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Genomic analysis offers insights into the evolution of the bovine TRA/TRD locus.

Connelley TK, Degnan K, Longhi CW, Morrison WI - BMC Genomics (2014)

Bottom Line: Both TRA and TRD selection have contributed to the evolution of the bovine TRAV/TRDV repertoire.However, our data suggest that due to homology unit duplication TRD selection for TRDV1 subgroup expansion may have substantially contributed to the genomic expansion of several TRAV subgroups.Such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine TRAV/TRDV repertoire.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian EH25 9RG, Scotland, UK. timothy.connelley@ed.ac.uk.

ABSTRACT

Background: The TRA/TRD locus contains the genes for V(D)J somatic rearrangement of TRA and TRD chains expressed by αβ and γδ T cells respectively. Previous studies have demonstrated that the bovine TRA/TRD locus contains an exceptionally large number of TRAV/TRDV genes. In this study we combine genomic and transcript analysis to provide insights into the evolutionary development of the bovine TRA/TRD locus and the remarkable TRAV/TRDV gene repertoire.

Results: Annotation of the UMD3.1 assembly identified 371 TRAV/TRDV genes (distributed in 42 subgroups), 3 TRDJ, 6 TRDD, 62 TRAJ and single TRAC and TRDC genes, most of which were located within a 3.5 Mb region of chromosome 10. Most of the TRAV/TRDV subgroups have multiple members and several have undergone dramatic expansion, most notably TRDV1 (60 genes). Wide variation in the proportion of pseudogenes within individual subgroups, suggest that differential 'birth' and 'death' rates have been used to form a functional bovine TRAV/TRDV repertoire which is phylogenetically distinct from that of humans and mice. The expansion of the bovine TRAV/TRDV gene repertoire has predominantly been achieved through a complex series of homology unit (regions of DNA containing multiple gene) replications. Frequent co-localisation within homology units of genes from subgroups with low and high pseudogene proportions suggest that replication of homology units driven by evolutionary selection for the former may have led to a 'collateral' expansion of the latter. Transcript analysis was used to define the TRAV/TRDV subgroups available for recombination of TRA and TRD chains and demonstrated preferential usage of different subgroups by the expressed TRA and TRD repertoires, indicating that TRA and TRD selection have had distinct impacts on the evolution of the TRAV/TRDV repertoire.

Conclusion: Both TRA and TRD selection have contributed to the evolution of the bovine TRAV/TRDV repertoire. However, our data suggest that due to homology unit duplication TRD selection for TRDV1 subgroup expansion may have substantially contributed to the genomic expansion of several TRAV subgroups. Such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine TRAV/TRDV repertoire.

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Usage of TRAV/TRDV subgroups in the expressed TRA and TRD chain repertoires. cDNA from CD3+γδ- (αβ T cells) and CD3+γδ+ (γδ T cells) populations isolated from the PBMC of 2 animals were subjected to unbiased SMART TRAC+/ TRDC+ PCR amplification and representative amplicons sequenced. The subgroup of the TRAV/TRDV gene expressed by each TRA and TRD chain sequence that was predicted from in silico analysis to be functional (for TRA transcripts n = 60 and 61 and for TRD transcripts n = 68 and 63 for Animals A and B respectively) was identified. The percentage of the TRA transcripts (A) and TRD transcripts (B) that utilised genes from the different TRAV/TRDV subgroups is shown (grey and black bars) alongside the percentage of the genes in the genomic repertoire available for TRA and TRD rearrangement within each TRAV/TRDV subgroup (striped bars).
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Fig3: Usage of TRAV/TRDV subgroups in the expressed TRA and TRD chain repertoires. cDNA from CD3+γδ- (αβ T cells) and CD3+γδ+ (γδ T cells) populations isolated from the PBMC of 2 animals were subjected to unbiased SMART TRAC+/ TRDC+ PCR amplification and representative amplicons sequenced. The subgroup of the TRAV/TRDV gene expressed by each TRA and TRD chain sequence that was predicted from in silico analysis to be functional (for TRA transcripts n = 60 and 61 and for TRD transcripts n = 68 and 63 for Animals A and B respectively) was identified. The percentage of the TRA transcripts (A) and TRD transcripts (B) that utilised genes from the different TRAV/TRDV subgroups is shown (grey and black bars) alongside the percentage of the genes in the genomic repertoire available for TRA and TRD rearrangement within each TRAV/TRDV subgroup (striped bars).

