Download Gene conversion analysis of the mouse Pilr locus

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.