Organisation of the Hb 1 genes of the Antarctic skate Bathyraja eatonii: new insights into the evolution of globin genes

An extensive investigation of the organisation of globin genes has greatly contributed to the understanding of universal mechanisms of gene evolution and expression. Cartilaginous fish are the first organisms that have evolved the tetrameric form of hemoglobin (Hb). So far, there has been absolute lack of data about globin genes in chondrichthyans. Bathyraja is the dominant rajid south of 60 degrees S. In the framework of the investigations on globin genes of Antarctic red-blooded and Hb-less fish we obtained the cloning of the alpha- and beta-globin cDNAs of the main Hb (Hb 1) of the skate Bathyraja eatonii. Then, a genomic fragment of 6.2 kb was isolated where the Hb 1 alpha and beta genes are linked in a tail-to-head (3' to 5') orientation. The beta-globin gene promoter region and the chromosomal organisation of the Hb 1 genes of B. eatonii have been compared to their homologues in other vertebrates. The finding of a tail-to-head linkage of the Hb 1 alpha- and beta-globin genes in B. eatonii is the first characterisation of the organisation of globin genes in chondrichthyes; such finding offers a novel contribution to the understanding of the evolution of this class of genes. Moreover, the characterisation of chondrichthyan genes is very important for gaining insight into the ancestral state of vertebrate genomes.     

Gene duplication,genome duplication,and the functional diversification of vertebrate globins

The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α₂β₂). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

Antarctic notothenioid fishes: genomic resources and strategies for analyzing an adaptive radiation

The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species-flock in the cold Southern Ocean that surrounds Antarctica. To facilitate genome-level studies of the diversification of these fishes, we present estimates of the genome sizes of 11 Antarctic species and describe the production of high-quality bacterial artificial chromosome (BAC) libraries for two, the red-blooded notothen Notothenia coriiceps and the white-blooded icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families (e.g., the crown group Channichthyidae [icefishes]), was accompanied by genome expansion. Six species from the basal family Nototheniidae had C-values between 0.98 and 1.20?pg, a range that is consistent with the genome sizes of proposed outgroups (e.g., percids) of the notothenioid suborder. In contrast, four icefishes had C-values in the range 1.66-1.83?pg. The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprise 12× and 10× coverage of the respective genomes and have average insert sizes of 138 and 168?kb. Paired BAC-end reads representing ～0.1% of each genome showed that the repetitive element landscapes of the two genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The availability of these high-quality and well-characterized BAC libraries sets the stage for targeted genomic analyses of the unusual anatomical and physiological adaptations of the notothenioids, some of which mimic human diseases. Here we consider the evolution of secondary pelagicism by various taxa of the group and illustrate the utility of Antarctic icefishes as an evolutionary-mutant model of human osteopenia (low-mineral density of bones).     

The hemoglobin genes of Drosophila

We recently reported the unprecedented occurrence of a hemoglobin gene (glob1) in the fruitfly Drosophila melanogaster. Here we investigate the structure and evolution of the glob1 gene in other Drosophila species. We cloned and sequenced glob1 genes and cDNA from D. pseudoobscura and D. virilis, and identified the glob1 gene sequences of D. simulans, D. yakuba, D. erecta, D. ananassae, D. mojavensis and D. grimshawi in the databases. Gene structure (introns in helix positions D7.0 and G7.0), gene synteny and sequence of glob1 are highly conserved, with high ds/dn ratios indicating strong purifying selection. The data suggest an important role of the glob1 protein in Drosophila, which may be the control of oxygen flow from the tracheal system. Furthermore, we identified two additional globin genes (glob2 and glob3) in the Drosophilidae. Although the sequences are highly derived, the amino acids required for heme- and oxygen-binding are conserved. In contrast to other known insect globin, the glob2 and glob3 genes harbour both globin-typical introns at positions B12.2 and G7.0. Both genes are conserved in various drosophilid species, but only expression of glob2 could be demonstrated by western blotting and RT-PCR. Phylogenetic analyses show that the clade leading to glob2 and glob3, which are sistergroups, diverged first in the evolution of dipteran globins. glob1 is closely related to the intracellular hemoglobin of the botfly Gasterophilus intestinalis, and the extracellular hemoglobins from the chironomid midges derive from this clade.     

Hemoglobin structure/function and globin-gene evolution in the Arctic fish Liparis tunicatus

The importance of the Arctic, in contributing to the knowledge of the overall ensemble of adaptive processes influencing the evolution of marine organisms, calls for investigations on molecular adaptations in Arctic fish. Unlike the vast majority of Antarctic Notothenioidei, several Arctic species display high hemoglobin multiplicity. The blood of four species, the spotted wolffish of the family Anarhichadidae and three Gadidae, contains three functionally distinct major components. Similar to many Antarctic notothenioids, Arctic Liparis tunicatus (suborder Cottoidei, family Liparidae) has one major hemoglobin (Hb 1) accompanied by a minor component (Hb 2). This paper reports the structural and functional characterisation of Hb 1 of L. tunicatus. This hemoglobin shows low oxygen affinity, and pronounced Bohr and Root effects. The amino-acid sequence of the beta chain displays an unusual substitution in NA2 (beta2) at the phosphate-binding site, and the replacement of Val E11 (beta67) with Ile. Similar to some Antarctic fish Hbs, electron paramagnetic resonance spectra reveal the formation of a ferric penta-coordinated species even at physiological pH. The amino-acid sequences have also been used to gain insight into the evolutionary history of globins of polar fish. L. tunicatus globins appear close to the notothenioid clades as predicted by teleostean phylogenies. Close phylogenetic relationships between Cottoidei and Notothenioidei, together with their life style, seem to be the main factor driving the globin-sequence evolution.     

