full-length plasmodium falciparum maf1 coding sequence  (GenScript corporation)

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Few components of the TORC1 pathway remain in the P. falciparum genome. (A) A simplified representation of the major TOR complex 1 ( TORC1 ) components, regulators, targets, and converging pathways across several eukaryote lineages. Solid circles (●) indicate the presence of the gene coding for the component in representative members of the lineage, and hollow circles (O) indicate the absence of the corresponding component in the lineage. (B) An illustration of the generalized animal PI3K-TORC1 signaling cascade used to regulate cellular growth in the presence of amino acids and other growth factors (left) and a projection of this pathway in Plasmodium spp. based on the conserved components (right). Plasmodium spp. lack a class I PI3K enzyme and the other components ( PTEN , PDK , an Akt homolog containing a PH domain) typically associated with this signaling cascade. Plasmodium spp. do encode a class III PI3K enzyme (PF3D7_0515300) whose ortholog has been implicated in TORC1 signaling in human cells, as well as a PH domain lacking PKB family kinase (PF3D7_1246900) resembling human S6K . The genomes of Plasmodium parasites also encode an apparent ortholog of the TORC1 -dependent RNA polymerase III regulator Maf1 (PF3D7_0416500).

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques:

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Functional complementation of Maf1-knockout yeast cells with a chimeric P. falciparum Maf1. (A) Schematics showing the key structural features of the yeast and Plasmodium Maf1 orthologs (Nt, N terminus; Ct, C terminus; NLS, nuclear localization signal). Vertical lines indicate the homologous region exchanged to generate the chimera for complementation. *P, known site of phosphorylation in the yeast protein. (B) Fivefold serial dilutions of from ~5,000 to ~8 yeast cells transformed with the indicated complementation vectors were plated on normal rich media or on rich media supplemented with 10 nM sirolimus, which is lethal to Maf1 knockout yeast cells. Images are representative of one of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Functional Assay, Knock-Out, Phospho-proteomics, Transformation Assay

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: The PB-11 mutant carries a piggyBac insertion in the 5′UTR of the Maf1 gene. (A) Schematics of the Maf1 genomic locus and piggyBac insertion in wild-type and mutant parasites. Numbers below the chromosomal line indicate the reported and verified transposon insertion positions relative to the Maf1 start codon. Right-angled arrows and numbers above the chromosome line indicate the transcription start sites in wild-type (−93 nt) and PB-11 mutant (−395 nt) cells as determined by 5′-RACE. The Maf1 transcript in the PB-11 mutant arises from within the piggyBac transposon and is likely due to bidirectional promoter activity of the calmodulin promoter fragment used for drug selection in the transposon. (B) The upstream insertion site was confirmed by whole-genome sequencing (top). Coverage depth plots indicate the total number of reads mapping to each base pair around the two termini of the piggyBac insertion at the −53 TTAA site upstream of the Maf1 start codon. The maximum read coverage for each end of the transposon is indicated by the axes on the left and right (bottom). Raw reads aligned to the termini of the transposon surrounding the −53 insertion site. Each read is 100 bp in length. Colored spots indicate base mismatches within a given read and the genomic sequence.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Activity Assay, Selection, Sequencing

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: The PB-11 mutant displays an abnormal Maf1 mRNA expression profile. An analysis of the time course of Maf1 mRNA expression was performed using qRT-PCR at seven time points across the intraerythrocytic cycle and synchronous wild-type (NF54) and mutant (PB-11) parasites. Maf1 expression was quantified relative to that of the seryl-tRNA ligase transcript (PF3D7_0717700). Points represent individual biological replicates (three in total), and curves represent LOESS smoothed models fitted to the data, with the 95% confidence interval indicated by shading. ΔΔCT, threshold cycle method; hpi, hour post-red blood cell (RBC) invasion.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Maf1 mutant parasites cannot recover from a prolonged dormancy-like state induced by isoleucine starvation. Synchronous young ring-stage parasites (approximately 4 h postinvasion) were washed repeatedly and transferred to medium lacking isoleucine (Ile) for the indicated times. Recovery data denote transfer back to normal culture medium (containing isoleucine) for 72 h of growth. Parasitemia was quantified by flow cytometry. Growth was measured relative to the final level of parasitemia of a control culture incubated in normal culture medium for 72 h. P values were calculated using t tests of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Flow Cytometry, Control, Incubation

