Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

Evolution of MHC class I in the order Crocodylia.

The major histocompatibility complex (MHC) is a dynamic genomic region with an essential role in the adaptive immunity of jawed vertebrates. The evolution of the MHC has been dominated by gene duplication and gene loss, commonly known as the birth-and-death process. Evolutionary studies of the MHC have mostly focused on model species. However, the investigation of this region in non-avian reptiles is still in its infancy. To provide insights into the evolutionary mechanisms that have shaped the diversity of this region in the Order Crocodylia, we investigated MHC class I exon 3, intron 3, and exon 4 across 20 species of the families Alligatoridae and Crocodilidae. We generated 124 DNA sequences and identified 31 putative functional variants as well as 14 null variants. Phylogenetic analyses revealed three gene groups, all of which were present in Crocodilidae but only one in Alligatoridae. Within these groups, variants generally appear to cluster at the genus or family level rather than in species-specific groups. In addition, we found variation in gene copy number and some indication of interlocus recombination. These results suggest that MHC class I in Crocodylia underwent independent events of gene duplication, particularly in Crocodilidae. These findings enhance our understanding of MHC class I evolution and provide a preliminary framework for comparative studies of other non-avian reptiles as well as diversity assessment within Crocodylia.

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis.

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, alpha-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.

Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding.

Little is known about the structure of major histocompatibility complex (MHC) molecules outside of mammals. Only one class I molecule in the chicken MHC is highly expressed, leading to strong genetic associations with infectious pathogens. Here, we report two structures of the MHC class I molecule BF2*2101 from the B21 haplotype, which is known to confer resistance to Marek's disease caused by an oncogenic herpesvirus. The binding groove has an unusually large central cavity, which confers substantial conformational flexibility to the crucial residue Arg9, allowing remodeling of key peptide-binding sites. The coupled variation of anchor residues from the peptide, utilizing a charge-transfer system unprecedented in MHC molecules, allows peptides with conspicuously different sequences to be bound. This promiscuous binding extends our understanding of ways in which MHC class I molecules can present peptides to the immune system and might explain the resistance of the B21 haplotype to Marek's disease.

Various domains of the B-cell regulatory molecule CD72 has diverged at different rates in mammals: cloning, transcription and mapping of porcine CD72.

We report the cloning of the porcine B-cell co-receptor CD72, as well as genomic mapping and examination of transcription. The B-cell receptor (BCR) complex mediates signalling upon antigen recognition by the membrane bound BCR. Several co-receptors modulate this signal positively or negatively. CD72 has been shown to be a negatively regulating BCR co-receptor. We isolated and sequenced three porcine CD72 transcript variants. Using a pig radiation hybrid panel we found the porcine CD72 gene to be located on chromosome 1q21-28 in a region syntenic to human chromosome 9. The porcine CD72 gene is highly transcribed in lymph node, thymus and lung tissues as well as in pulmonary alveolar macrophages. The predicted porcine CD72 polypeptide shows conservation of immunoreceptor tyrosine-based inhibitory motifs and an extracellular C-type lectin domain. Compared to CD72 sequences from other mammals as well as from chicken, the polypeptide is highly conserved in the intracellular part and much less conserved in the extracellular part. We suggest that this difference might be due to the different nature of ligands and the constrains on these to co-evolve.

Cloning, characterization and mapping of porcine CD14 reveals a high conservation of mammalian CD14 structure, expression and locus organization.

The cell surface protein CD14 plays a central role in innate immunity as a pattern recognition receptor. CD14 is part of a receptor complex also including toll-like receptor 4 and MD2 proteins. Binding of the ligand lipopolysaccharide to the complex on myeloid cells leads to release of pro-inflammatory cytokines and mediators from the cell. In this study, we present the cloning, characterization and tissue expression pattern of a porcine CD14 encoding cDNA, and the chromosomal localization of the porcine CD14 gene. The open reading frame is predicted to encode a protein of 373 amino acids, which shows conservation of structural as well as functional regions when compared to other mammalian species. The CD14 gene was localized to porcine chromosome 2 in a region syntenic to human chromosome 5q. Transcription analysis shows that CD14 is widely expressed in tissues examined in this study, which correlates well with expression primarily on myeloid cells.

Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens.

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a "minimal essential MHC" for chickens, in strong contrast to typical mammals.

Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC.

CD1 molecules play an important role in the immune system, presenting lipid-containing antigens to T and NKT cells. CD1 genes have long been thought to be as ancient as MHC class I and II genes, based on various arguments, but thus far they have been described only in mammals. Here we describe two CD1 genes in chickens, demonstrating that the CD1 system was present in the last common ancestor of mammals and birds at least 300 million years ago. In phylogenetic analysis, these sequences cluster with CD1 sequences from other species but are not obviously like any particular CD1 isotype. Sequence analysis suggests that the expressed proteins bind hydrophobic molecules and are recycled through intracellular vesicles. RNA expression is strong in lymphoid tissues but weaker to undetectable in some nonlymphoid tissues. Flow cytometry confirms expression from one gene on B cells. Based on Southern blotting and cloning, only two such CD1 genes are detected, located approximately 800 nucleotides apart and in the same transcriptional orientation. The sequence of one gene is nearly identical in six chicken lines. By mapping with a backcross family, this gene could not be separated from the chicken MHC on chromosome 16. Mining the draft chicken genome sequence shows that chicken has only these two CD1 genes located approximately 50 kb from the classical class I genes. The unexpected location of these genes in the chicken MHC suggests the CD1 system was present in the primordial MHC and is thus approximately 600 million years old.

The properties of the single chicken MHC classical class II alpha chain ( B-LA) gene indicate an ancient origin for the DR/E-like isotype of class II molecules.

In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) alpha chain from outbred chickens as the basis for the cloning and sequencing of the cDNA. We found only one class II alpha chain transcript, which bears the major features of a classical class II alpha sequence, including the critical peptide-binding residues. The chicken sequence is more similar to human DR than to the DQ, DP, DO or DM isotypes, most significantly in the peptide-binding alpha(1) domain. The cDNA and genomic DNA sequences from chickens of diverse origins show few alleles, which differ in only four nucleotides and one amino acid. In contrast, significant restriction fragment length polymorphism is detected by Southern blot analysis of genomic DNA, suggesting considerable diversity around the gene. Analysis of a large back-cross family indicates that the class II alpha chain locus ( B-LA) is located roughly 5.6 cM from the MHC locus, which encodes the classical class II beta chains. Thus the chicken class II alpha chain gene is like the mammalian DR and E isotypes in three properties: the presence of the critical peptide-binding residues, the low level of polymorphism and sequence diversity, and the recombinational separation from the class II beta chain genes. These results indicate that the sequence features of this lineage are both functionally important and at least 300 million years old.

The chicken CD4 gene has remained conserved in evolution.

CD4 has a central role in thymocyte differentiation and cell-mediated immunity. We isolated and analyzed chicken CD4. The gene spans 11.5 kb and is composed of ten exons. The promoter is TATA-less and similar to the mouse and human CD4 promoters, with two transcription start sites as determined by 5'RACE analysis. In general the introns are short, although the 5'untranslated region includes a large intron of 5.6 kb containing binding sites of the putative CD4 silencer. The single-strand conformation polymorphism technique was used to identify a polymorphism to map the gene, which lies on chicken Chromosome 1 in a position showing conserved synteny to mouse and human. This is the first report describing the organization of CD4 from a non-mammalian species. The structure and localization of chicken CD4 and many sequence motifs important in its regulation have remained conserved during evolution.