A and its posttranslational modifications play an important role in proliferation and potentially in differentiation of the human enteric protozoan parasite Entamoeba histolytica
Fig 7
Silencing of EheIF5A2 caused growth retardation and up regulation of EheIF5A expression.
(A) Evaluation of gene expression by semi-quantitative RT-PCR analysis of EheIF5A2 gene silenced transformant. The steady-state levels of transcripts of EheIF5A1, EheIF5A2, and EhRNA pol II genes were measured in trophozoites of G3 strain transfected with either empty vector (psAP2G) or the EheIF5A2 gene silencing plasmid (psAP2G-eIF5A2). cDNA from the generated cell lines (psAP2G and EheIF5A2gs strains) was subjected to 25 cycles of PCR using specific primers for the eIF5A1, eI5FA2 and RNA pol II genes. RNA polymerase II served as a control. PCR analysis of samples without reverse transcription was used to exclude the possibility of genomic DNA contamination. (B) Growth kinetics of EheIF5A2 gene-silenced (EheIF5A2gs) and control (pSAP2G) transformants. Approximately 60,000 amoebae in the logarithmic growth phase were inoculated into 6 ml fresh culture medium and amoebae were then counted every 24 hr. Data shown are the means ± standard deviations of five biological replicates. (C) An immunoblot analysis of EheIF5A2gs and control (pSAP2G) transformants. Total cell lysates were electrophoresed on a 15% SDS-PAGE gel and subjected to an immunoblot assay with antibodies raised against EheIF5A1 and EheIF5A2.