Progress 05/01/06 to 04/30/10
Outputs
OUTPUTS: Over the life of this award we made significant progress toward all three aims. These achievements have led to a new view of the chicken MHC. We have a desire to apply the knowledge gained. We hope to be able to use the genetic information gathered in further work to guide genetic selection for disease resistance, to define the genetic basis of antigen selection by class I molecules, and to link additional presently unmapped genes into this region of chicken chromosome 16 (GGA 16). The first objective was to extend the gene map of the chicken MHC defining the boundaries of this region with respect to adjacent segments of GGA 16. We completed a 92-kb map of the MHC-B region in the Red Jungle Fowl genome in collaboration with Takashi Shiina and our colleagues at Tokai University (see 2007 publication for the full list of collaborators). The map encompasses 46 genes and defined the locations of recombination breakpoints within the series of MHC-B recombinant lines isolated by W.E. Briles (only a portion of these breakpoints are currently published). The map helped, as will be described below, to define the genetic basis of MHC-linked resistance to Marek's disease (MD). In collaboration with Mary Delany and Charmaine Robinson (UC Davis) and using fully sequenced BAC clones for MHC-B and MHC-Y, we mapped MHC-Y, MHC-B and the NOR on GGA 16 (a microchromosome) using multicolor fluorescence in situ hybridization (FISH). Gonadal meiotic cells provided decondensed bivalents that allowed the order of the closely positioned Y and B regions to be resolved. Assembly of the MHC-Y region remains to be completed. Currently the assembly contains three contigs made from MHC-Y BAC clones, several of which are fully sequenced. Clones for the BG gene family have been isolated and await analysis. The second objective was to define genetic polymorphism across the MHC-B region. We completed experiments directed at this goal. In collaboration with Takashi Shiina and Kazu Hosomichi and others in their lab we produced sequences and analyses across 59 kb for 14 MHC-B haplotypes. The third objective was to make chicken MHC data and knowledge available to the research community through development of databases and a wiki site. We have put part of the data up on Gallus Gbrowse (http://128.175.126.109/cgi-bin/gbrowse/MHC/). We are continuing to work with Carl Schmidt (U. Delaware) to upload the additional haplotype sequence data in a form where the variability will be readily accessible. We are also working with Janet Weber, NCBI archivist, to correct nomenclature errors and miss-attributions currently present in GenBank notation for chicken MHC-B genes. Production of a wiki site elementary guide to the chicken MHC is just getting underway. PARTICIPANTS: Individuals who worked on this project are nearly all listed as authors in the publications. Our recent undergraduate interns include Nydia Ekasumara, Pomona College (Spring quarter intern) and Shaira Bhanji, Harvard University (current summer intern). TARGET AUDIENCES: This work is relevant to academics in poultry science and to breeders of poultry for eggs and meat. PROJECT MODIFICATIONS: There were not major changes from the initial project description.
