In Silico Analysis of Cu-Zn Superoxide Dismutase and Mn Superoxide Dismutase Genes in Fugu (Takifugu rubripes)

Mehtap Bayir*

Department of Agricultural Biotechnology, Faculty of Agriculture, Atatürk University, 25240, Erzurum, Turkey

ABSTRACT

Superoxide dismutases are the best-known enzymatic antioxidants because of their central role in the antioxidant defense system. In this study, the phylogeny, gene structure, and conserved gene synteny of sod1 and sod2 genes in fugu—a model organism—were determined. Maximum amino acid similarity identity was found between putative fugu Sod1 and Sod2 proteins and their orthologs from teleost fish and tetrapods. Phylogenetic clustering was seen between sod genes in fugu and their orthologs. Finally, highly conserved gene synteny was determined between fugu sod genes and their orthologs from teleost fish and human.

Article Information

Received 22 January 2019

Revised 22 May 2019

Accepted 27 August 2019

Available online 02 April 2020

Key words

Bioinformatics, Fugu, Model organism, sod1, sod2

DOI: https://dx.doi.org/10.17582/journal.pjz/20190122060142

* Corresponding author: mehtap.bayir@atauni.edu.tr

0030-9923/2020/0004-1377 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

INTRODUCTION

Fugu (Japanese pufferfish, Takifugu rubripes) is an ideal model organism for vertebrate genome research and developmental biology studies (Uji et al., 2011). Japanese pufferfish has more advantages for genomic studies compared with other vertebrates. Its 400-Mb genome size is quite small compared with the 3,000-Mb human genome size (Watabe and Ikeda, 2006). It is also known that the pufferfish genome has more genes than those of coelacanths and air-breathing fish due to teleost-specific whole genome duplication (tsWGD) event (Van de Peer, 2004). The small size of the genome, which is a factor that facilitates the detection and analysis of genes, has regular sequences like those of the other vertebrates, requiring less work to obtain comparable data. Green spotted pufferfish (Tetraodon nigroviridis) can also be used as model organisms for vertebrate genomes (Close et al., 2016). Many human genes have been uncovered by comparing these two pufferfish genomes with the human genomes (Brenner et al., 1993).

Aerobic biological systems generate reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and superoxide anion (O2.−). ROS attacking essential cellular components may result in irreversible damage in their structure. However, organisms can initiate ROS removal and restrict generation of ROS via their well-developed antioxidant defense system (ADS). The superoxide dismutases (SODs) have an important role in ADS due to their ability to prevent the generation of hydroxyl radical (OH.) and removing O2.− and catalyzing the dismutation of O2.− into H2O2 and molecular oxygen. SODs using metal ions in their activities have two forms of eukaryotic systems, namely, Cu+2/Zn+2 and Mn+2 SOD. While the first isoform (Cu+2/Zn+2 SOD) is found in cytosols and its activity is not affected by oxidative stress, the second isoform (Mn+2 SOD) is located in mitochondria, and its activity is increased in proportion to oxidative stress (Babior, 1997; Davies, 2000). Several studies have been carried out on SOD enzymes in aerobic organisms (Lopes et al., 2001; Fink et al., 2002; van der Oost et al., 2003; Ken et al., 2003; Farombi et al., 2007; Cho et al., 2009). Limited work, however, is available on the genomic organization and gene structure of teleost sods (Cho et al., 2009) and regulatory region of the sod genes in teleost fish (Maehara et al., 1999; Mao et al., 2006). It is known that transcription of the Mn+2 SOD gene can be regulated by environmental factors (Valavanidis et al., 2006; Kim et al., 2007; Cho et al., 2009), and the function and structure of SOD2 are well conserved in variant organisms (Fink et al., 2002). Therefore, the goals of the current study are determining sod genes in the fugu genome using bioinformatic tools, leading to future molecular works on antioxidant enzyme genes in teleost fishes.

 

MATERIALS AND METHODS

Fugu sod1 and sod2 gene sequences were obtained by performing BLAST (http://useast.ensembl.org/Multi/blastview) searches with an identical orthologous zebrafish (Danio rerio) Sod protein sequence as an inquiry to the Ensembl genome sequence database (http://useast.ensembl.org/index.html). Zebrafish sod cDNA sequences provided from the Ensembl genome database were used to identify ESTs coded by specific fugu sod1 and sod2 genes in a BLAST search of the NCBI database (http://blast.ncbi.nlm.nih.gov).

Teleost fish exhibits strict evolutionary conservation for the gene structure in the same gene family. Therefore, the exon-intron junctions of the sod1 and sod2 genes were determined using zebrafish sod1 and sod2 gene structures as a reference. To confirm whether sod1 and sod2 genes were transcriptionally active or served as pseudogenes or non-functional genes, tBLASTn searches in the NCBI database were conducted to identify their ESTs using their Ensembl-derived amino acid sequences as queries (Table I). Superoxide dismutase genes in fugu with their corresponding zebrafish query sequence IDs, NCBI cDNA IDs, chromosomal locations and length of Sod polypeptides were also determined using Ensembl and the NCBI genome databases (Table II).

 

Table I. Ensembl gene ID and expressed sequence tags (EST) coded by fugu and zebrafish sod1 and sod2 genes.

