**2.3.1 The opossum MHC**

10 Bacterial Artificial Chromosomes

paternally expressed *SNRPN* gene and maternally expressed *UBE3A.* A cross-species comparison of the arrangement of these two genes across vertebrates uncovered an unexpected finding. A wallaby BAC clone containing the *SNRPN* gene mapped to wallaby chromosome 1, whereas the BAC containing the *UBE3A* localized to the short arm of chromosome 5. Furthermore, a fully sequenced platypus BAC clone containing *UBE3A* identified the gene adjacent to be *CNGA3*, a human chromosome 2 gene (Rapkins et al., 2006). Subsequent analysis of the chicken, zebrafish and opossum genome sequence assemblies unequivocally showed this to be the ancestral arrangement, with *UBE3A* adjacent to *CNG3A* while *SRNPN* is located elsewhere in the genome. Both *UBE3A* and *SRNPN* were found to be biallelic expressed in marsupials and monotremes. It appears that the other imprinted genes found in this region in eutherians do not exist in marsupials and originated from RNA copies of genes located in other parts of the genome. Rapkins et al (2006) concluded that these genes only became subject to genomic imprinting when the region was assembled in the eutherian lineage. This study also provided the first evidence that genomic imprinting was acquired by different loci at different times during mammalian

The Callipyge locus, so named after a muscle trait observed in sheep, contains a cluster of three paternally expressed genes (*DIO3, DLK1, RTL1*). In order to carry out a comprehensive analysis of this locus, seven platypus and 13 wallaby overlapping BAC clones were fully sequenced and assembled into a single contig for each species (Edwards et al., 2008). Comparative genome analysis revealed that the genomic landscape of this locus has undergone a number of changes during mammalian evolution. In marsupials, the locus is twice the size of the orthologous region in eutherians as a result of an accumulation of LINE1 repeats. In addition, there has been selection against SINE repeats in eutherians along with an increase in GC and CpG island content. Over 140 evolutionary conserved regions were found by phylogenetic footprinting but none of these regions corresponded to the imprint control element identified in eutherians. These findings were consistent with the absence of imprinted expression for this locus both in monotremes and marsupials. Similar to the situation described above for the Prader-Willi/Angelman locus, it appears that a retrotransposition event resulted in the formation of a novel gene in eutherians and it was suggested that this may have been the driving force behind the evolution of imprinting at

One the most studied regions of the vertebrate genome is the Major Histocompatibility Complex (MHC), a region central to the vertebrate immune response. In humans, the MHC is a large, gene dense region, spanning 3.6Mb and containing 224 genes divided into three regions; Class I, II and III (MHC Sequencing Consortium, 1999). Classes I and II encode genes involved in endogenous and exogenous antigen presentation respectively. Class III contains immune genes, involved in the inflammatory, complement and heat-shock responses, as well as a number of non-immune genes. This organization is in stark contrast to the chicken MHC consisting of only 19 genes within a 92kb region (Kaufman et al., 1999), making it difficult to establish the evolutionary history of the MHC. The position of marsupials and monotremes in vertebrate phylogeny ideally situates them to bridge the gap

evolution.

**2.2.3 Evolution of the Callipyge imprinted locus** 

this locus (Edwards et al., 2008).

**2.3 Major Histocompatibility Complex** 

The opossum MHC was the first multi-megabase region to be annotated for the opossum genome project. Annotation of this region was performed on preliminary genome assemblies MonDom1 and MonDom2. The MHC region in MonDom1 was distributed across five sequence scaffolds. Previous mapping localized MHC Class I genes to different locations on opossum chromosome 2, with genes *UB* and *UC* located at the telomeric end of the short arm (Belov et al., 2006) and *UA1* located near the centromere on the long of arm (Gouin et al., 2006). Thus, it was imperative that this assembly of MHC scaffolds was accurately determined to establish whether the separation of these genes was the result of a chromosomal rearrangement or a transposition event. This was achieved by isolating BAC clones corresponding to the ends of the MHC scaffolds. All BACs from these scaffolds, with the exception of one containing *UB* and *UC,* mapped to the centromeric region of chromosome 2. As a result of this information, the MHC was assembled into a single scaffold in the MonDom2 assembly (Belov et al., 2006). Furthermore, mapping of BAC clones from the genes at either end of this large scaffold enabled the orientation of the MHC on the chromosomes to be determined.

The complete annotation of this region provided the necessary information required to start piecing together the changes which have occurred throughout vertebrate evolution. In contrast to the chicken, the MHC of the opossum spans almost 4Mb and contains at least 140 genes, making it similar in size and complexity to the human MHC (Belov et al., 2006). However, the opossum has a very different gene organization with Class I and II genes found interspersed rather than separated by the Class III region as they are in eutherian mammals. This organization is similar to that of other vertebrates, such a shark and frog, suggesting that the marsupial organization is similar to that of the vertebrate ancestor and the eutherian organization is derived.
