Wang X et al. (2016), A Comprehensive Analysis of the Phylogeny, Geno...

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PLoS One 2016 Jan 01;112:e0147704. doi: 10.1371/journal.pone.0147704.

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A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

Wang X , Cheng G , Lu Y , Zhang C , Wu X , Han H , Zhao Y , Ren L .

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Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

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Species referenced: Xenopus
Genes referenced: eif2ak3 gnaz hira igl mrpl40 rpia rpl19 rpl31 slc9a4 suclg1

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	Fig 1. Schematic map of the Alligator sinensis immunoglobulin light chain gene loci.(A) Schematic map of the Alligator sinensis immunoglobulin light chain λ gene loci. (B) Schematic map of the Alligator sinensis immunoglobulin light chain κ gene loci. V: variable gene segments; J: joining gene segments; C: constant region gene; pseudo-variable gene segments were not be shown in the figure.
	Fig 2. Southern blotting detection of the Alligator sinensis Ig light chain C gene segments.Genomic DNA was digested with restriction endonucleases, which are indicated above each lane, and hybridized with probes for Cλ and Cκ, respectively.
	Fig 3. Phylogenetic tree analysis of the 86 Alligator sinensis Vλ genes.A phylogenetic tree of the nucleotides of 86 Alligator sinensis Vλ segments was constructed. The 19 Vλ gene families are labeled with numbers on the right. Phylogenetic trees were constructed using MrBayes3.1.2 [58] and viewed in TREEVIEW [59].
	Fig 4. Phylogenetic tree analysis of the 62 Alligator sinensis Vκ genes.A phylogenetic tree of the nucleotides of the Alligator sinensis Vκ segments was constructed. The 12 Vκ gene families are labeled with numbers on the right. Phylogenetic trees were constructed using MrBayes3.1.2 [58] and viewed in TREEVIEW [59].
	Fig 5. Phylogenetic analysis of the IgL chain C genes in jawed vertebrates.The phylogenetic tree was constructed using C domains. The scale shown as a bar represents the genetic distance (number of nucleotide changes at the given scale). The credibility value for each node is shown. Phylogenetic trees were constructed using MrBayes3.1.2 [58] and viewed in TREEVIEW [59].
	Fig 6. Phylogenetic analysis of the IgL chain V genes in jawed vertebrates.The phylogenetic tree was constructed using V domains. The scale shown as a bar represents the genetic distance (number of nucleotide changes at the given scale). The credibility value for each node is shown. Phylogenetic trees were constructed using MrBayes3.1.2 [58] and viewed in TREEVIEW [59].
	Fig 7. Chromosomal locations of the λ genes in different species and type III genes in X. tropicalis.Arrows indicate the transcriptional orientation of the genes. Chr: chromosome; IGLC: immunoglobulin λ chain constant region gene; GNAZ: guanine nucleotide-binding protein, α z subunit; HIRA: histone cell cycle regulation defective homologue A; MRPL40: mitochondrial ribosomal protein L40; RTDR1: rhabdoid tumor deletion region gene 1. The figure was modified from Ref. [31].
	Fig 8. Chromosomal locations of the κ genes in different species and ρ genes in X. tropicalis.Arrows indicate the transcriptional orientation of the genes. Chr: chromosome; IGκC: immunoglobulin κ chain constant region gene; RPIA: ribose-5-phosphate isomerase A; EIF2AK3: eukaryotic translation initiation factor 2-α kinase 3; SUCLG1: succinate-CoA ligase, GDP-forming, α subunit; SLC4A11: solute carrier family 4, sodium borate transporter, member 11; SLC9A4; solute carrier family 9, sodium borate transporter, member 4; RPL31: ribosomal protein L31; RPL19-PS: ribosomal protein L19, pseudogene 8; RPL19: ribosomal protein L19. SLC4A1: solute carrier family 4, sodium borate transporter, member 1. The figure was modified from Ref. [31].
	Fig 9. Usage frequency of Vλ and Jλ genes in the Alligator sinensis.The number behind the Vλ indicates the number of the family.
	Fig 10. Usage frequency of Vκ and Jκ genes in the Alligator sinensis.The number behind the Vκ indicates the number of families.

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