Impacts
The MHC-B 92-kbp map provided important insights into the content of the MHC-B subregion that confers resistance to MD. Initially, genetic resistance to MD was linked by serological typing to polymorphic BF (class I antigens) in 1983 using the recombinant haplotype BR5 isolated by Elwood Briles. Other genes in the subregion inherited in linkage with BF were unknown until the current gene map was completed. By mapping the recombination breakpoint along the 92 kb sequence we determined that 16 genes including five loci with significant polymorphism (Trim27.1, B-BTN-2 and Blec 1) are linked with BF in the BR5 recombinant haplotype. Hence all of these then became candidates for conferring the resistance to MD disease assigned to BF by low resolution serological mapping. Sequencing of an additional pair of highly similar MHC-B recombinant haplotypes (BR2 and BR4) was also supported by this award. These are two highly similar haplotypes that confer different levels of resistance to Marek's disease. The sequences assigned the basis of this difference in MD to the BG1 gene. BR2 and BR4 differ only at the BG1 locus. The two BG1 alleles differ only by a 225 bp insertion into the 3'UTR of BR4. The BG1 locus, with intriguing variability in its structure beyond this one difference, encodes an inhibitory signaling molecule that likely modulates cell activation. This is the first time MD resistance has been definitively mapped to a single locus. Whether BF2 antigen presentation contributes to MD resistance remains to be determined. Finding a significant role for BG1 will have an impact on thinking of how MHC-B genes influence MD and other diseases. The results of the FISH study will also have a significant impact. FISH revealed the order on GGA 16 q-arm to be -- starting from the centromere -- the NOR, MHC-Y, a GC-rich insert in which a secondary constriction is present, MHC-B and telomere. The crossover which allows MHC-Y and MHC-B alleles to assort independently must occur within the newly recognized GC-rich region. Judging from genes found in the MHC in other organisms, the genes residing in this unmapped region may be important for their contributions to reproduction, as well as immunity. The sequences of the fourteen haplotypes will have a big impact on the understanding of how MHC haplotypes evolve. To my knowledge there is not another organism for which such extensive haplotype data are available. The large number of sequences reveals several different mechanisms contributing to MHC diversity. These data are very helpful for a number of purposes. They contribute to ongoing investigations of MHC-B class I presentation and the contribution of BG1 to MD disease resistance. This study also provides a benchmark with which to compare MHC organization and diversity in other vertebrates, including other birds of agricultural importance, such as the turkey. The overall understanding of the organization of the chicken MHC-B gene region and how it functions has advanced significantly through the work performed over the life of this award.
Publications
- Hee, C.S., Gao, S., Miller, M.M., Uchanska-Ziegler, B., Daumke, O., and Ziegler, A. (2010). Structure of a classical MHC class I molecule that binds non-classical ligands. In revision.
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Progress 05/01/08 to 04/30/09
Outputs
OUTPUTS: For Objective 1 (to complete an extended map of the chicken MHC -B and -Y region defining the boundaries relative to adjacent segments of chicken chromosome 16 (GGA 16) including the nucleolar organizer region, centromere, and telomeres). One output was completion of the analysis of the order of MHC genes on GGA 16. We began to define genes that might be present in the newly revealed G/C-rich region of GGA 16. Another output was work defining the sequence difference between of two highly similar recombinant MHC-B haplotypes that show highly significant differences in the incidence of Marek's disease tumors in standardized trials with the very virulent RB1B strain of Marek's disease virus. We continued to work on assembling a gene map for the MHC-Y region. We explored the possibility Solexa sequencing technology as a means for gathering additional sequences for the assembly of this region. For Objective 2 (to assess MHC variability identifying polymorphic sequences and the positions of crossover breakpoints that contribute to making new haplotypes within the regions) we completed the investigation with our collaborators at Tokai University Kazuyoshi Hosomichi, Takashi Shiina, and others into sequence variability across a region of 59 kb (from BG1 through BF2) in fourteen different MHC-B haplotypes. The sequence data were analyzed for gene structural changes, synonymous and non-synonymous polymorphisms, insertions, deletions and allelic gene rearrangements or exchanges that contribute to MHC-B haplotype diversity. Evidence for crossover breakpoints was looked for, as was evidence for whole and partial allelic gene conversion events. The sequences were closely examined for evidence of variation in copy number and gene rearrangements. In particular BG1 genes were closely examined for how alleles differ. For Objective 3 (to assemble these data into a comprehensive, useful public database to share MHC gene sequence data) we continued to work with Carl Schmidt at the University of Delaware to make the data from these studies available in Gallus Gbrowse. The availability of MHC gene data on this website is one very definite way, in addition to publications, in which the findings have been disseminated to the community of investigators and breeders interested in genetics and disease resistance in poultry. PARTICIPANTS: We trained undergraduates as part of this project. These include: Courtney Maloney, UC Berkeley; Alex Faggen, Pomona College; Thanh Nguyen, Emory University; and Rie Ohta, Brown University. Others are noted in the report above and in the list of publications. TARGET AUDIENCES: Target audience are those engaged in research into the genetics of disease resistance and those interested in evolution of the immune response. The findings, while very basic in nature, provide underpinnings for more investigations into the how to produce better chickens grown for food by genetic selection and into how to make better vaccines to protect against major diseases in poultry. PROJECT MODIFICATIONS: We made no major changes in direction.
