**2.4 Tracing the evolutionary history of globin genes**

12 Bacterial Artificial Chromosomes

tammar wallaby MHC, including the 'core' MHC located on chromosome 2 and many of the dispersed Class I genes found elsewhere in the genome. A BAC-based approach was taken, with the idea of constructing a BAC-contig across the core MHC, as well as

After finding Class I genes dispersed across the genome, a thorough screening of the wallaby BAC library was performed in order to isolate as many Class I genes as possible. As a result four additional BAC clones containing Class I genes were isolated, with FISHmapping of these BACs localizing these genes to the core MHC region on chromosome 2 (Siddle et al., 2009). Complete sequencing of these BACs identified six Class I genes within the core MHC, which were interspersed with antigen processing genes and a Class II gene. Sequencing of ten BACs mapping outside this region identified nine Class I genes with open reading frames. In depth sequence analysis of these BACs revealed a tendency for Kangaroo Endogenous Retroviral Element (KERV) to flank these dispersed Class I genes, suggesting that this element may be implicated in the movement of these genes to regions outside the

A BAC contig across the core MHC on wallaby chromosome 2 was constructed for sequencing purposes (Siddle et al., 2011). Unfortunately, despite extensive library screening with overgo probes designed from BAC end sequence, a single contig spanning the entire region was not obtained. Instead, the isolated BACs assembled into nine contigs and three 'orphaned' BACs. The order of these contigs and orphaned BACs was determined using BAC clones as probes for FISH on metaphase chromosome spreads and interphase nuclei. The resulting 4.7Mb sequence contained 129 predicted genes from all three MHC Classes. A comparison of the gene arrangement between wallaby, opossum and other vertebrates indicated that the wallaby MHC has a novel MHC gene arrangement, even within the core MHC. The wallaby Class II genes have undergone an expansion, residing in two clusters either side of the Class III region. Once again, KERV sequences are prominent in this region and may have contributed to the overall genomic instability of the wallaby MHC region

Although the platypus genome has been sequenced, the high GC and repeat content hampered this sequencing effort, leaving the assembly with many more gaps than other mammalian genomes sequenced to a six-fold depth by Sanger sequencing (Warren et al., 2008). As a result, complete annotation of the platypus MHC as a region was impossible because MHC genes were found on many sequence contigs and/or scaffolds. However, three BAC clones were completely sequenced and mapped to platypus chromosomes (Dohm et al., 2007). One of these BACs, localized to chromosome 3, only contained a processed class I pseudogene. Of the remaining two BACs, one contained two Class I genes and two Class II genes as well as antigen processing genes, while the other contained mainly Class III genes. The most surprising result came from FISH-mapping, which revealed that platypus MHC is not contiguous and maps to the pseudoautosomal region of two pairs of sex chromsomes. The Class I and II genes were located on X3/Y3 and the Class III region on X4/Y4. Subsequent FISH-mapping of BACs containing these same genes in the echidna demonstrated that this separation of the MHC onto two different pairs of sex chromosomes was a common feature for monotremes. Monotremes are the only mammals known to date

to have the MHC reside on sex chromosomes (Dohm et al., 2007).

sequencing the dispersed Class I genes.

core MHC (Siddle et al., 2009).

(Siddle et al., 2011).

**2.3.3 The MHC in monotremes** 

Haemoglobin is essential for oxygen transportation in vertebrates. The haemoglobin molecule is encoded by members of the - and -globin gene clusters. These gene clusters were presumed to have arisen from a single globin gene that duplicated to form a combined - and -globin gene cluster as is seen in amphibians (Jeffreys et al., 1980). It was proposed that either a fission event or a chromosome duplication event, followed by independent evolution of the duplicate copies, gave rise to the separate - and -globin gene clusters observed in amniotes (Jeffreys et al., 1980). Determining the gene content of the marsupial and monotreme globin gene clusters has had a tremendous impact in this field. This work was facilitated by sequencing and mapping of BACs containing globin genes.

The discovery of a novel -like globin gene called *HBW* residing adjacent to the wallaby globin cluster provided support for the chromosome duplication hypothesis (Wheeler et al., 2004). Further support was provided when BAC clones from the dunnart (*S.macroura*) spanning the separate - and -globin gene clusters were sequenced and it was found that, like the wallaby, the *HBW* was adjacent to the -globin cluster (De Leo et al., 2005). The next obvious step in testing the chromosome duplication hypothesis was to determine the organization of the platypus - and -globin gene clusters. The fragmented nature of the platypus genome meant that a BAC-based approach was required to obtain a more complete sequence of the alpha and beta globin gene clusters in this species (Patel et al., 2008). Analysis of the sequence obtained from these BAC clones was instrumental in the formation of a new hypothesis for the evolution of these gene clusters.

The platypus -globin cluster also contained a copy of *HBW,* which taken on its own would support the chromosome duplication hypothesis. However, an examination of the genes flanking the two clusters revealed that the combined /-globin cluster in amphibians was flanked by the same genes as the -globin cluster in all amniotes, whereas the -globin cluster in amniotes was surrounded by olfactory receptors. This led to a hypothesis where the -globin cluster in amniotes was proposed to correspond to the original /-globin cluster present in other vertebrates. The -globin cluster was proposed to have evolved after a copy of the original -globin gene (*HBW*) was transposed into an array of olfactory receptors (Patel et al., 2008).
