The evolution of haemoglobin gene loci in amniotes

The genes in alpha ({u03B1})- and beta ({u03B2})- globin clusters constitute a functional haemoglobin molecule, crucial for oxygen transportation. In most fish and amphibians, {u03B1}- and {u03B2}-globin genes are located together, whereas in amniotes (birds and mammals), there are two distinct clusters. Several complex models have been proposed to explain the evolution of these gene clusters. However, there was a lack of data for key positions in amniote phylogeny to discern which one was most parsimonious. Therefore, the main aims of this project were to characterise {u03B1}- and {u03B2}-globin clusters and their regulatory regions in a monotreme Ornithorhynchus anatinus (Australian duck-billed platypus) and two reptilian species Pogona vitticeps (Australian bearded dragon) and Anolis carolinensis (green anole lizard), to gain insight into globin loci evolution. This thesis is presented as a collection of research papers covering each topic, and a review and discussion that summarises my research. The first paper (Chapter 2) reports a comprehensive study on the characterisation, expression and evolution of {u03B1}- and {u03B2}-globin gene clusters in the platypus, using a combination of molecular and bioinformatics approaches. The most important findings from this work leading to the development of a new and simple model for globin gene evolution concerned the discovery of a {u03B2}-like globin gene within the a-globin cluster and genomic context analysis of {u03B1}- and {u03B2}-globin clusters across vertebrates. I showed that the amniote a-globin cluster is in fact the same as the a-{u03B2} cluster found in fish and amphibians, and both clusters share common flanking genes (C16orf35 and LUC7L). I proposed a transposition model in which a copy of {u03B2}-globin gene was inserted into a cluster of olfactory receptors (flanked by RRMl, CCKBR and ILK) in the ancestor of amniotes, thus originating the amniote {u03B2}-globin cluster. To elaborate this model further, my second paper (Chapter 3) reviews some events that could have led to this transposition, and their effects on the current fate of regulation. Information on the organization of globin genes in reptiles was required to test this transpositional model. I looked into the globin gene organization in the green anole using a bioinformatics approach and in the bearded dragon using a molecular approach. The results are reported in Chapter 4 and my third paper, which describe how fragmentary data from the green anole genome sequence assembly and mapping data from bearded dragon provided further evidence to support my proposed model for the evolution of the {u03B2}-globin gene cluster in amniotes. I also studied the evolution of regulatory regions of the platypus {u03B1}- and {u03B2}-globin clusters to address the question whether the translocation of the {u03B2}-globin locus resulted in a transposition of its regulatory region, or whether a new regulatory region evolved as a result of this translocation (reported in the fourth paper, Chapter 5). By using some novel techniques, I showed that the platypus a-globin has a major regulatory element that is conserved with other jawed vertebrates, whereas the regulatory regions of their {u03B2}-globin cluster do not show any conservation at the sequence level to those of birds and therian mammals. This suggested that the regulatory regions of amniote {u03B2}-globin genes evolved either more rapidly (more substitutions) or more extensively (e.g. more rearrangements) from a common ancestral regulatory region. Alternatively, these regulatory regions may have independent origins in different amniote lineages. In my final chapter, I discuss the overall implications of my findings on this area of research. I highlight the special value of studying non-model species mammals and reptiles, by which researchers are able to gain novel information about globin evolution and regulation

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

Background: Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. Results: The platypus α-globin cluster (chromosome 21) contains embryonic and adult α- globin genes, a β-like ω-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-ζ-ζ'-αD-α3-α2-α1-ω-GBY-3'. The platypus β-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-ε-β-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate α-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal β-globin clusters are embedded in olfactory genes. Thus, the mammalian α- and β-globin clusters are orthologous to the bird α- and β-globin clusters respectively. Conclusion: We propose that α- and β-globin clusters evolved from an ancient MPG-C16orf35-α-β-GBY-LUC7L arrangement 410 million years ago. A copy of the original β (represented by ω in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of β-globin genes with different expression profiles in different lineages.Vidushi S. Patel, Steven J.B. Cooper, Janine E. Deakin, Bob Fulton, Tina Graves, Wesley C. Warren, Richard K. Wilson and Jennifer A.M. Grave