Impacts
With Mary Delany and Charmaine Robinson at UC Davis we defined the architecture and organization of chicken chromosome 16 (GGA 16). Molecular, cytogenetic and genomic data gathered established the order of MHC (B and Y) with respect to the centromere, q-arm telomere and the nucleolar organizing region (NOR). The order is centromere, NOR, MHC-Y, a newly revealed G/C-rich region with a secondary restriction, MHC-B, and telomere. This work, a significant advance, was published as the lead, cover story article in the J. Heredity September/October 2009. Additional experiments to identify genes within this newly recognized intervening region are underway. We found the sequence difference between two recombinant haplotypes that contribute significantly differently to the incidence of tumors induced by the RB1B strain of Marek's disease virus. The only difference is the insertion of a 225 bp sequence into the 3'UTR of the BG1 locus. This finding, published in September 2009 in PNAS, is a major breakthrough in that previously resistance and susceptibility could be resolved only to the level of MHC-B haplotype. We have screened the Red Jungle Fowl (RJF) BAC libraries for inserts containing MHC-Y region genes. We have assembled these into three contigs. The sequences of the cloned segments are particularly difficult to assemble since they are filled with many similar and closely spaced MHC-Y class I genes and genes encoding C-type lectin-like sequences. A spin off project from our study of MHC-Y has resulted in the solving of the crystal structure of YF1*7.1 which has revealed interesting details for these poorly defined MHC class I genes. This finding is included in the list of publications below. A 59 kb region from BF2 (MHC class I) to BG1 (disease resistance gene) was fully sequenced in fourteen haplotypes. The sequences revealed evidence for whole and partial-allelic gene conversion events, and for homologous reciprocal recombination, as well as nucleotide mutations, but no evidence of gene copy number variation. BF2 genes may occasionally be duplicated, but the duplications are replacements for BF1 and so change in copy number. The BG1 show very intriguing variation. Variation takes the form of not only the 225 bp insertion in the 3'UTR, but also variation in the number of exons encoding intracellular domains of these molecules. This work, published in the J. Immunol. in late 2008, has had an impact on the newly emerging turkey MHC gene map. A spinoff is collaboration on the turkey MHC with K. Reed and M. Bauer, U Minn. We are using Gallus Gbrowse to provide detailed data for the chicken MHC. The sequence data from the 242 kb MHC-B map was been entered by Carl Schmidt (U. Delaware) into http://128.175.126.109/cgi-bin/gbrowse/MHC/. This Data Source contains the MHC-B Red Jungle Fowl (RJF) sequence from Shiina et al. 2007. Positions of tRNA, SNPs, microsatellites, and GC content are graphically displayed. We will add the fourteen MHC-B haplotypes. We are working with an NCBI archivist to resolve the errors that abound in the MHC-B assembly provided by the chicken genome project.
Publications
- Goto, R.M., Wang, Y., Taylor, Jr., R.L., Wakenell, P.S., Hosomichi, K., Shiina, T., Blackmore, C.S., Briles, W.E., and Miller, M.M. (2009). BG1 has a major role in MHC-linked resistance to malignant lymphoma in the chicken. Proc Natl Acad Sci U S A. 106: 16740-16745.
- Hee, C.S., Gao, S., Miller, M.M., Goto, R.M., Ziegler, A., Daumke, O., and Uchanska-Ziegler, B. (2009). Expression, purification and preliminary X-ray crystallographic analysis of the chicken MHC class I molecule YF1*7.1. Acta Cystallographica F65: 422-425.
- Delany, M.E., Robinson, C.M., Goto, R.M., and Miller, M.M. (2009). Architecture and organization of chicken micro-chromosome 16: Order of the NOR, MHC-Y and MHC-B subregions. J. Heredity 100: 507-514.
- Hosomichi, K., Miller, M.M., Goto, R.M., Wang, Y., Suzuki, S., Kulski, J.K., Nishibori, M., Inoko, H., Hanzawa, K., and Shiina, T. (2008). Contribution of mutation, recombination and gene conversion to chicken Mhc-B haplotype diversity. J. Immunol. 181: 3393-3399.
