1. Figure 1. substitution rate of purine and pyrimidine
Molecular evolution of the HSP70 gene family in cattle J. Ahmadpanah1, M. Moridi1, A. A. Masoudi 2*, R. Vaez Torshizi2 Student1 and Assistant Professor2, Department of Animal Science, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran   * Corresponding author:
ABSTRACT
Heat shock proteins (HSPs) are distributed across the genome and divided to various groups according to the own molecular weights. One of the members of HSPs, HSP70, shows differentiated expression against the unusual environmental changes such as stress, feed restriction and high temperature. Therefore, the genes encoding these proteins have been identified as candidate genes in view of animal sciences. Current study was conducted to identify nucleotide substitutions in the evolutionary path of the HSP70 gene family in cattle comparing to the other animal species. Genomic sequences of the HSP70 gene family were identified by in silico screening of the animals' genomic information at NCBI. Multi alignment analysis, estimation of the pattern of nucleotide substitution and selection analysis of the sequences were carried out applying GENETYX and MEGA4 software. The overall frequencies of pyrimidine and purine nucleotides in HSP70 gene family were and 0.534, respectively. However the most substitution rate has been occurred between thymine and cytosine bases (16.15), but this rate was 9.64 for purines. Furthermore, evidence from the comparisons of the nonsynonymous to synonymous substitutions and distribution of the mutation sites suggested that HSP70 gene family evolved under purifying selection in cattle. Altogether suggested that purifying selection plays an important role in the evolution of this gene family. The analysis reveals that the structure of HSP70 gene family has changed little during decades of evolution.
Key words: Heat shock protein, Selection, Evolution
INTRODUCTION
Heat shock proteins (HSPs) are present in all cells of the body and function as molecular chaperons to protect the other proteins from degradation and malfunction [6]. In animals, occurrence of some SNPs in the structure of these genes was associated with resistance to mastitis and infectious bursal disease, conservation of homeostasis, adaptation to hot regions and increasing the immunity systems. Expression of heat shock proteins increases under such conditions and they are rapidly produced and act to solve these problems [3]. HSPs expression takes place in mRNA production level after transcription and in translation stage. Among reasons for lack of structural changes in high temperatures in heat shock proteins are better hydrogen bonds, enhanced intramolecular hydrophobic packaging, improved secondary structure and stabilization of bipolar chain in these protein molecules. HSP expression increases in various stressful conditions such as infection, inflammation, chemicals, starvation and water shortage, making function of these proteins in confronting such unfavorable conditions to maintain cell viability [2]. HSPs are distributed across the genome and divided to various groups according to the own molecular weights including HSP10, HSP40, HSP60, HSP70, HSP90, HSP104 and HSP110 [3, 6]. HSP70 gene family has a high polymorphism, and many changes have been caused by gene derivation during evolution process. HSP70 is very important due to big range of functions for maintaining protein structures in different stages and cellular sites and covering the function of other heat shock protein groups, and knowledge of amount and pathways of mutations in these genes during evolution is of importance.
MATERIALS AND METHODS
In order to identify nucleotide substitutions in the evolutionary path of the HSP70 gene family in cattle comparing to the other animal species, Genomic sequences of the HSP70 gene family were identified by in silico screening of the animals' genomic information at NCBI. The species under study included human, cow, mouse, rat, hen, dog, zebrafish and finch. Percent similarity between HSP70 gene family members was determined by Genetyx software. Protein sequence of each gene family member was predicted using Genetyx software and took the format of MEGA4 software. To compare and analyze the resulting sequences, the coding regions were used. Genetyx and MEGA4 software were used to determine similarity and nucleotide substitutions [7]. Evaluation of synonymous to nonsynonymous variations was used to evaluate the selection process for HSP70 family genes. Also, the sequence classification was done based on HSP types and clusters in each HSP. In initial and final parts of arranged sequences, disorders were seen but due to similarity of disorder elimination results to results of their inclusion, the nucleotides present in these regions were not eliminated for analysis. To determine the substitution rate of nucleotides in purine and pyrimidine bases the equation 1 was used.
R= (A×G×k1 + T×C×k2)/((A+G)×(T+C)) (1)
A, G, T and C indicate of adenine, guanine, thymine and cytosine, respectively, and k1 shows transition/transversion ratio for purine bases and k2 is the same ratio for pyrimidine bases [5].
RESULTS AND DISCUSSION
Evaluation of synonymous to nonsynonymous changes is an easy method to recognize natural selection in protein level. If this ratio is higher than one, the selection is positive, if lower than one the selection is purifying and if equal to one the selection is neuter [1]. Nucleotide frequency in sequences for purine and pyrimidine bases and k1 and K2 ratio were estimated 0.466, 0.534, and 2.267, respectively. The results of synonymous and nonsynonymous evaluations showed that selection in the levels of these family proteins in cow has been purifying selection, and this selection type in protein level plays an important role in evolution of these family genes. Finally, it is expected that some changed be made in structure of HSP70 gene family during decades of evolution. As shown in Figure 1, the vertical row shows substitution rate and horizontal row also shows Substitution type. Substitution rate is related to pyrimidine bases including thymine to cytosine (16.63%) and cytosine to thymine (11.91%), and the lowest substitution is guanine to cytosine (6.74%) and guanine or adenine to thymine (4.83%) compatible with substitution rate and SNP in gene sequences of other sources mentioning the highest SNP for pyrimidine bases [4, 5].
Figure 1. substitution rate of purine and pyrimidine
CONCLUSION
Regarding the presence of various mutations during gene evolution, selection in protein level of this gene family in cow has been purifying selection. Existence of purifying selection shows minor changes in this gene family incapable of causing new varieties and as a consequence production of proteins with novel functions, but it has played an important role in during their evolution.
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