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals-3

Tantly related globin gene, (green), which are flanked by --on the 5' end and ---on the 3' end (black). The platypus β-globin cluster contains only two genes, ε and β (blue), which are flanked on both sides by genes (black). (B) Relative positions of the putative transcription factor binding sites in the 200 bp promoter region located upstream of the predicted platypus, marsupial (ζ and ψζ', and α, ψα, α, α, ω, ε and β) and human α- and β-like globin genes. For the platypus no data was available from other species, including , for comparisons.<p><b>Copyright information:</b></p><p>Taken from "Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals"</p><p>http://www.biomedcentral.com/1741-7007/6/34</p><p>BMC Biology 2008;6():34-34.</p><p>Published online 25 Jul 2008</p><p>PMCID:PMC2529266.</p><p></p

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals-8

Encies in an unlinked analysis using MrBayes (v. 3.1.2). Numbers adjacent to branches refer to % posterior probabilities. GenBank accession numbers for sequences are: Fat-tailed Dunnart () β [], ε [], ω []; Stripe-faced Dunnart () β, ε []; Virginian Opossum () β [], ε []; Brazilian Opossum () β [], ε [], ω []; Tammar Wallaby () β [], ε [], ω []; African clawed frog () larval β [], larval βII []; Western clawed frog () β [], larval ε1 []; Chicken () β (β) [], ε [], γ (β) []; Duck () β [], ε []; Human () β [], γ [], ε []; Mouse () β (β1) [], γ (β h0) [], ε (ε) []; Goat ()(β) [], ε (ε) [], γ []; Rabbit () β, γ, ε []; Echidna (β []; Pufferfish () β []; Zebrafish () ε1 []; Platypus β, ε [], ω [].<p><b>Copyright information:</b></p><p>Taken from "Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals"</p><p>http://www.biomedcentral.com/1741-7007/6/34</p><p>BMC Biology 2008;6():34-34.</p><p>Published online 25 Jul 2008</p><p>PMCID:PMC2529266.</p><p></p

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals-5

D the β-globin cluster on chromosome 2q5.1 (red). The chromosomes are counterstained with DAPI (blue).<p><b>Copyright information:</b></p><p>Taken from "Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals"</p><p>http://www.biomedcentral.com/1741-7007/6/34</p><p>BMC Biology 2008;6():34-34.</p><p>Published online 25 Jul 2008</p><p>PMCID:PMC2529266.</p><p></p

Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals-0

Seen in the amphibian lineage. This region further duplicated and underwent some gene silencing in teleost fish. In an amniote ancestor of reptiles, birds and mammals (>315 MYA), a copy of an ancestral β-globin gene from this region was inserted into a different chromosome within a region replete with multiple copies of genes. The original amniote β-globin gene survives as the ω-globin gene (β1) in the α-globin cluster of marsupials and monotremes, whereas the transposed β-globin gene (β2) duplicated several times to form different clusters in the different lineages. (B) Tandem duplications of the ancestral amniote α-globin gene produced a three-gene (π-α-α) cluster in the avian lineage. In the mammalian lineage, further duplications gave rise to a six-gene (ζ-ζ'-α-α-α-α) cluster with ongoing gene conversion events homogenising the embryonic and adult genes. In monotremes, the ancestral ω (β1) and are retained. After the divergence of monotreme and therian mammals, there was an additional duplication of αto form θ, giving rise to the seven-gene cluster (ζ-ζ'-α-α-α-α-θ) in marsupials and eutherians. Marsupials also retain the ancestral ω but may have lost gene; eutherians retain no identifiable remnant of either gene. Furthermore, the ancestral transposed β2-globin gene duplicated independently in birds and mammals. Before the mammalian radiation, we propose that the ancestral β2 gene duplicated to form a two-gene β-globin cluster (ε-β) as seen in monotremes and marsupials, except that ongoing gene conversion events homogenised platypus ε to group with monotreme β genes. After the divergence of marsupial and eutherian mammals, there were further tandem duplications of these two genes to produce complex β-globin cluster (ε-γ-η-δ-β) in eutherians.<p><b>Copyright information:</b></p><p>Taken from "Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals"</p><p>http://www.biomedcentral.com/1741-7007/6/34</p><p>BMC Biology 2008;6():34-34.</p><p>Published online 25 Jul 2008</p><p>PMCID:PMC2529266.</p><p></p