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dc.contributor.advisor Czerny, Claus-Peter Prof. Dr. Dr.
dc.contributor.author Walther, Stefanie
dc.date.accessioned 2016-06-10T08:06:06Z
dc.date.available 2016-06-10T08:06:06Z
dc.date.issued 2016-06-10
dc.identifier.uri http://hdl.handle.net/11858/00-1735-0000-0028-8776-A
dc.language.iso eng de
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc 630 de
dc.title Organization and transcription analyses of the immunoglobulin genes in cattle and horses de
dc.type doctoralThesis de
dc.contributor.referee König, Sven Prof. Dr.
dc.date.examination 2016-05-02
dc.description.abstracteng Initial studies on genetic aspects of immunoglobulins were performed on humans and mice but were successfully applied to various other animals such as chicken, rabbit, swine, cattle, and horses, too. Especially in cattle and horses, fundamental research in immunoglobulin genetics still needs more attention to complete previous information such as the number of available gene segments, gene families, and allotypes of different isotypes of the immunoglobulin heavy and light chains. Results will enable the analysis and generation of synthetic recombinant antibodies, as well as an alternating treatment of infectious diseases to prevent resistance to antibiotics. As reviewed in the first publication, the understanding of the organization of equine immunoglobulin genes has increased significantly in the recent years. For equine heavy chains, 52 immunoglobulin heavy chain variable gene segments (IGHV), 40 immunoglobulin heavy chain diversity gene segments (IGHD), 8 immunoglobulin heavy chain joining gene segments (IGHJ) and 11 immunoglobulin heavy chain constant region genes (IGHC) are present. Seven of these IGHCs are gamma chain genes. Sequence diversity is increasing between fetal, neonatal, foal and adult age. The kappa light chain contains 60 immunoglobulin kappa light chain variable gene segments (IGKV), 5 immunoglobulin kappa light chain joining gene segments (IGKJ) and 1 immunoglobulin kappa light chain constant region gene (IGKC), whereas there are 144 immunoglobulin lambda light chain variable gene segments (IGLV), 7 immunoglobulin lambda light chain joining gene segments (IGLJ), and 7 immunoglobulin lambda light chain constant region genes (IGLC) for the lambda light chain, which is expressed predominantly in horses. A decrease in IGLVs is noted during age development, although nucleotide diversity and significant differences in gene usage increased. A standardization of the existing nomenclature of immunoglobulin genes is suggested. The first experimental study focused on the identification of allotypic variants of equine IGLC and differences in the expression of IGLV within and between the two horse breeds Rhenish German Coldblood (RGC) and Hanoverian Warmblood (HW). The two breeds differ in stud book size and breeding goals. After PCR amplification of cDNA and subcloning, 120 samples per breed were isolated and sequenced. Statistical analysis of transcription frequencies were performed applying non-parametric tests. The significant majority of the sequences represented IGLC6/7 in both breeds, whereas IGLC1, IGLC4, and IGLC5 occurred in significant different frequencies per breed. Five allotypic IGLC1 variants, four allotypic IGLC5 variants, and three allelic as well as two allotypic IGLC6/7 variants were identified in breed specific proportions. Eleven out of 144 known IGLV segments were transcribed of which IGLV15 and IGLV17 were preferred significantly. IGLV25 displayed significant differences in the rearrangement between both breeds. In addition, the pseudogenes IGLV101ψ and IGLV74ψ were also identified. Rearrangements with IGLC genes showed significant differences for IGLV15 in both breeds, whereas IGLV25 also revealed significant differences between the breeds. The transcriptional orientation of the functional segments had no influence on the occurrence of the IGLV. The second experimental study carried out in cattle dealt with two main topics. On the one hand it focused on the third complementarity determining region of the bovine heavy chain (CDR3H) whose exceptional length previously was described as a specificity of bovine IgG and IgM. On the other hand, the genomic organization of the immunoglobulin