- Miller, M.M., Wang, Y., Goto, R.M., Wakenell, P.S., and Taylor, R.L. (2008). Genetic Resistance to GaHV-2 induced lymphoma in the chicken model. 11th International Conference on Malignancies in AIDS and Other Acquired Immunodeficiences. October 6-7, 2008, NIH, Bethesda, Maryland.
- Miller, M.M. (2009). Genes, variability and linkage in the chicken MHC B and Y regions. CSREES, USDA National Research Initiative Animal Genome Annual Investigator Meeting, January 9, 2009, San Diego, CA
- Miller, M.M. (2009). Genetic factors contributing to chicken MHC-B haplotype diversity. Station Report. Poultry Workshop. Plant and Animal Genome XVII, January 10 - 11, 2009, San Diego, CA.
- Bauer, M.M., Miller, M.M. Briles, W. and Reed, K.M. (2009). Assessing haplotype variation in turkey MHC class II genes using PCR/RFLP and Southern blot analysis. Plant & Animal Genome XVII, January 10 - 14, 2009, San Diego, CA.
- Miller, M.M. (2009). Microbes and genetic resistance in cancer biology. City of Hope Comprehensive Cancer Center, Cancer Biology Research Program Retreat, March 21, 2009, Industry Hills, CA.
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Progress 05/01/07 to 04/30/08
Outputs
OUTPUTS: This project is devoted to defining the genes present within the chicken major histocompatibility complex (MHC), their allelic variability, and the frequency with which they recombine to form new haplotypes. During this (second) year of the award, we completed, in collaboration with our colleagues Takashi Shiina, Kazuo Hosomichi and others at Tokai University, sequence determinations for fourteen different MHC-B haplotypes over a region of 59 kb containing 14 genes. This work has allowed not only the full breadth of the sequence diversity in these haplotypes to be seen, but also it has allowed us to recognize that variability is introduced by meiotic recombination and gene conversion, as well as mutation. A manuscript describing these results has been submitted for publication. We have made significant progress in mapping the positions of MHC-B and MHC-Y relative to the NOR. This work, done in collaboration with Mary Delany and Charmaine Robinson at UC Davis, has revealed an unexpected order of these three gene regions. High resolution in situ hybridization analysis of mitotic prometaphase and pachytene chromosomes was carried out with BAC probes specific for MHC-B and MHC-Y. The cytogenetic results revealed the presence of a heretofore unknown element on chromosome 16 and an unanticipated order of the NOR, MHC-B and MHC-Y. Consistent with our MHC-Y BAC contig assembly MHC-Y is adjacent to the nucleolus organizer region (NOR). Both are located proximally on the q arm. MHC-B is located distally on q. It is not the NOR, but rather a large GC-rich chromosomal region that separates NOR/MHC-Y from MHC-B. An abstract describing these findings has been accepted for the 2008 Poultry Science Association meeting. Another part of our sequencing efforts has focused on BG1. This locus is of interest since other work in our lab has been identified BG1 as a candidate locus affecting the incidence of Marek's disease. Allelic variability of this locus takes many forms including variations in exon number encoding the coiled-coil region, loss of terminal exons, and differences in 3'-untranslated regions as the result of genetic recombination. These variations are intriguing given the apparent role of BG1 in Marek's disease. We also determined that BAC 165I18, a candidate clone for the MHC-B region distal to CD1, does not correspond to this region, even though it presented as a likely candidate clone in Southern hybridization analyses. Sequencing revealed that this clone contained errant plasmid DNA. Our third objective in this project is to assemble data on the chicken MHC and make it publically available in a database on the web. Rather than develop this database from the ground up, as we originally envisioned it, we are providing data to Carl Schmidt at U. Delaware for inclusion in the Chicken (Gallus gallus) Genome Browser. PARTICIPANTS: Training Courtney Maloney, High School Student, cloning elements of MHC-Y. Andrea Dillon, College Senior, cloning of elements of MHC-B. Roshni Patel, College Senior, database development Dr. Yujun Wang, Research Fellow, Functional analysis of BG1 Ronald M. Goto, Senior Research Associate, Contig building, sequencing, and gene analysis TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work is going to have an ongoing impact in poultry science since it is providing significant quantities of sequence data and detailed genetic information for the MHC, a gene region that is particularly important in immune responses and to which disease resistance has been mapped.
