Polymorphic Analysis And Detecting Positive Selection Of SLA Classical Class Ⅰ Genes

Major histocompatibility complex(MHC) antigen was such gene cluster that linked with certain chromosome encoding a group of cell membrance glycoprotein that correlated with immune response. MHC classⅠgenes encode the MHC classⅠmolecule that take responsibility of the presentation of endogenous antigen,correlated with the organism immune response,and act in graft rejective reaction, which have been investgated much in Human and Mouse.In this article,these methods of RT-PCR, cloning,PCR-SSCP and DNA sequencing were used to study the polymorphism of the second exon and third exon of three SLA classⅠgenes in Congjiangxiang(CJ) pig and Dahuabai(DHB) pig,the phylogenetic and selective mechanisms were analysised.Further,some antigen reconized sites(ARSs) were proposed by detecting positive selection at single amino acid sites in SLA classⅠgenes,which provide the rationale bases for the studies of polymorphism,adaptive evolution,antigen presentation of SLA classⅠgenes.The results were as follows:1.By aligning with alleles of SLA classⅠgenes from Genbank,53 new alleles of SLA-1,30 new alleles of SLA-2,31 new alleles of SLA-3 were found in the 222 sequences obtained by DNA sequencing and 12 alleles on Genbank were confirmed in this study,.Each of the new alleles was given a systematical naming following the principles of systematic nomenclature system for SLA classⅠalleles by the ISAG Nomenclature Committee,and the phylogenetic trees of alleles were constructed.2.Exons 2 and 3 of the three SLA classic classⅠgenes are highly polymorphic.71 variable sites(57 parsim-information sites and 14 singleton sites),64 variable sites(50 parsim-information sites and 14 singleton sites) were respectively found in the amino acid sequences of 169 amino acid residues that encoded by the entire exon 2 and 5'part of exon 3 of SLA- 1,SLA-2 in CJ pig,while 75 variable sites(55 parsim-information sites and 20 singleton sites) were found in the sequences of 182 amino acid residues that encoded by the entire exon 2 and exon 3 of SLA-3.A insert of 9 nucleotides (AGTGGCAGG) was found between the 238 and 239 site in CDS of SLA-3*0702dh01 allele,while a insert of 3 nucleotides(TGT) was found between the 881 and 882 site in CDS of SLA-3~*05dh01 allele. Three nucleotides deletion just like SLA-3~*04sc 19(AY135586) were not detected.The sixth exon was found deleted in a certain SLA-1 allele of CJ pig,also in SLA-3 alleles of both CJ and DHB pig.3.The nucleotide content in exons 2 and 3 of the three SLA classic classⅠgenes were as follows: G＞C＞A＞T.Nucleotide contents at the 1st,2nd,3rd codon between the three genes were similar,and the 3rd codon was abundant of GC with its G+C content(87.6%) much higher than that of A+T(12.4%). Overall,No.of transversions was more than that of transitions in the three genes.Frequency of the four kinds of transversion:T-A,T-G,A-C,C-G were close to each other,but frequency of C-T was obviously less than that of A-G.Nucleotide substitution at each codon in exons 2 and 3 of the three genes were different from each other.In SLA-1,substitutions at the 2nd codon was the most,the 1st codon next, and the 3rd least.In SLA-2 and SLA-3,the most substitutions was at the 1st codon,the 2nd less,and the 3rd least.The highest transition/transversion ratio was at the 2nd codon,while that of the 1 st and 3rd codon were similar to each other.4.Comparing with the ligand sequence of NK cell receptor in HLA classⅠmolecules,the SLA classⅠmolecules of CJ and DHB pig were short of the critical amino acid residues that linking to human NK cell receptor.Theα2 domain of SLA classⅠmolecules had the same critical anmino acid redidues that recognizing and linking to human CD8 molecule compared with HLA classⅠmolecules,but variations were existed at sites of 119,121,123,126,127.And theα3 domain of SLA classⅠmolecules had variations at two critical amino acid residues:Ala-223-Val,Ala-235-Lys compared with that of HLA classⅠmolecules which recognizing and linking to CD8 molecule,also had many variations at other no-critical amino acid residues.5.Extremely significant and significant positive selection were respectively detected at the whole exons 2 and 3 of SLA-1 and SLA-2 but not SLA-3.For SLA-1,positive selection was detected by P-distance but not Jukes-cantor-distance at its exon 2,also positive selection was detected at exon 3. While for SLA-2,positive selection was detected at exon 3(0.01＜P＜0.05) but not exon 2.To consider theα-helices andβ-strands in exon 2 and exon 3 of the three genes,positive selection was also detected at theα-helices in exon 2(P＜0.01) and exon 3(0.01＜P＜0.05) of SLA-1,exon 3(0.01＜P＜0.05) of SLA-2 and exon 2(0.01＜P＜0.05) of SLA-3.No positive selection was detected at theβ-strands in exon 2 and exon 3 of both three genes(P＞0.5).Finally,to the binding cleft,TCR directed region and out directed region in exons 2 and 3,positive selection was only detected at binding cleft(P＜0.01).6.It was concluded that there was 23 positive selection sites(24,54,62,66,67,73,75,77,79,95, 97,114,116,124,138,147,151,152,155,156,163,167,169)along the SLAclassic class I molecules after combining the results of SLA-1,SLA-2,SLA-3 genes.Except 54,79,124,138 sites,the other 19 positive selection sites were as ARS sites in HLA classic classⅠmolecules.6(66,73,75,147,155,169) of the 20 sites could be as ARSs even though they were not detected to be positive selection sites in HLA classⅠmolecules,.