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Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

Ye Q, He K, Wu SY, Wan QH - PLoS ONE (2012)

Bottom Line: In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days.Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail.The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education and State Conservation Center for Gene Resources of Endangered Wildlife, College of Life Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

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Phylogenetic trees of IA (A), IIB (B), NK-Blec (C), DMB (D) and TAP (E) genes.For the turkey, chicken and quail, the coding regions of genes were extracted from genomic sequences of Mega-MHC (DQ993255), Gaga-MHC (AB268588) and Coja-MHC (AB078884), respectively. The following sequences were also included for tree building: For the IAs: AF033106 (Grus Canadensis, Grca-f51), AY387652 (Anser anser, Anan), and AB115241 (Anas platyrhynchos, Anpl-Du1); For the IIBs: L42335 (Lonchura striata, Lost), DQ490139 (Anas platyrhyncho, Anpl), AJ404372 (Acrocephalus arundinaceus, Acar-c01), AF170972 (Agelaius phoeniceus, Agph-DAB1), and FJ588549 (Pachytila belcheri, Pabe-DAB1); For the DMB: DQ268506 (Xenopus laevis, Xela); For the TAPs: AY885227 (Anas platyrhynchos, Anpl-TAP1 and Anpl-TAP2), XM_003230087 (Anolis carolinensis, Anca-TAP1), XM_003229647 (Anolis carolinensis, Anca-TAP2). The NK and Blec members of lection superfamily were all from the same genomic sequences of DQ993255, AB268588 and AB07888.
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pone-0032154-g005: Phylogenetic trees of IA (A), IIB (B), NK-Blec (C), DMB (D) and TAP (E) genes.For the turkey, chicken and quail, the coding regions of genes were extracted from genomic sequences of Mega-MHC (DQ993255), Gaga-MHC (AB268588) and Coja-MHC (AB078884), respectively. The following sequences were also included for tree building: For the IAs: AF033106 (Grus Canadensis, Grca-f51), AY387652 (Anser anser, Anan), and AB115241 (Anas platyrhynchos, Anpl-Du1); For the IIBs: L42335 (Lonchura striata, Lost), DQ490139 (Anas platyrhyncho, Anpl), AJ404372 (Acrocephalus arundinaceus, Acar-c01), AF170972 (Agelaius phoeniceus, Agph-DAB1), and FJ588549 (Pachytila belcheri, Pabe-DAB1); For the DMB: DQ268506 (Xenopus laevis, Xela); For the TAPs: AY885227 (Anas platyrhynchos, Anpl-TAP1 and Anpl-TAP2), XM_003230087 (Anolis carolinensis, Anca-TAP1), XM_003229647 (Anolis carolinensis, Anca-TAP2). The NK and Blec members of lection superfamily were all from the same genomic sequences of DQ993255, AB268588 and AB07888.

Mentions: MHC B fragment of gold pheasant has undergone frequent events and evolved two copies of NK gene (NK1 and NK2), three copies of MHC class II classical β gene (IIB1, IIB2 and IIB3), two copies of MHC class II non-classical β gene (DMB1 and DMB2), two copies of TAP gene (TAP1 and TAP2) and two copies of MHC class I gene (IA1 and IA2) (Fig. 3). However, the self-sequence dot plot of the golden pheasant MHC-B locus just revealed the duplication events of the NK, IIB and IA genes and had no evidence of recently duplicating the DMB and TAP genes (Fig. 4A), which suggested DMB and TAP genes duplicated in ancestors. Phylogenetic trees showed that the NK, IIB and IA genes all formed a highly-supportive clustering specific to the golden pheasant (>90% bootstrap values; Fig. 5). On the contrary, phylogenies of the DMB and TAP genes showed that the sequences were grouped according to gene categories rather than species; producing the DMB1 and DMB2 branches and the TAP1 and TAP2 clusters (Fig. 5D–E). As a result, multiple copies of the NK, IIB and IA genes were derived from younger intra-species duplication events, while the duplicated DMBs or TAPs were generated by ancestral triggering.


Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

Ye Q, He K, Wu SY, Wan QH - PLoS ONE (2012)

Phylogenetic trees of IA (A), IIB (B), NK-Blec (C), DMB (D) and TAP (E) genes.For the turkey, chicken and quail, the coding regions of genes were extracted from genomic sequences of Mega-MHC (DQ993255), Gaga-MHC (AB268588) and Coja-MHC (AB078884), respectively. The following sequences were also included for tree building: For the IAs: AF033106 (Grus Canadensis, Grca-f51), AY387652 (Anser anser, Anan), and AB115241 (Anas platyrhynchos, Anpl-Du1); For the IIBs: L42335 (Lonchura striata, Lost), DQ490139 (Anas platyrhyncho, Anpl), AJ404372 (Acrocephalus arundinaceus, Acar-c01), AF170972 (Agelaius phoeniceus, Agph-DAB1), and FJ588549 (Pachytila belcheri, Pabe-DAB1); For the DMB: DQ268506 (Xenopus laevis, Xela); For the TAPs: AY885227 (Anas platyrhynchos, Anpl-TAP1 and Anpl-TAP2), XM_003230087 (Anolis carolinensis, Anca-TAP1), XM_003229647 (Anolis carolinensis, Anca-TAP2). The NK and Blec members of lection superfamily were all from the same genomic sequences of DQ993255, AB268588 and AB07888.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293878&req=5

pone-0032154-g005: Phylogenetic trees of IA (A), IIB (B), NK-Blec (C), DMB (D) and TAP (E) genes.For the turkey, chicken and quail, the coding regions of genes were extracted from genomic sequences of Mega-MHC (DQ993255), Gaga-MHC (AB268588) and Coja-MHC (AB078884), respectively. The following sequences were also included for tree building: For the IAs: AF033106 (Grus Canadensis, Grca-f51), AY387652 (Anser anser, Anan), and AB115241 (Anas platyrhynchos, Anpl-Du1); For the IIBs: L42335 (Lonchura striata, Lost), DQ490139 (Anas platyrhyncho, Anpl), AJ404372 (Acrocephalus arundinaceus, Acar-c01), AF170972 (Agelaius phoeniceus, Agph-DAB1), and FJ588549 (Pachytila belcheri, Pabe-DAB1); For the DMB: DQ268506 (Xenopus laevis, Xela); For the TAPs: AY885227 (Anas platyrhynchos, Anpl-TAP1 and Anpl-TAP2), XM_003230087 (Anolis carolinensis, Anca-TAP1), XM_003229647 (Anolis carolinensis, Anca-TAP2). The NK and Blec members of lection superfamily were all from the same genomic sequences of DQ993255, AB268588 and AB07888.
Mentions: MHC B fragment of gold pheasant has undergone frequent events and evolved two copies of NK gene (NK1 and NK2), three copies of MHC class II classical β gene (IIB1, IIB2 and IIB3), two copies of MHC class II non-classical β gene (DMB1 and DMB2), two copies of TAP gene (TAP1 and TAP2) and two copies of MHC class I gene (IA1 and IA2) (Fig. 3). However, the self-sequence dot plot of the golden pheasant MHC-B locus just revealed the duplication events of the NK, IIB and IA genes and had no evidence of recently duplicating the DMB and TAP genes (Fig. 4A), which suggested DMB and TAP genes duplicated in ancestors. Phylogenetic trees showed that the NK, IIB and IA genes all formed a highly-supportive clustering specific to the golden pheasant (>90% bootstrap values; Fig. 5). On the contrary, phylogenies of the DMB and TAP genes showed that the sequences were grouped according to gene categories rather than species; producing the DMB1 and DMB2 branches and the TAP1 and TAP2 clusters (Fig. 5D–E). As a result, multiple copies of the NK, IIB and IA genes were derived from younger intra-species duplication events, while the duplicated DMBs or TAPs were generated by ancestral triggering.

Bottom Line: In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days.Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail.The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Conservation Biology for Endangered Wildlife of the Ministry of Education and State Conservation Center for Gene Resources of Endangered Wildlife, College of Life Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

Show MeSH
Related in: MedlinePlus