Molecular cloning and characterization of two sets of alpha-theta genes in the rat alpha-like globin gene cluster

The highly heterogeneous rat hemoglobin system was investigated at the gene level. Two regions of the alpha-like globin gene cluster from a Wistar rat were isolated. Four lambda Dash recombinant clones carrying rat alpha-like globin genes were localized on two distinct gene regions. A region of approximately 16kb was found to contain the 5'-IIalpha1-psi theta 1-3' loci, and another of approximately 24kb the 5'-IIalpha2-psi theta2-psiI alpha3-3' loci. Both IIalpha1 and IIalpha2 are considered to be active, coding the IIalpha-globin chain. The nt sequences of IIalpha1 and IIalpha2 are identical except for six nt in the non-coding region. The psiI alpha3 locus is a truncated pseudogene. The putative promoter region of an alpha-like globin gene is joined directly to the third exon, homologous to that of Ialpha-globin cDNA. psi theta1 and psi theta2 are also pseudogenes, as evidenced by several deletions located in the protein-coding regions of these loci. The psi theta1 and psi theta2 loci exhibit extensive homology, but the restriction maps of these genes and their flanking regions differ considerably. Genomic Southern blot analyses of the total liver DNA from six rats showed the existence of three theta-globin-related genes, including psi theta1 and psi theta2. These results indicate that the two gene regions investigated are not allelic variants, but may be generated by block duplication. This is the first report of the existence of rodent theta-globin genes.     

Purification, characterization and sequence analyses of the extracellular giant hemoglobin from Oligobrachia mashikoi

We purified an extracellular hemoglobin with the molecular mass of ca. 440 kDa from the whole homogenates of Oligobrachia mashikoi (phylum Pogonophora) by a one-step gel-filtration. The preparation was pure to be crystallized. The P50 values of the hemoglobin and the fresh blood prepared from O. mashikoi were about 0.82 Torr and 0.9 Torr, respectively, which were much lower than the P50 value of human hemoglobin. However, the n values of the hemoglobin and the blood were about 1.2 and 1.1, respectively.Using the improved tricine SDS-PAGE, we could separate O. mashikoi hemoglobin into four kinds of the globin chains, A1, A2, B1 and B2, and succeeded for the first time in cloning and sequencing of the complete cDNA encoding B1 globin gene, in addition to A1, A2 and B2 globin genes in full length. We found that all globin genes have the extracellular signal sequences in each molecule and the distal His of the B1 globin chain is replaced to Gln. Finally, we constructed phylogenetic trees of the hemoglobins from Pogonophora, Vestimentifera and Annelida.     

Demonstration of an enhancer activity in a fragment of DNA located at the 3' of the adult alpha globin gene in the duck

We found an enhancer element placed at the 3' side of the adult duck alpha A globin gene. The duck alpha globin gene cluster contains three genes from the 5' to 3' side: the pi embryonic gene, the alpha D minor adult gene and the alpha A adult major gene. We analyzed a 16 kb genomic domain extending from 2 kb upstream of the pi gene to 5 kb downstream of the alpha A gene. This enhancer is active in AEV transformed chicken erythroblasts. Its is inactive both in HeLa cells and in the human erythroid cells K562 which express only embryonic genes. These findings are discussed in relation to previous results concerning the duck beta globin enhancer located at the 3' side of the beta A globin gene.     

The organization of the tadpole and adult alpha globin genes of Xenopus laevis

Adult erythrocytes of X. laevis contain six electrophoretically resolvable globin polypeptides while tadpole erythrocytes contain four polypeptides, none of which comigrates with an adult protein. We show that three of the adult proteins are alpha globin polypeptides (alpha 1, alpha 2, alpha 3) and three are beta globin polypeptides (beta 1, beta 2, beta 3). We find that a tadpole alpha globin gene (alpha T1) is linked to the major adult locus in the sequence 5'-alpha T1-alpha 1-beta 1-3' with 5.2 kb separating alpha T1 from alpha 1. Another tadpole alpha globin gene (alpha T2) is linked to the minor adult locus in the sequence 5'-alpha T2-alpha 2-beta 2-3' with 10.7 kb separating alpha T2 from alpha 2. These linkage relationships are consistent with the major and minor loci having arisen by tetraploidization but the different separation of larval and adult globin genes at the two loci indicates the occurrence of some additional chromosomal rearrangement. Two alternative models are presented.