MOESM1 of Identification of a PH domain-containing protein which is localized to crystalloid bodies of Plasmodium ookinetes
Creators: name: Rachaneeporn Jenwithisuk
name: Niwat Kangwanrangsan
name: Tachibana, Mayumi
name: Amporn Thongkukiatkul
name: Otsuki, Hitoshi
name: Jetsumon Sattabongkot
name: Tsuboi, Takafumi
name: Motomi Torii
name: Ishino, Tomoko
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: 29999 Physical Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: FOS: Physical sciences
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Evolutionary Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology
subject: Computational Biology

description: Additional file 1: Figure S1. Construction of transgenic parasites with CryPH gene modifications. (a) Schematic representation of the generation of GFP-tagged CryPH expressing parasites. The native PyCryPH gene locus was replaced with a coding sequence of CryPH tagged by GFP at the C-terminal by double-crossover homologous recombination. The vector contains two homologous regions, the coding region of the C-terminal of CryPH (striped orange box, CryPH-C), which is connected in frame to GFP coding sequence (green box, GFP), and the 3′-UTR of CryPH (grid orange box, CryPH-3′). For selection of DNA integrated parasites, human DHFR coding sequence (dotted green box, hDHFR) is inserted between them. The resulting integrated locus is shown in the bottom panel. (b) Schematic representation of the targeted gene disruption of CryPH. The CryPH-coding region in the genome is replaced with the human DHFR expression cassette (dotted green box) by homologous recombination at the sites corresponding to the 5′- and 3′-UTR of CryPH (grid orange box). (c) Generation of CryPH-control (CryPH-cont) parasites. By homologous recombination, human DHFR expression cassette (dotted green box) is inserted next to the CryPH gene with the transgenic vector containing two homologous recombination sites corresponding to CryPH-C and 3′-UTR of CryPH (striped and grid orange boxes). Parasites with integrated DNA (bottom panel) were selected by drug treatment. (d) PCR genotyping of transgenic parasites. Correct DNA insertion into the PyCryPH locus of ∆CryPH (cl1 and cl2) and CryPH-cont transgenic parasites was confirmed by PCR using specific primer sets. The amplicons were diagnostic for: lane 1, integrated form; lane 2, episomal form; lane 3, wild-type. The expected sizes of the amplified fragments (lanes 1–3) were 1,000 bp, 1,235 bp, and 1,030 bp, respectively. Episomal form was not detected in any clones. Figure S2. Alignment of amino acid sequences of CryPH orthologues in apicomplexan parasites. Amino acid sequences of CryPH orthologues in Plasmodium yoelii (PY17X_0705200), Toxoplasma gondii (TGME49_219160), Theileria equi (BEWA_008110), Babesia microti (BMR1_02g03021), and Cryptosporidium meleagridis (CmeUKMEL1_06995) are aligned using CLUSTALW algorithm ( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html ). Underbars indicate the signal peptide of each sequence. Strand-specific RNA-seq data suggested that translation of predicted Toxoplasma CryPH may start from the internal methionine (marked in orange). Approximately 3% of residues including all cysteine residues are conserved among CryPH orthologues. Similarity is higher in the PH domain predicted by the secondary structure of PyCryPH, indicated by a box. Figure S3. Molecular phylogenetic analysis by the Maximum Likelihood method. The evolutionary history was inferred using the Maximum Likelihood method [69]. The initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. Evolutional analyses were conducted in MEGA7 [70].