GENETIC ANALYSIS OF P53 GENE USING UNWEIGHTED PAIR GROUP METHOD WITH ARITHMETIC MEAN AND NEIGHBOR JOINING METHODS.

The p53 molecular network is a master regulator of cellular process and control various signaling pathway, including cell apoptosis, senescence, cellular growth and DNA repair mechanism. Many species have evolved with unique environmental and genetic responses, we tested the related hypothesis that the evolutionary dynamics of, and mode of selection on, genes within the p53 network differs between human and other species, and that these differences may underlie the evolution of p53 in different species. The sequence of human tumor protein p53 (TP53), mRNA, complete cds sequence was retrieved from the NCBI in FASTA format and was studied for its relationship and percent similarity within human and others species. Variation in p53 gene in human and comparative analysis were studied in detail. The phylogenetic analysis was performed using unweighted pair group method with arithmetic mean (UPGMA) and neighbor joining (NJ) methods and revealed the relationships and percent similarity of Homo sapiens TP53, mRNA, complete cds among different organisms especially with monkey, guinea pig, golden hamster, tupaia, whale, rabbit, pig, cat, sheep and cattle. We observed that positive selection of TP53 gene during the divergence of different species during evolution. clusters showing their evolution relationship with each other and NJ tree reveals different clade showing their evolutionary distance within different species. The sequences which lie in the same cluster are closely related.

The p53 molecular network is a master regulator of cellular process and control various signaling pathway, including cell apoptosis, senescence, cellular growth and DNA repair mechanism. Many species have evolved with unique environmental and genetic responses, we tested the related hypothesis that the evolutionary dynamics of, and mode of selection on, genes within the p53 network differs between human and other species, and that these differences may underlie the evolution of p53 in different species. The sequence of human tumor protein p53 (TP53), mRNA, complete cds sequence was retrieved from the NCBI in FASTA format and was studied for its relationship and percent similarity within human and others species. Variation in p53 gene in human and comparative analysis were studied in detail. The phylogenetic analysis was performed using unweighted pair group method with arithmetic mean (UPGMA) and neighbor joining (NJ) methods and revealed the relationships and percent similarity of Homo sapiens TP53, mRNA, complete cds among different organisms especially with monkey, guinea pig, golden hamster, tupaia, whale, rabbit, pig, cat, sheep and cattle. We observed that positive selection of TP53 gene during the divergence of different species during evolution. 559 as DNA damage, exposure to radiation or chemical, genomic instability and others. The p53 controls the development of cancerous cell and suppresses tumor development which is the basic characteristic of tumor suppressor. p53 was first discovered in 1979 as the main interacting partner of the viral SV40 T-antigen (Chang et al., 1979;Kress et al., 1979;Lane and Crawford, 1979;Linzer and Levine, 1979), and rose to reputation of a tumor suppressor gene in late 1980s (Weisz et al., 2007). Since then, p53 has been one of the most intensively studied proteins worldwide, it is mainly due to the evidence that most of human malignancies bear the abovementioned alterations in its signaling pathway. Indeed, genetic mutations inactivating normal p53 functions can be found in various human tumors, with percentages that vary from nearly 50% in ovary cancer to 5.8% in cervical tumors (Bouaoun et al., 2016).
The p53 protein is encoded by the tumor protein p53 (TP53) gene. Its expression is modulated by different physiologic and pathologic stimuli to orchestrate adequate cellular responses in order to maintain genomic stability. p53 is actively involved in multiple signaling pathways and has diverse functional output at cellular level (Chumakov et al., 2007). Studies delving into this relationship between p53 and lifespan have focused on one of the pathways within the multi-pathway p53 signaling network. Additionally, previous observations have demonstrated that mutant p53 with increased activity of the protein can lead to shortened life span and early aging in mice with aging phenotypes such as reduced longevity, osteoporosis, organ atrophy, and reduced stress tolerance (Tyner et al., 2002).
The excessive p53 cell fate decisions can lead to tissue atrophy and degeneration caused by apoptosis and loss of tissue renewal or regeneration caused by senescence. However, it is important to note that mice with super-p53 or, extra copies of p53, display increased tumor suppression without consequence and may live longer (Matheu et al., 2007). Therefore, there is a fine balance between too little p53 and too much p53, and the data on the regulation of the p53 network and aging are equivocal. It has been proposed that since p53 is beneficial at an early age, it can drive aging phenotypes later in life (Campisi 2005). This theory holds optimal as the effects of alleles that are beneficial for an organism early in life can become deleterious at a later stage. Therefore, p53 responses can protect an organism from cancer early in life but can also promote aging phenotypes later. It also indicates that function of p53 could modulated in different species during evolution and divergence of species from same ancestors.
The current study was therefore designed with the following objectives: 1. To understand the evolutionary pattern of p53 gene in various species, including human 2. To identify the evolutionary dynamics and molecular evolution of the genes contained within the p53 network.
We performed this analysis by generating comparative transcriptomes across various species, and by comparing pathway evolution between different species of mammals and others. We also examined genes in the pathway individually and the pathway as a whole using a large multi-gene dataset. We queried the sequences for signatures of selectionboth purifying and positive, to determine if the ancestral species branch and whether the entire species clade experienced different rates and forms of evolutionary history between the species. Additionally, the evolution of the p53 network in human was examined and we compared it to other human as well as other species because p53 network signaling also has implications in broad range of cellular functions.

