Project description:ChIP-seq experiments were performed for the putative telomere repeat-binding factor (PfTRF) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRF at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRF was enriched at seven additional, intra-chromosomal sites and called in the PfTRF-HA ChIP-seq only. Plasmodium falciparum 3D7 parasites were generated with -GFP or -3xHA C-terminal tagged TRF (PF3D7_1209300). Nuclei were isolated from formaldehyde cross-linked schizont-stage transgenic parasites and used to prepare chromatin. Chromatin immunoprecipitations were performed using mouse anti-GFP (Roche Diagnostics, #11814460001) or rat anti-HA 3F10 (Roche Diagnostics, #12158167001). Sequencing libraries were prepared according to a Plasmodium-optimized library preparation procedure including KAPA polymerase-mediated PCR amplification.
Project description:Purpose: this study is to analyze the change of overal transcriptome after disruption of DNA methyltransferase (DNMT) in Plasmodium falciparum. Methods: In this study, the transcriptomes of a PfDNMT gene knockout (KO) parasite line with its wildtype control, its complementation (adding back the DNMT expression by episomal expression of DNMT in the DNMT KO parasite), and overexpression (episomal expression of DNMT in the wildtype parasite) were analyzed by RNAseq. Total RNA were harvested from the asexual parasites at three developmental stages (ring, trophozoite, and schizont) using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Results: Using an optimized data analysis workflow, we mapped about 5 million sequence reads per sample to the malaria parasite genome (pf3D7_V3.0.) and identified 5712 transcripts with high mapping rate at the range between 80% and 95. PfDNMT KO profoundly disturbed the global transcription pattern,especially at trophozoite stage, causing 1732 (30.3%) genes to be differentially expressed at trophozoite.Complementation restored the expression pattern and overexpression of PfDNMT caused nearly 2000 gene ro be differentially expressed at schizont stage. Conclusions: Collectively, transcriptomic analysis of DNMT KO, complemetation and overexpression shows PfDNMT plays important role in gene regulation.
Project description:ChIP-seq experiments were performed for the putative Telomere Repeat-binding Zinc finger protein (PfTRZ) in the malaria parasite Plasmodium falciparum strain 3D7. The gene encoding this factor (PF3D7_1209300) was endogenously tagged with either a GFP- or a 3xHA-tag and these transgenic parasite lines were used in ChIP-sequencing experiments. Sequencing of the ChIP and input libraries showed enrichment of PfTRZ at all telomere-repeat containing chromosome ends (reference genome Plasmodium falciparum 3D7 from PlasmoDB version 6.1) as well as in all upsB var promoters.In addition,PfTRZ was enriched at seven additional, intra-chromosomal sites and called in the PfTRZ-HA ChIP-seq only.
Project description:We compared transcript levels at several points along the asexual blood cycle between 21 P. falciparum lines. These 21 parasite lines are organized in 4 sets of parasite lines, with all parasite lines within a set sharing a clonal origin. We compared transcript levels within each set. We also performed comparative genome hybridization (CGH) for all 21 parasite lines.
Project description:Liver stage of malaria parasite exports SLTRiP and PB268 to the cytosol of parasite infected host cell. To know the host genes perturbed by WT-PBANKA, SLTRiP-KO and PB268-KO parasite growth, we did transcriptomic sequencing of infected host cells. We did mRNA sequencing of four samples for comparative analysis of WT and PB-knockout parasites infected host cells at 22 hours of post sporozoites infection. mRNA profiles of Plasmodium PBANKA, PBSLTRiP-KO, PB268-KO parasite infected and uninfected HepG2 cells after 22hrs of sporozoites infections were generated by deep sequencing using Illumina GAIIx.
Project description:Purpose: this study is to analyze the change of RNA splicing events after disruption of Protein Arginine Methyltranferase 5 (PRMT5) in Plasmodium falciparum. Methods: In this study, the transcriptomes of a PfPRMT5 gene knockout (KO) parasite line with its wildtype control were analyzed by RNAseq.Total RNA were harvested from the asexual parasites at four stages (ring, early trophozoite, late trophozoite, and schizont)(12h, 24h, 36h, and 46h post-invasion) using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Results: This RNAseq analysis with strand-specific mRNA libraries from both ΔPfPRMT5 and WT lines showed that >90% of sequencing reads were of high quality for mapping to the P. falciparum genome with nearly 40 times of coverage. This allows us to analyze the change of alternative splicing events (alternative 5' splice site, alternative 3' splice site, retained intron and skipped exon). 800, 1056, 479, and 1158 alternative splicing events (alternative 5' splice site, alternative 3' splice site, retained intron and skipped exon) were altered in the DPfPRMT5 parasite line as compared to the WT line at four development stages, respectively (unpublished data). Conclusions: Collectively, this RNAseq provide a dataset for analysis of abnormal RNA splicing events in the ΔPfPRMT5 parasites.
Project description:During red-blood-cell-stage infection of Plasmodium falciparum, the parasite undergoes repeated rounds of replication, egress, and invasion. Erythrocyte invasion involves specific interactions between host cell receptors and parasite ligands and coordinated expression of genes specific to this step of the life cycle. We show that a parasite-specific bromodomain protein, PfBDP1, binds to chromatin at transcriptional start sites of invasion-related genes and directly controls their expression. Conditional PfBDP1 knockdown causes a dramatic defect in parasite invasion and growth and results in transcriptional downregulation of multiple invasion-related genes at a time point critical for invasion. Conversely, PfBDP1 overexpression enhances expression of these same invasion-related genes. PfBDP1 binds to acetylated histone H3 and a second bromodomain protein, PfBDP2, suggesting a potential mechanism for gene recognition and control. Collectively, these findings show that PfBDP1 critically coordinates expression of invasion genes and indicate that targeting PfBDP1 could be an invaluable tool in malaria eradication. ChIPseq mapping of the genome wide enrichment profile of the P. falciparum bromodomain protein PfBDP1 in two parasite stages and correlation with RNAseq expression data
Project description:Liver stage of malaria parasite exports SLTRiP and PB268 to the cytosol of parasite infected host cell. To know the host genes perturbed by WT-PBANKA, SLTRiP-KO and PB268-KO parasite growth, we did transcriptomic sequencing of infected host cells. We did mRNA sequencing of four samples for comparative analysis of WT and PB-knockout parasites infected host cells at 22 hours of post sporozoites infection.
Project description:This experiment details the gene expression data from the small intestine from a set of parasite resistant and a set of parasite susceptible sheep. Keywords = parasite resistance Keywords: other