Mentions: To determine at what frequencies the different TRAV/TRDV subgroups were utilised in the expressed TRA and TRD repertoires, we used a SMART RACE-PCR system that enables unbiased amplification of TR chain sequences [24] to analyse TRA and TRD transcripts from αβ and γδ T cells of 2 MHC-disparate animals. TRA chain analysis showed utilisation of a diverse range of TRAV/TRDV subgroups (19 and 23 subgroups) in both animals (Figure 3A). Although most subgroups were represented at low frequencies (<5%), in each animal there was a limited number of subgroups (4 or 5) that constituted a higher proportion of the repertoire. Although these highly represented subgroups generally differed between the 2 individuals, both animals showed a particularly high representation of TRAV3 (11.3% and 8.2%) and TRAVX (23% and 22.6%). Comparison with the functional genomic TRAV/TRDV gene repertoire revealed that these two TRAV/TRDV subgroups, as well as TRAV17, 27 and 29, were relatively over-represented in the expressed TRA chain repertoire in both animals, whilst TRAV26 and TRDV1 were under-represented (Figure 3A). With reference to the phylogenetic groups discussed above, Groups 1 and 2 each accounted for approximately ~45% of the expressed TRA chain sequences whilst the remaining ~10% used genes from Group 4. Thus, in comparison to the genomic repertoire there is a preference for use of Group 1 and 2 genes and bias against use of Group 4 genes.Figure 3


Genomic analysis offers insights into the evolution of the bovine TRA/TRD locus.

Connelley TK, Degnan K, Longhi CW, Morrison WI - BMC Genomics (2014)

Usage of TRAV/TRDV subgroups in the expressed TRA and TRD chain repertoires. cDNA from CD3+γδ- (αβ T cells) and CD3+γδ+ (γδ T cells) populations isolated from the PBMC of 2 animals were subjected to unbiased SMART TRAC+/ TRDC+ PCR amplification and representative amplicons sequenced. The subgroup of the TRAV/TRDV gene expressed by each TRA and TRD chain sequence that was predicted from in silico analysis to be functional (for TRA transcripts n = 60 and 61 and for TRD transcripts n = 68 and 63 for Animals A and B respectively) was identified. The percentage of the TRA transcripts (A) and TRD transcripts (B) that utilised genes from the different TRAV/TRDV subgroups is shown (grey and black bars) alongside the percentage of the genes in the genomic repertoire available for TRA and TRD rearrangement within each TRAV/TRDV subgroup (striped bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289303&req=5