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: The PB-11 mutant remains viable but loses the ability to recover within the first 72 h of starvation. (A) Parasitemia of Maf1 mutant parasites decreases more rapidly during prolonged isoleucine starvation. Synchronous young ring-stage NF54 (WT) and Maf1 insertion mutant (PB-11) parasites were washed repeatedly and transferred to medium lacking isoleucine. Samples of each parasite line were taken at 8-h intervals over the course of a 9-day (216-h) period, and parasitemia was determined by flow cytometry. Lines represent LOESS curves fitted to the data for each of three biological replicates. (B) Maf1 mutant parasites display minimal differences in death rate over the first 72 h of starvation. Regression models were fitted to the 216-h isoleucine starvation data to determine the rate of death of NF54 (WT) and Maf1 mutant (PB-11) parasites over the full 216 h or for only the first 72 h of the same data set (t 1/10 = time required to reach 1/10 the starting level of parasitemia). (C) Maf1 mutant parasites lose the ability to recover after (on average) 43 h of isoleucine starvation. Synchronous young ring-stage PB-11 parasites were washed and transferred to medium lacking isoleucine. Every 3 h, samples were transferred to normal medium for a 72-h recovery period. The final level of parasitemia after recovery was quantified by flow cytometry. A logistic regression fitted to the data shows that each hour of starvation decreases the parasitemia level to 93% of the level seen the previous hour (β o = −0.076, P < 2.00 × 10 −16 ). The logistic model fitted to the data predicts that the time point corresponding to 43 h of starvation is the point at which 50% of the PB-11 parasites are able to recover and 50% are not (t 50 ). Data shown are the results of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Flow Cytometry

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: PB-11 Maf1 mutant parasites display defects in recovery from fosmidomycin exposure and low-temperature treatment. (A) Young ring-stage NF54 (WT) and Maf1 mutant (PB-11) parasites were incubated in the presence of 5 μM fosmidomycin (fosm.) for 72 h and were then “recovered” by incubation for a further 72 h in the presence of 5 μM fosmidomycin and 5 μM geranylgeraniol. Growth was measured relative to that of parasites incubated for 72 h in normal medium treated with both fosmidomycin and geranylgeraniol. (B) Young ring-stage parasites were incubated at 18°C for 72 h and were recovered by 72 h of growth at 37°C. Growth was measured relative to that of parasites incubated for 72 h at 37°C. P values were calculated using t tests of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Incubation

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: PB-11 Maf1 parasites display a decreased growth rate under low-isoleucine conditions and at elevated temperatures. Growth curves were measured for NF54 (WT) and Maf1 mutant (PB-11) parasites cultured in normal culture medium at 37°C ( P < 0.0001) (A), 8 μM isoleucine (approximately 2% the concentration of normal medium) ( P < 0.001) (B), normal culture medium at 39°C ( P = 0.003) (C), and 20% (0.4 g/liter) of the glucose level of normal medium (2.0 g/liter) ( P = 0.08) (D). P values represent results of tests of the parasite line (i.e., the wild type versus PB-11) as a factor in the regression analysis. t D , doubling time.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Cell Culture, Concentration Assay

Journal: mBio

Article Title: Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation

doi: 10.1128/mBio.02317-16

Figure Lengend Snippet: Maf1 mutant parasites display elevated pre-tRNA expression and elevated global translation under normal and isoleucine starvation conditions. (A) An alignment of the genomic pre-tRNA Tyr sequence and the mature tRNA Tyr sequence reveals an 11-nucleotide intron adjacent the anticodon. (B) Stem-loop RT-qPCR profiling of pre-tRNA expression in NF54 (WT) and Maf1 mutant (PB-11) parasites. Synchronous young ring-stage parasites were incubated in normal medium or medium lacking isoleucine for 24 h prior to RNA isolation. Pre-tRNA Tyr expression was quantified relative to 5.8S rRNA levels. (C) ELISA of puromycin incorporation relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene levels in wild-type and mutant parasites under conditions of the indicated treatments. Synchronous young ring-stage parasites were incubated in normal medium or medium lacking isoleucine for 24 h prior to the 1-h puromycin pulse and subsequent harvesting. Translational arrest by cycloheximide (CHX) treatment was used as a negative control. P values were calculated using t tests of three biological replicates.

Article Snippet: A full-length Plasmodium falciparum Maf1 coding sequence was synthesized with codon usage optimized for S. cerevisiae expression (GenScript).

Techniques: Mutagenesis, Expressing, Sequencing, Quantitative RT-PCR, Incubation, Isolation, Enzyme-linked Immunosorbent Assay, Negative Control