Figure 1.
Structures within NCX, NCKX, and CCX proteins, and an overview of the pipeline used to detect orthologs of these proteins in twelve species of nematodes.
(A) Cartoon depicting structures within the NCX, NCKX, and CCX (NCLX) proteins. (B) Overview of a pipeline used to detect orthologous sodium calcium exchanger genes in twelve different species of nematodes.
Figure 2.
Phylogenetic analysis of NCX and NCKX exchangers in various nematodes.
(A) Phylogenetic analysis of NCX type exchangers from Strongyloides ratti, Haemonchus contortus, Heterorhabditis bacteriophora, Caenorhabditis elegans, Caenorhabditis brenneri, Caenorhabditis japonica, Caenorhabditis briggsae, Caenorhabditis remanei, Pristionchus pacificus, Brugia malayi, Loa loa, and Ascaris suum. Inferred phylogeny was constructed using PhyML [38] and derived from amino acid alignments using MUSCLE [35]. The NCKX5 exchanger from human and mouse was used as an outgroup. (B) Phylogenetic analysis of NCKX type exchangers from S. ratti, H. contortus, H. bacteriophora, C. elegans, C. brenneri, C. japonica, C. briggsae, C. remanei, P. pacificus, B. malayi, L. loa, and A. suum. Inferred phylogeny was constructed using PhyML [38] using the model WAG+G+F determined from Prottest [36] and derived from amino acid alignments using MUSCLE [35]. The NCKX type exchangers from human and mouse served as an outgroup.
Figure 3.
Sequence and structural analysis of nematode exchangers.
(A) Sliding window analysis of nucleotide diversity using DnaSP version 5 [47] with 100 bp windows and 25 bp steps for CCX (upper graph), NCKX (middle graph), and NCX exchanger (lower graph) gene pairs between C. elegans and C. briggsae. (B) Box plot analysis of molecular diversity for each ncx gene (ncx-1 to ncx-10) between C. elegans and C. briggsae. Whiskers extend to data points that are less than 1.5 × interquartile range away from 1st/3rd quartile; center lines show the medians and outliers are shown as dots. (C) Ribbon model of NCX-1 from C. elegans and an NCX-3 ortholog from A. suum (GS_14034). N and C termini are indicated, and red arrowheads refer to structural differences between each NCX. Numbers refer to the transmembrane (TM) domains. Extracellular side is up in left views. The position of two candidate sites in NCX-1 undergoing episodic diversifying selection are indicted - codon 455 and codon 925. In each case the right view is rotated by 90°. Structural predictions were made using Phyre [48] and visualized using RasMol [49]. In each case the single highest scoring modelling template was the resolved NCX (NCX_Mj) structure (PDB: 3V5U) from Liao et al. [8].
Figure 4.
Predicted gene structure of CCX proteins from Caenorhabditis japonica and Heterorhabditis bacteriophora.
(A) Predicted gene structures from Cja11479 and Cja38547 on contig 17913 from Caenorhabditis japonica. Translation of each predicted gene produces approximately half an NCLX-like protein containing the α1 repeat sequence GNGAPD and the α2 domain sequence SNSIGD. Blue arrows indicate the approximate position of the predicted coding sequence mapped to the translation. (B) Predicted gene structure from Hba_19835 and Hba_19836 on contig 1352 from Heterorhabditis bacteriophora. Translations generate approximately half an NCLX-like protein containing the α1 repeat sequence GNGAPD and the α2 domain sequence SNSIGD. Blue arrows indicate the approximate position of the predicted coding sequence mapped to the protein translation. (C) PCR Primers were designed that spanned the final exon of the upstream predicted gene Cja11479 and the first exon of the downstream predicted gene Cja38547. Using genomic DNA as template for PCR we observed a band at 2443 bp (third lane in gel inset), and using reverse transcribed RNA as template for PCR we observed a band at 813 bp (second lane in gel inset). Primers are indicated by red arrowheads, and resulting cDNA sequence mapped to gene structure is indicated by the blue rectangles. First lane in the gel electrophoresis image is a GenRuler DNA ladder mix (Thermo Scientific - SM0334). Purple arrowhead at inset indicates the position of the gDNA band corresponding to the 2443 bp fragment. (D) Primers were designed that spanned the third to last exon of the upstream predicted gene Hba_19836 and the third exon of the downstream predicted gene Hba_19835. Using genomic DNA as template for PCR we observed a band at 1623 bp (third lane in gel inset), and using reverse transcribed RNA as template for PCR we observed a band at 674 bp (second lane in gel inset). Primers are indicated by red arrowheads, and resulting cDNA sequence mapped to the gene structure is denoted by blue rectangles. First lane in the gel electrophoresis inset image is a GenRuler DNA ladder mix (Thermo Scientific - SM0334).
Figure 5.
Phylogenetic analysis of NCLX (CCX) exchangers from various nematodes.
(A) Phylogenetic analysis of NCLX type exchangers from Strongyloides ratti, Haemonchus contortus, Heterorhabditis bacteriophora, Caenorhabditis elegans, Caenorhabditis brenneri, Caenorhabditis japonica, Caenorhabditis briggsae, Caenorhabditis remanei, and Pristionchus pacificus. Inferred phylogeny was constructed using PhyML [38] using the model WAG+I+G+F determined from Prottest [36] and derived from amino acid alignments using MUSCLE [35]. ‘CCX div’ denotes a divergent CCX cluster that does not group with the Caenorhabditis CCX protein clusters (i.e. NCX-6 to NCX-10). The NCLX exchanger from human and mouse were used as an outgroup. (B) Alignment of α repeat domains within CCX (NCLX) proteins from various nematodes and also human NCLX. Alignments were generated using MUSCLE [35] of NCLX type exchangers from Human, S. ratti, H. contortus, H. bacteriophora, C. elegans, C. brenneri, C. japonica, C. briggsae, C. remanei, and P. pacificus.