New views of evolution and regulation of vertebrate beta-like globin gene clusters from an orphaned gene in marsupials.

F if any, proteins have been studied in as many organisms as have the hemoglobins. They are found in bacteria, fungi, plants, and animals, serving physiological roles ranging from oxygen transport in the blood of vertebrates to catalyzing the combination of oxygen and nitric oxide to form nitrate in bacteria, yeast, and worms (1, 2). Hence hemoglobins and the globin genes encoding them have been an important system for investigating many biochemical and evolutionary issues. Furthermore, the tissuespecific and developmental regulation of globin gene expression has been studied intensively in mammals and birds. This led to the discovery of many cis-regulatory sequences such as promoters, enhancers, locus control regions (LCRs) (3), and insulators (4). If any model system were well understood both in terms of gene evolution and regulation, this should be the one. However, by their discovery of an orphaned b-like globin gene in marsupials, Wheeler et al. (5) show in a recent issue of PNAS that previously inferred relationships between the b-globin gene clusters in eutherian mammals and birds were oversimplified. The new data force a reconsideration of the evolutionary course of this gene cluster, how these regulatory elements evolved, and how they may be acting today. In all jawed vertebrates (gnathostomes) studied, distinctive forms of hemoglobin are produced during primitive erythropoiesis in embryonic life, followed by different hemoglobins produced during definitive erythropoiesis in fetal and adult life. These hemoglobins are tetramers of two a-like globins and two b-like globins, each with an associated heme to which oxygen can bind reversibly. The genes encoding the globins are clustered, with the a-like globin gene cluster on a different chromosome from the b-like globin gene cluster in birds and mammals. The human b-like globin gene cluster has five active globin genes, encoding the embryonic «-globin, two fetal g-globins, the adult d-globin produced at low levels, and the abundant adult b-globin (Fig. 1). Extensive comparisons with homologous genes from other eutherian mammals deduced a parsimonious pathway for the evolution of this gene cluster by repeated gene duplications (6, 7), including a predicted cluster containing the ancestor to «-globin and bglobin genes (Fig. 1). This prediction was verified by the discovery of linked «-globin and b-globin genes in two marsupial species, the American opossum, Didelphis virginiana (8), and the Australian dunnart, Sminthopsis crassicaudata (9). Chickens also have a developmentally regulated b-like globin gene cluster, but it is rearranged relative to that in eutherians (Fig. 1), with two embryonic genes, encoding r-globin and «-globin, at the ends of the cluster and two b-globin genes, one expressed at hatching (encoding bH-globin) and one expressed abundantly in adult red blood cells (encoding bA-globin). Given that the avian r-b-«globin gene cluster and the eutherian «-gd-b-globin gene clusters are the only b-like globin gene clusters known in these two taxa, it was natural to consider them to be orthologous, i.e., they were thought to be derived from the same gene cluster in the last common ancestor to birds and mammals. The discovery of the marsupial v-globin (5) argues against this. Rather, the eutherian «-g-d-b-globin gene cluster appears to be descended from a different gene cluster from that which led to the avian r-b-« cluster. Wallabies are medium-sized relatives of the kangaroos in Australia. The v-globin was discovered as a component of a novel hemoglobin found in the blood of neonatal tammar wallabies (Macropus eugenii). The v-globin gene is expressed just before and after the birth of the joey (5). A partial amino acid sequence of the tammar vglobin indicated that it was more closely related to bird b-globin than to the eutherian, marsupial, or monotreme b-globins (10, 11). The new studies from Wheeler et al. (5) show that the v-globin gene is widely distributed in marsupials and provide the DNA sequence of the v-globin gene from two species of marsupials, the tammar and the dunnart. This allows a more rigorous and complete phylogenetic analysis. The marsupial v-globin genes form a sister group with the avian b-, «-, and r-globin genes, clearly separated from the group containing the eutherian, marsupial, and monotreme b-like globin genes. Finally, Wheeler et al. (5) show that the v-globin gene is found on a separate chromosome from the «and b-globin gene cluster; hence it is an ‘‘orphaned’’ b-like gene. These new data argue for a significant change in the view of hemoglobin gene evolution in the warm-blooded vertebrates (Fig. 1). Marsupial mammals have both a bird-like v-globin gene and a b-like gene cluster similar to that of eutherian mammals. If they inherited both gene clusters from an ancestor common to birds and mammals, then the b-like globin gene clusters of eutherian mammals and avians are not orthologous. By this model, at a time preceding the divergence of birds and mammals, an ancestral gene (or more likely, gene cluster) duplicated. One gene cluster diverged to generate the «-g-d-bglobin gene clusters found in eutherian mammals and the «-b-globin gene clusters found in marsupials. The other gene cluster diverged to form the avian r-b-«globin gene cluster and the orphaned v-globin gene found in marsupial species. The eutherian b-like globin gene cluster