heavy chain locus was analyzed with a special focus on the number of IGHV. After isotype-specific cDNA PCR, subcloning of 20 DNA plasmids per immunoglobulin isotype and sequence analyzes of the variable regions, we proved the existence of exceptionally long CDR3H in all five bovine isotypes. The sequences of CDR3H belong to three distinct groups and possess ≤10, 12 to 31 or ≥48 amino acid residues. Hydrophilic amino acid residues dominated in long and intermediate long CDR3H, while short CDR3H possessed hydrophobic amino acid residues, too. All sequences with exceptionally long CDR3H were related to the germline IGHV10. Further, the germline IGHD2, with 148 bp in size, contributes to those CDR3H. The genomic organization of the bovine immunoglobulin heavy-chain locus was analyzed using the current genome assembly, Bos taurus NCBI build 6.1. A main locus was identified on BTA21. Additional exons coding for immunoglobulin heavy chain variable (IGHV), diversity (IGHD), and joining (IGHJ) segments, as well as for the constant regions of different isotypes, were localized on BTA7, BTA8, BTA20, and on unplaced contigs, too. Altogether, 36 IGHV were detected of which 13 are putatively functional. For the phylogenetic analysis, the complete nucleotide sequences of the 36 bovine IGHV segments were aligned with one member of the human IGHV families 1 to 7. Results proved the existence of two bovine IGHV families (boVH1, boVH2). The boVH1 comprises all functional segments. This study substantially improved the understanding of the generation of immunoglobulin diversity in cattle. The third study aimed to gain more insight into the combinatorial diversity, somatic hypermutations and putative gene conversions of IgG in the four cattle breeds Aubrac, German Simmental, German Black Pied, and Holstein Friesian. For the more detailed analysis of rearranged bovine heavy chain immunoglobulin variable regions, a new bioinformatics framework was developed by combining and refining widely used alignment algorithms. Immunoglobulin heavy chains possessing exceptionally long CDR3Hs can now be analyzed specifically, as well as the dominantly transcribed IGHV, IGHD, and IGHJ segments and their recombination. The use of 15 different IGHV segments, 21 IGHD segments, and 2 IGHJ segments was investigated with significant different transcription levels within the breeds. There are preferred rearrangements within the 3 groups of CDR3H lengths. In sequences of group 1 (≤10 aa) and 3 (≥48 aa) a lower number of recombinations were observed than in sequences of group 2 (11-47 aa). The combinatorial diversity revealed 162 significantly different rearrangements of germline IGHV, IGHD, and IGHJ segments. The few preferably rearranged gene segments within group 3 CDR3H regions are supposed to indicate specialized antibodies because this length is unique in cattle. The main result of this study enabled by the new bioinformatics framework, is the strong evidence for gene conversion as a rare event using pseudogenes fulfilling all definitions for this particular diversification mechanism. In conclusion, this thesis contributes to a more detailed understanding of the expressed immunoglobulin repertoire in cattle and horses. Breed and husbandry conditions are supposed to influence the repertoire significantly. This thesis also highlights that the bovine heavy chain diversity is not restricted to the use of a limited number of germline genes although there are preferred rearrangements within the three groups of CDR3H lengths. These results will be of future importance in analyzing seroconversion data after infection or vaccination, as well as determining breed specific differences to select healthy, robust animals. de
dc.contributor.coReferee Knorr, Christoph Prof. Dr.
dc.subject.eng immunoglobulins de
dc.subject.eng cattle de
dc.subject.eng horse de
dc.subject.eng heavy chain de
dc.subject.eng light chain de
dc.subject.eng antibody diversity de
dc.subject.eng gene conversion de
dc.subject.eng transcription analysis de
dc.identifier.urn urn:nbn:de:gbv:7-11858/00-1735-0000-0028-8776-A-1
dc.affiliation.institute Fakultät für Agrarwissenschaften de
dc.subject.gokfull Land- und Forstwirtschaft (PPN621302791) de
dc.identifier.ppn 861060466

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