Phylogenetic analysis:-
In the phylogenetic analysis, alignment of nucleotide or amino acid sequences is a major consideration, particularly in studies of genes from divergent taxa (e.g. human). It seems obvious to state that the phylogenetic analysis of sequences begins with the appropriate alignment of the data themselves, yet alignment remains one of the most difficult and poorly understood facets of molecular data analysis. Alignment of the genomic sequences is required to analyze the phylogenetic tree. Phylogenetic analysis often includes the search for evidence of directional selection in molecular evolution (Hsu et al., 2005;Hofmann et al., 2003). Evolution of the TP53 was studied in different organisms and adaptive changes were in the sequences. The phylogenetic analysis of the p53 gene dataset resulted in a tree consistent with modern systematic understanding of the relatedness among different species, mainly based on DNA sequences homology. This provides strong support for the quality of our sequences and bioinformatics methods. The posterior probability values at the nodes indicate strong support for the branch splits, providing further support for this tree (demonstrated in Figure 1 and 2).
The phylogenetic tree based on UPGMA and NJ method for human and other species were clustered into the two major clades: with human and monkey species grouped together and other species such as whale, rabbit, pig, cat, sheep and cattle, as expected. In additional, we observed the species of hamster, tupaia and guinea pig made separate lineages. Phylogenetic analysis of Homo sapiens TP53, mRNA sequence was performed through the MEGA7 software using Tamura-Nei algorithm (Tamura et al., 1993). The UPGMA rooted tree diagram of Homo sapiens TP53, transcript mRNA sequences showed different clusters formation. Organism that originated from same ancestors having same e-value and 100% pair wise identity, are placed in same clusters whereas those which are distant from each other are placed in separate clusters ( Figure 1).
The optimal tree with the sum of branch length = 0.24911474 is shown. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 43 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 31 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Figure 1).
Our observation based on phylogenetic tree using UPGMA method revealed that the majority of human p53 sequences are lying in the same clusters, as expected. Additionally, Macaca species p53 nucleotide sequences are also grouped with human cluster, represented as Group A. However, the p53 gene sequence from different species such as whale, rabbit, pig, cat, sheep and cattle lie mainly in second cluster, named as Group B. In addition, some species are separated from both groups, these species are as follows: Mesocricetus auratus TP53, mRNA (U07182.1), Tupaia belangeri chinensis TP53 mRNA, complete cds (AF175893.1), and Cavia porcellus TP53, mRNA (AJ009673.1). Observations suggest that human and monkey p53 gene are evolved nearly similar species or 567 sharing common ancestor. Results also suggest that Group A members p53 gene may be evolved from Group B or both the species have evolved it from the same ancestor (Figures 1 and 2).
The evolutionary history was inferred using the NJ method. The optimal tree with the sum of branch length = 0.26666605 is shown in Figure 2. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 43 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There was a total of 31 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.
We have also analyzed phylogenetic tree using NJ method to determine the evolutionary distance between the TP53 genes in the different species. The principle of NJ method is to find pairs of operational taxonomic units (OTUs; neighbors) that minimize the total branch length at each stage of clustering of OTUs starting with a star like tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method (Saitou et al., 1987). We have retrieved TP53 gene of selected species human, monkey, Guinea pig, Golden hamster, Tupaia, whale, rabbit, pig, cat, sheep and cattle. NJ tree also indicated similar pattern observed using UPGMA based phylogenetic tree. Results revealed that human and monkey have more similar p53 nucleotide sequence compared to other species. However, these both species p53 nucleotide sequences are distinct to Group B member's p53 sequence, including whale, rabbit, pig, cat, sheep and cattle. Interestingly, p53 gene sequence belongs to the species Mesocricetus auratus (U07182.1), Tupaia belangeri chinensis (AF175893.1), and Cavia porcellus (AJ009673.1) are represented homology between both groups. While Mesocricetus auratus TP53, nucleotide sequence is demonstrated more homology with group A, suggesting that Group A member species and Mesocricetus auratus p53 gene are evolved from common ancestor. Observation also suggest that the TP53 genes of human and monkey encoded appropriate similar protein depicted as Group A in figure 2. However, TP53 gene of group B is variable compare to Group A. Group B cluster indicate homology of TP53 gene in whale, rabbit, pig, cat, sheep and cattle. During the study, our finding showed that, Group A cluster member species had more conserved TP53 gene sequence compare to other species.
The observations based on phylogenetic analysis of TP53 gene using UPGMA and NJ method revealed the relationships and percent similarity of TP53 gene within human and others species. Genetic variation among TP53 found in human beings and other organisms were studied in detail. Phylogenetic analysis and multiple sequence alignment of the Homo sapiens TP53, mRNA, complete cds sequence through various phylogenetic tree were performed which showed its pattern of variations and relationship among different organisms especially with rat, mouse and chimpanzee. Our study demonstrated that positive selection of TP53 gene during the divergence of different species during evolution. It is known that homoiothermy and viviparity first appeared among synapsids. These evolutionary acquirements have made necessary changes in the genetic control of ontogeny, and this, in turn, might have caused adaptive changes in the p53 gene.
The findings from the current study will help in modern research strategies through the manipulation and exploitation of p53, as its pathways are promising and one can predict its extensive clinical to control the regulation of p53 in cancer development and biological use in the future for the human benefit worldwide. Furthermore, this study will allow a better understanding of evolutionary aspect of p53 gene in different species. Further study of p53 superfamily proteins in human and other species will further promote our understanding of not only how functional p53 and their functions have evolved in different species but will also contribute to our understanding of the p53 function and evolutionary history.