Affinity Of The Zα Domain From A CaPKR-like To Nucleic Acids And Adaptive Evolution In Several Proteins As Chemokines

The Carassius auratus protein kinase PKR-like (CaPKR-like), belongs to the Z-DNA binding protein family, contains an N-terminal which is composed of two Z-DNA binding domains (Zα1 and Zα2). The CαPKR-like Zαpeptides, wild type (P_Zα1, P_Zα2 and P_Zα) and mutant type (P_Zα^38N and P_Zα^60W), were expressed by a prokaryotic expression system and then purified by affinity chromatography. Bandshift assay analyzed the affinity of Zαpeptides bind to pMD18-T plasmid, d(GC)₁₃ recombined plasmid and Poly I:C. The results indicate that none of P_Zα1, P_Zα1 and P_Zα can bind to pMD18-T. But P_Zα, rather than P_Zα1 and P_Zα2, can bind to d(GC)₁₃ recombined plasmid and Poly I:C. Site-directed mutant peptides P_Zα^38N and P_Zα^60W restrict P_Zα bind with nucleotide acids, suggest that 38N and 60W two amino acids are important to the process of P_Zα bind to nucleotide acids. In addition, ethidium bromize (EB) is much to damage the binding of P_Zα to nucleotide acids.Codon-Substitution Models are statistical methods for detecting molecular adaptation. The models not only can indicate whether the genes of phylogeny under the positive selection or not, but also forecast these codon sites under the positive selection, which promote gene divergence and polymorphism. For the protein coding genes, the detecting and identifying of positive selection sites is importance to understanding the structure and function of protein. Here, used the models to analyze the adaptation evolution of Zαhomology sequences and didn't find positive selection sites in Zαdomain. It showed Zαsequences have high conservation in evolution.Used the models to analyze the molecular adaptation of interferon genes, found that interferon genes suffered the positive selection, and then located the positive selection sites of interferon sequences.Here also used the models to analyze the molecular adaptive evolution of transferrins. Althrough be conservative, the result showed transferrins suffered the positive selection effect during the molecular evolution. Most of the residues identified as likely to be under positive selection were focus on the C-lobe of transferrins. And their secondary structures are not the alpha helix or the beta sheet. These positive selection sites may influence the difference of bind iron between the N-lobe and C-lobe of transferrins.Chemokines are chemotactic cytokines that can induce immune cells migration, conducting their functions via chemokine receptors. In the test, the molecular adaptation of chemokines and chemokines receptors genes had been analyzed by models. The results showed that only a few of genes, such as RANTES and CCRS, are suffered the positive selection. Several CCR5 positive selection sites are located in a region that involved in binding to receptor and its chemokine.