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Download Gene conversion analysis of the mouse Pilr locus
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Gene conversion analysis of the mouse Pilr locus. Gene conversion has played a role in shaping the mouse Pilr locus and is detected by a variety of phylogenetic and statistical methods. Phylogenetic analysis of a conserved 3 kb region (the promoter through to intron 3) suggests that Pilrb1 and Pilrb2 are more related to each other than they are to Pilra (Fig. 1B). This would be expected from a region duplicated approximately 5 MYA. However, analyses of three distinct sub-regions show different evolutionary relationships between these genes (Fig. 1C, D, and E). The 1600 bp found upstream of exon 1 of Pilra (containing the putative Pilra promoter, which we can infer by alignment with the previously described putative human PILRA promoter (1)), is more similar to Pilrb2 than Pilrb1 is to Pilrb2 (Table 1). Pair-wise alignments and phylogenetic analysis of the region comprising the coding portion of mouse exon 1 and exon 2 demonstrate that this region of Pilra is more closely related to either Pilrb1 or Pilrb2 than the latter two genes are to themselves (Fig. 1 and Table 1). The most convincing evidence of gene conversion comes from intron 1 as only one base pair distinguishes intron 1 of Pilra from Pilrb1, while 11 base pairs distinguish Pilra from Pilrb2. Overall, this strongly suggests gene conversion events have played a role in the evolution of the mouse Pilr genes. Fig. 1. Genomic structure and phylogenetic analysis of mouse Pilra, Pilrb1, Pilrb2 and Pilrp1. A) Scale diagram of Pilra, Pilrb1, Pilrb2, and Pilr-ps1 (pseudogene) gene sequences and repetitive elements. The Pilrb2 transcript (AK036467) is supported by cDNA evidence, while Pilrb2 (predicted) was deduced from alignments of Pilrb1 to our mouse 129/Sv genomic DNA. Exons 1 to 4 are homologous between Pilra, Pilrb1, and Pilrb2 (predicted). Note that simple repeats flank the repeat-free region of similarity shared by the Pilr genes. B-E) Phylogenetic trees based on alignments from various segments of a naturally occurring repeat free region shared between Pilra, Pilrb1, Pilrb2 and the Pilrb1 related pseudogene Pilr-ps1. Trees were created using the neighbor-joining method with Jukes-Cantor distances and consisted of 3041 sites in B, 1558 sites in C, 524 sites in D, and 267 sites in E. Table 1. Percent identity between specific regions of Pilra, Pilrb1 and Pilrb2. Regions of Pilra cDNA, Pilrb1 and Pilrb2 sequences were globally aligned. The EMBOSS program Needle (Implementing Needleman-Wunsch global alignment with a gap penalty 10 and Gap extension of 0.5) was used to perform pairwise global alignments. Region of gene aligned gene Pilra Pilrb1 Repeat-free region Upstream of exon 1 Exon 1 and exon 2 Intron 1 Exon 3 Repeat-free region Upstream of exon 1 Exon 1 and exon 2 Intron 1 Exon3 Percent identity (nucleotide) Pilrb1 Pilrb2 83.9 82.5 97.6 93.8 94.0 94.1 99.7 96.5 38.3 45.3 91.6 93.5 91.2 96.8 97.4 We looked for evidence of gene conversion in the same multiple alignment using the GENECONV program. Several statistically significant gene conversion events between Pilra and Pilrb1 and Pilra and Pilrb2 were detected in the upstream genomic region as well as in intron 1. The Pilr-ps1 pseudogene was retained in the alignment to add statistical power to the analysis and default GENECONV parameters were used. We assume that Pilrb1 and Pilrb2 were recently duplicated (supported by the 97% identity of the 15 kb duplicon including Pilrb1/Pilrb2) and that the duplication that created the Pilrb genes occurred anciently (all mammalian species have one PILRA gene and at least one PILRB gene) with sufficient time for divergence of non coding regions (supported by low percent identity between regions of Pilrb homologous to Pilra intron 2 and intron 3). GENECONV identified the phylogenetically-predicted intron 1 gene conversion event between Pilra and Pilrb1 as two distinct segments. The first segment begins in the last 14 bp of exon 1 and extending 64 bp into intron 1. The second segment begins after the one base difference that distinguishes Pilra intron 1 from Pilrb1 intron1 and extends 261 covering the remainder of intro one and the first 15 bp of exon 2 (shown as 71 and 261 bp respectively in Table 2). It is likely this region represents one conversion event followed by a base pair mutation in Pilrb1. The overlapping (Pilra/Pilrb1 and Pilra/Pilrb2) gene conversion events detected in intron 1 and the upstream genomic region detection suggests that a gene conversion event occurred in intron 1 before the duplication of Pilrb1 followed by a specific Pilra/Pilrb1 conversion event after the duplication. Nonoverlapping conversion events of Pilra/Pilrb2 are detected in the upstream genomic region supporting the phylogeny obtained using the upstream genomic region and suggesting post duplication conversion events occurred in this region as well (Table 2). We also used GENECONV to look for gene conversion events in exon 2 sequences between multiple species. Since GENECONV makes multiple-comparison corrections for all possible sequence pairs, we grouped the sequences by species and limited the comparisons for events occurring within a species. We performed the analysis using silent polymorphic sites only as well as using all non-monomorphic sites. We could not detect globally significant gene conversion events (corrected for multiple comparisons), although significant pair-wise gene conversion events (not corrected for multiple comparisons) were detected for Chimp (p=0.0188 length =190 bp), Mouse Pilrb1/Pilra (p=0.0164 length=173 bp), Rat Pilra/XM_344095.1 (p=0.0175 length = 64bp) and Rat Pilra/XM_222062.2 (p=0.0170 length = 69bp). The gene conversion prediction for Chimp is globally significant after correcting for multiple sequence comparisons (p=0.0213) if the rodent sequences are excluded from the analysis. Table 2. Statistically significant gene conversion events detected by GENECONV between Pilra and Pilrb1/Pilrb2. Predicted gene conversion events between Pilrb1 and Pilrb2 and Pilrb1/Pilrb2 and Pilr-ps1 are not shown as our genomic analysis suggests that the similarity is due to recent gene duplication and not gene conversion. Gene pair Region of gene involved Position in alignment Length (bp) P-value Pilra/Pilrb1 Pilra/Pilrb1 Pilra/Pilrb1 Pilra/Pilrb2 Pilra/Pilrb2 Pilra/Pilrb2 Pilra/Pilrb2 Pilra/Pilrb2 Pilra/Pilrb2 upstream genomic intron 1 intron 1 upstream genomic upstream genomic upstream genomic upstream genomic intron 1 intron 1 1433-1570 2021-2281 1934-2004 1475-1683 141-287 336-443 576-673 1889-1958 2164-2275 138 261 71 209 147 108 98 70 112 0.0008 0.0000 0.0109 0.0000 0.0000 0.0013 0.0131 0.0178 0.0006 References 1. Trinklein ND, Aldred SJF, Saldanha AJ, and Myers RM. Identification and Functional Analysis of Human Transcriptional Promoters. Genome Res 13: 308-312, 2003.