Gene	Ensembl gene ID	EST
Fugu sod1	ENSTRUG00000021322	Not detected
Fugu sod2	ENSTRUG00000005242	Not detected
Zebrafish sod1	ENSDARG00000043848	EH544614.1
Zebrafish sod2	ENSDART00000062556.4	EH537787.1

 

Phylogenetic analysis

CLUSTALW (Thompson et al., 1994) at BioEdit software (http://www.mbio.ncsu.edu/bioedit/page2.html) was used for sequence alignment of the sod1 and sod2 genes. The protein sequence of fugu Sod1 and Sod2 was aligned with Sod/SOD protein sequences from fugu, tetraodon, medaka, zebrafish, human, and mice. The pairwise alignment of the BLOSUM62 matrix (Gromiha, 2010) was used for sequence identity and similarity. A maximum-likelihood tree with the Poisson correction distance model based on amino acid substitution per site was built using MEGA6 (Tamura et al., 2013) to determine the phylogenetic relationships of the fugu, tetraodon, medaka, zebrafish, human, and mouse Sod sequences. A bootstrapped neighbor-joining tree was also constructed before construction of the maximum-likelihood tree to confirm the phylogeny of sod genes (data not shown). As in a previous study, the protein sequence of human lymphocyte cytosolic protein LCP2) was used as an external group (Kell et al., 2018).

Conserved gene synteny

The conserved gene synteny of fugu sod genes with the sod/SOD of zebrafish, medaka and human was arranged manually using the region conceptus selection of Ensembl database to recognise co-localized gene (Thirumaran and Wright, 2014).

 

Table II. Superoxide dismutase genes of fugu with their corresponding zebrafish query sequence ID, NCBI cDNA ID, locations of chromosome and length of sod polypeptides.

Gene	Zebrafish query sequence ID	NCBI cDNA ID	Location	Number of amino acids
sod1	NP_571369.1	XP_003971372.1	Chromosome 10: 25 694 395-25 699 454	154
sod2	NP_956270.1	XP_003971923.1	Chromosome 16: 9 495 906-9 498 317	227

 

RESULTS AND DISCUSSION

Bioinformatics and computational analysis of fugu sod genes

As a known procedure in bioinformatics studies, first, statistical knowledge is collected using biological data; then, a model is generated for solving any computational modeling problem. Finally, testing and evaluation of a computational algorithm for solving a bioinformatics problem are conducted (Can, 2014). In this study, I retrieved some statistics using the Ensembl genomic database, NCBI database, BioEdit software, pairwise alignment of the BLOSUM62 matrix program and MEGA6 program.

 

 

The cDNA sequence was obtained from the Ensembl genomic database to determine the exon–intron structure of the fugu sod genes, and it was found that these genes had five exons separated by four introns. It was determined that introns of both genes followed the gt-ag rule. Moreover, they have putative TATA and CAAT boxes and polyadenylation signals. The results clearly show that sod genes in fugu exhibit a highly conserved gene structure (Figs. 1 and 3).

I searched the ESTs of sod1 and sod2 by BLAST searches in the NCBI genomic database using cDNA nucleotide sequences obtained from the Ensembl genomic database (Table I). However, as shown in Table I, there was no EST for these genes. The sequence identity and similarity among the fugu and human, mouse, zebrafish, and medaka sod genes are given in Figures 2 and 4. The highest identity and similarity rates for fugu sod1 and sod2 genes were determined with their orthologs (tetraodon, medaka, and zebrafish). After that, a maximum-likelihood phylogenetic tree was built based on identity and similarity results (Fig. 5). At the end of the phylogeny analysis, high phylogenetic clustering was seen between the sod genes in fugu and their orthologs. Conserved gene synteny evidence was determined for sod genes of fugu and Sod/sod genes of other teleost fishes and human (Fig. 6). The syntenic genes of fugu sod1 and sod2, located on chromosomes 15 and 16, exhibit conserved gene synteny with human SOD1 and SOD2 located on chromosomes 21 and 6, medaka sod1 and sod2 located on chromosomes 14 and 24, and zebrafish sod1 and sod2 located on chromosomes 10 and 20. The results of conserved gene synteny between sod genes in fugu and their orthologs in medaka, zebrafish, and human show that teleost fish lost duplicated copies of the sod1 and sod2 genes after tsWGD because of mutations. This would be an interesting result for future studies on whether all teleost species have single copies of sod genes and their transcriptional controls.

 

 

CONCLUSION

Antioxidant enzymes are gaining more importance, and they have been studied more in recent years. When antioxidant levels and production of ROS are in balance in normal conditions, the harmful effect of free radicals can be observed with oxidative stress-induced diseases (Scandalios, 1993). Stress responses in fishes can manifest multifaceted levels involving the actions of different sets of genes and their products. Understanding the genetic characteristics associated with model organisms that exhibit a stress response is important for molecular studies. Identification and characterization of the stress genes that are differentially expressed between stress-tolerant and intolerant fish will provide important genetic markers that may be used in aquaculture selection programs to help improve stress tolerance, as well as serving as a model for other vertebrates, including human (Iwama et al., 1999). However, it is known that oxidative stress has a big role in more than 100 diseases in human as their reason or effect (Halliwell et al., 1992; Gutteridge, 1993; Poljsak et al., 2013). For this purpose, I identified sod genes in fugu—serving as a model organism—using bioinformatic tools; the results can illuminate the path for future studies on molecular stress responses in fish.

 

Statement of conflict of interest

The authors declare there is no conflict of interest.

 

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