Fig3: Usage of TRAV/TRDV subgroups in the expressed TRA and TRD chain repertoires. cDNA from CD3+γδ- (αβ T cells) and CD3+γδ+ (γδ T cells) populations isolated from the PBMC of 2 animals were subjected to unbiased SMART TRAC+/ TRDC+ PCR amplification and representative amplicons sequenced. The subgroup of the TRAV/TRDV gene expressed by each TRA and TRD chain sequence that was predicted from in silico analysis to be functional (for TRA transcripts n = 60 and 61 and for TRD transcripts n = 68 and 63 for Animals A and B respectively) was identified. The percentage of the TRA transcripts (A) and TRD transcripts (B) that utilised genes from the different TRAV/TRDV subgroups is shown (grey and black bars) alongside the percentage of the genes in the genomic repertoire available for TRA and TRD rearrangement within each TRAV/TRDV subgroup (striped bars).
Mentions: To determine at what frequencies the different TRAV/TRDV subgroups were utilised in the expressed TRA and TRD repertoires, we used a SMART RACE-PCR system that enables unbiased amplification of TR chain sequences [24] to analyse TRA and TRD transcripts from αβ and γδ T cells of 2 MHC-disparate animals. TRA chain analysis showed utilisation of a diverse range of TRAV/TRDV subgroups (19 and 23 subgroups) in both animals (Figure 3A). Although most subgroups were represented at low frequencies (<5%), in each animal there was a limited number of subgroups (4 or 5) that constituted a higher proportion of the repertoire. Although these highly represented subgroups generally differed between the 2 individuals, both animals showed a particularly high representation of TRAV3 (11.3% and 8.2%) and TRAVX (23% and 22.6%). Comparison with the functional genomic TRAV/TRDV gene repertoire revealed that these two TRAV/TRDV subgroups, as well as TRAV17, 27 and 29, were relatively over-represented in the expressed TRA chain repertoire in both animals, whilst TRAV26 and TRDV1 were under-represented (Figure 3A). With reference to the phylogenetic groups discussed above, Groups 1 and 2 each accounted for approximately ~45% of the expressed TRA chain sequences whilst the remaining ~10% used genes from Group 4. Thus, in comparison to the genomic repertoire there is a preference for use of Group 1 and 2 genes and bias against use of Group 4 genes.Figure 3

Bottom Line: Both TRA and TRD selection have contributed to the evolution of the bovine TRAV/TRDV repertoire.However, our data suggest that due to homology unit duplication TRD selection for TRDV1 subgroup expansion may have substantially contributed to the genomic expansion of several TRAV subgroups.Such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine TRAV/TRDV repertoire.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian EH25 9RG, Scotland, UK. timothy.connelley@ed.ac.uk.

ABSTRACT

Background: The TRA/TRD locus contains the genes for V(D)J somatic rearrangement of TRA and TRD chains expressed by αβ and γδ T cells respectively. Previous studies have demonstrated that the bovine TRA/TRD locus contains an exceptionally large number of TRAV/TRDV genes. In this study we combine genomic and transcript analysis to provide insights into the evolutionary development of the bovine TRA/TRD locus and the remarkable TRAV/TRDV gene repertoire.

Results: Annotation of the UMD3.1 assembly identified 371 TRAV/TRDV genes (distributed in 42 subgroups), 3 TRDJ, 6 TRDD, 62 TRAJ and single TRAC and TRDC genes, most of which were located within a 3.5 Mb region of chromosome 10. Most of the TRAV/TRDV subgroups have multiple members and several have undergone dramatic expansion, most notably TRDV1 (60 genes). Wide variation in the proportion of pseudogenes within individual subgroups, suggest that differential 'birth' and 'death' rates have been used to form a functional bovine TRAV/TRDV repertoire which is phylogenetically distinct from that of humans and mice. The expansion of the bovine TRAV/TRDV gene repertoire has predominantly been achieved through a complex series of homology unit (regions of DNA containing multiple gene) replications. Frequent co-localisation within homology units of genes from subgroups with low and high pseudogene proportions suggest that replication of homology units driven by evolutionary selection for the former may have led to a 'collateral' expansion of the latter. Transcript analysis was used to define the TRAV/TRDV subgroups available for recombination of TRA and TRD chains and demonstrated preferential usage of different subgroups by the expressed TRA and TRD repertoires, indicating that TRA and TRD selection have had distinct impacts on the evolution of the TRAV/TRDV repertoire.

Conclusion: Both TRA and TRD selection have contributed to the evolution of the bovine TRAV/TRDV repertoire. However, our data suggest that due to homology unit duplication TRD selection for TRDV1 subgroup expansion may have substantially contributed to the genomic expansion of several TRAV subgroups. Such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine TRAV/TRDV repertoire.

Show MeSH