Publications
- Iglesias, G.M., Huguet, M., Goto, R.M., Miquel, M.C., Miller, M.M. 2007. Report of new alleles of B-G or class IV genes in Camperos chickens. J. Basic and Applied Genetics 18: 29-30.
- Sherman, M., Goto, R.M. Moore, R.E., Hunt, H.D., Lee, T.D., and Miller, M.M. 2008. Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-B haplotypes associated with opposite responses to Marek's disease (Pending).
- Hosomichi, K., Miller, M.M., Goto, R.M., Wang, Y., Suzuki, S., Kulski, J.K., Nishibori, M., Inoko, H., and Shiina, T. 2008. Contribution of mutation, recombination and gene conversion to chicken Mhc-B haplotype diversity (Pending).
- Miller, M.M. 2007. MHC genes and their relationship to disease resistance. Report Contributing to the USDA NE-1016 Project. Annual Meeting September 29-30, 2007.
- Miller, M.M. 2008. Genes, Variability and Linkage in the Chicken MHC B and Y Regions. Report and Poster for the NRI Animal Genome Annual Investigator Meeting, January 11, 2008.
- Wang Y, Goto RM, Miller MM. 2008. BG1, a candidate gene affecting the occurrence of herpesvirus-associated avian lymphomas. FASEB J. 22: 856.10
- Zhang, L., Katselis, G.S, Goto, R.M., Goebel, T.W., Yokoyama, W.M., Lee, T.D., and Miller, M.M.. 2008. Proteomic Approaches to Defining Avian Natural Killer Cell Surface Proteins. FASEB J. 2008 22:620.3
- Delany, M., Robinson, C.M., Goto R. and Miller, M. 2008. A new cytogenetic model for the order of the nucleolus organizer region (NOR), major histocompatibility complex (MHC) -B and -Y on microchromosome 16 in
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Progress 05/01/06 to 04/30/07
Outputs
This project is devoted to defining the genes present within the chicken major histocompatibility complex (MHC), their allelic variability, and the frequency with which they recombine to form new haplotypes. During the first year of this award we completed a 242 kb gene map for the MHC-B haplotype in the Red Jungle Fowl. In addition to defining 42 genes and their locations, we have placed the crossover breakpoints for a number of MHC-B recombinant haplotypes on the map. We published the map in June with the position of the BR5 breakpoint. The position of the BR5 breakpoint establishes at the level of the genome which genes within the MHC-B map into the subregion conferring resistance to Marek's disease as defined by the landmark study of Elwood Briles and colleagues in 1983. Even though the MHC-B is structured differently from that in other species, many of the genes are related to loci typical of the MHC or to genes that are found in MHC paralogous regions. In
addition to classical MHC I and MHC II genes, the MHC subregion conferring disease resistance contains TRIM, C-type lectin-like, and Ig superfamily genes any of which may contribute resistance to Marek's disease. Work can now move forward to identify which gene or genes are responsible for this unusual, distinctive feature of the MHC in chickens.
Impacts
This project, devoted to building a full gene map for the MHC, will fill a gap in genome sequence data for the chicken that is not likely to be completed without concentrated effort. The strong association of the MHC with infectious disease and production traits places the MHC among the gene regions with significant potential for improving poultry. Greater knowledge of the variability within the MHC-B and MHC-Y regions and evidence of where crossover occurs will allow more intelligent selection for desired traits.
Publications
- Shiina, T., Briles, W.E., Goto, R.M., Hosomichi, K., Yanagiya, K., Shimizu, S., Inoko, H., and Miller, M.M. 2007. Extended gene map reveals TRIM, C-type lectin and Ig superfamily type genes within a sub-region of the chicken MHC-B affecting infectious disease. J. Immunol. 178: 7162-7172.
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