Lasergene 7

The AmpG secondary structure was predicted using a program at http://www.predictprotein.org/, and a Kyte-Doolittle algorithm from Lasergene 7 (DNASTAR, Madison, WI) [9 (link)] was used to predict AmpG transmembrane (TM) helices. The parameters of TOPPRED-transmembrane topology prediction were as follows: Full window size: 21, Core window size: 11, Wedge window size: 5, Using hydrophobicity file: GES-scale, Cutoff for certain transmembrane segments: 1.00, Cutoff for putative transmembrane segments: 0.60, Critical distance between 2 transmembrane segments: 2, Critical loop length: 60.

The assessment of the sequencing results has demonstrated a random point mutation in nucleotide 1541, a point mutation from guanosine to adenosine, in the active domain of tPA (Supplementary Fig. S1). To study protein structure, the consensus sequence has been translated into the protein by using Editseq software (Lasergene 7, DNASTAR Inc., Madison, WI, USA). MODELLER 9.14 (http://salilab.org/modeller/) software has been used to construct 10 homology models by employing the crystal structure of the tPA catalytic domain (PDB 1RTF) as template. To select the best model, the structure validation has been carried out by utilizing Structure Analysis and Verification Server (SAVES) (http://services.mbi.ucla.edu/SAVES/) via VERIFY3D^{38 (link)} and ERRAT^{39 (link)} programs. Then, GROMACS is applied as module to minimize energy resulting in the protein stability, by which the model is tested in water solvent to simulate the real and biological environment. The molecular docking of tPA and mtPA and plasminogen (PDB 4DCB) has been conducted using the HADDOCK web server) http://www.nmr.chem.uu.nl/haddock) to carry out comparative analysis between native and mutant forms. Dependent upon the quality of docking results based on the highest-ranking cluster, the native and mutant forms have been compared utilizing LigPlot v.1.4.5^{40 (link)} to ensure their poses are biologically acceptable.

Total RNA was extracted from samples following the CTAB method [39 (link)]. RNase-free DNase I (TaKaRa, Tokyo, Japan) was added to the CTAB extract to remove genomic DNA. After removing the genomic DNA, the RNA purity and integrity were assessed based on the A260/A280 ratio using Nanodrop and agarose gel (1.0%) electrophoresis. Then, 1 µg of the total RNA was used to synthesize the single-stranded cDNA using SMARTer™ PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA).
The 5′ and 3′ end cDNA sequences of AmPRX1, AmPRX2, AmPL, and AmPSK were obtained via rapid amplification of cDNA ends (RACE) performed using the total RNA extracted from leaves of A. marina with the SMARTer^TM RACE Kit (Clontech, Mountain View, CA, USA). The first round of PCR was performed using the gene-specific primer (GSP) (Table S1) and the universal primer mix (UPM), and a second nested PCR was performed with the product of the first PCR using a nested gene-specific primer (NGSP) (Table S1) and the nested universal primer A (NUP). The final PCR products were analyzed on agarose gels, cloned into the pMD-18T vector (Takara, Tokyo, Japan), and sequenced. Finally, the 5′ and 3′ end sequences were assembled with an overlapping fragment to obtain the full-length cDNA sequences using Lasergene 7 (DNASTAR, WI, USA).

The open reading frame (ORF) was predicted by the Open Reading Frame Finder program (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 22 September 2021), and the amino acid sequences (AASs) were deduced from the CDSs of buffalo Smads 1, 4, and 5 via EditSeq program of Lasergene 7 software package (DNAStar, Inc., Madison, WI, USA). Codon usage bias analysis was performed by codonW (http://codonw.sourceforge.net/, accessed on 22 September 2021). The genetic structure analysis of buffalo and its comparison with other Bovidae species are as follows: The transcript information of Smads 1, 4, and 5 of buffalo and other Bovidae species were obtained from the GTF (General Transfer Format) files which downloaded from NCBI datasets (https://www.ncbi.nlm.nih.gov/datasets/, accessed on 22 September 2021). Then, the mRNA and UTR information was added to these GTF files by the GXF fix function of TBtools software [16 (link)]. After calculating the relative position of each transcript and each exon in Excel, the information was summarized into a text file in the BED (Browser Extensible Data) format, and then submited to the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/, accessed on 22 September 2021) [17 (link)] for visualization of the gene structures, including untranslated regions, exons, and introns.

Electropherograms were inspected with Chromas Lite (Technelysium Pty. Ltd., Tewantin, Queensland). We used the term haplotype to refer to the unique allele from sequencing each of the three (E6-8, E20-21 and E31-32) regions. Unknown haplotypes were identified by aligning sequences with known haplotypes using the MegAlign and EditSeq applications of Lasergene 7 (DNAStar, Madison, WI). Haplotypes present in heterozygous samples were identified either by cloning and sequencing (a minimum of two clones) or were inferred by comparison the known haplotypes in same populations on the electropherograms. All inferred haplotypes were observed in more than one individual.
We used the term “pattern sequences” to refer to specific concatenated sequences that were assembled from the sequence of E6-8, the codon from V410L (GTA for V, TTA for L), the sequence of E20-21, the codon from F1534C (TTC for F, TGC for C) and the sequence of E31-32 in each individual mosquito (Fig 1). Pattern sequences from heterozygous individuals were identified by comparison of known pattern sequences from the homozygous samples in the same population. Unique pattern sequences were numbered in the order they were discovered.

Full nucleotide sequences of pomsp4 genes from all clinical isolates were translated to the deduced amino acid sequences using the MegAlign module of Lasergene 7 software package (DNAstar) and then aligned with reference sequences to assess conservation within PoMSP4. Amino acid sequences of the segments of PocMSP4 and PowMSP4 isolates were aligned with those of the PocGH01_04023000 and LT594508.1 reference strains, respectively. Sequence alignment for all P. ovale isolates was performed using MEGA v.7.0 software.
Statistical analysis and graphing were conducted using GraphPad Prism software version 5.0 (Graph Pad software, Inc.). SPSS v.16.0 was performed to analyse cross-reaction and antibody responses. Student’s t-test with probability (P) value of < 0.05 indicated a significant difference.