A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

A survey of type I interferons from a marsupial and monotreme: implications for the evolution of the type I interferon gene family in mammals

Sequence data for type I interferons (IFNs) have previously only been available for birds and eutherian ('placental') mammals, but not for the other two groups of extant mammals, the marsupials and monotremes. This has left a large gap in our knowledge of the evolutionary and functional relationships of what is a complex gene family in eutherians. In this study, a PCR-based survey of type I IFN genes from a marsupial, the tammar wallaby (Macropus eugenii), and a monotreme, the short-beaked echidna (Tachyglossus aculeatus), was conducted. Along with Southern blot and phylogenetic analysis, this revealed a large number of type I IFN genes for the wallaby, rivalling that of eutherians, but relatively few type I IFN genes in the echidna. The wallaby genes include both IFNA and IFNB orthologues, indicating that the gene duplication leading to these subtypes occurred prior to the divergence of marsupials and eutherians some 130 million years ago. Results from this study support the idea that the expansion of type I IFN gene complexity in mammals coincides with a concomitant expansion in the functionality of these molecules. For example, this expansion in complexity may have, at least partially, facilitated the evolution of viviparity in marsupials and eutherians. Other evolutionary aspects of these sequences are also discussed.     

Isolation of the chicken beta-globin gene and a linked embryonic beta-like globin gene from a chicken DNA recombinant library.

A library of random chicken DNA fragments, 15-22 kb long, has been prepared in the vector lambda Charon 4A. This library was screened with combined adult and embryonic globin cDNA, and several independent globin gene-containing recombinants were isolated. One of these recombinants, lambda Chicken beta-globin 1 (lambda C beta G1), contains the adult chicken beta-globin gene and a closely linked embryonic beta-like globin gene. Both genes are transcribed in the same direction with the adult gene located 5' to the embryonic gene. Electron microscopic visualization of R loop structures generated by hybridization of globin RNA to lambda C beta G1 demonstrates that both globin genes contain major intervening sequences about 800 bp long, similar to those present in mammalian beta-globin genes. The adult beta-globin gene also contains a minor (approximately 100 bp long) intervening sequence analogous to the one observed in mammalian beta-globin genes. Restriction enzyme analysis of the adult beta-globin gene on lambda C beta G1 is consistent with the hypothesis that its two intervening sequences occur in the same positions with respect to the beta-globin amino acid sequence as do the corresponding mammalian intervening sequences.     

Type I interferon genes from the egg-laying mammal, Tachyglossus aculeatus (short-beaked echidna)

The type I IFN are an important group of multifunctional cytokines that have, for whatever reason, evolved to a high level of complexity in eutherian mammals such as humans and mice. However, until recently, little was known about the type I IFN systems of the other two groups of extant mammals, the marsupials and the egg-laying monotremes. Preliminary partial type I IFN sequences from the short-beaked echidna were previously found to cluster only with the IFN-beta subtype in phylogenetic analyses, but a lack of sequence information made interpretation of these results tenuous. Here, we report cloning of the full-length genes of representatives from the two previously defined groups of echidna type I IFN by genomic walking PCR. Along with analysis of conserved cysteine placement and promoter elements, phylogenetic analysis incorporating these sequences strongly suggest that the two groups of echidna type I IFN genes are in fact homologous to IFN-alpha and IFN-beta, confirming that the duplication leading to these two major classes of type I IFN occurred prior to the divergence of eutherians and monotremes some 180 million years ago. Thus, even though there are major differences in gene copy number and heterogeneity, separate IFN-alpha and IFN-beta gene families are a feature of the cytokine networks of all three groups of living mammals.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Structure, sequence and function of a marsupial LIF gene: conservation of IL-6 family cytokines.

Leukaemia Inhibitory Factor (LIF) is a multifunctional cytokine with an obligate role in the mouse in embryonic implantation. In this paper we demonstrate the existence of a functional LIF gene in the marsupial Sminthopsis crassicaudata, and the presence of LIF-related sequences in the monotreme Tachyglossus aculeatus (Australian echidna). Isolation of genomic and cDNA clones from S. crassicaudata, indicated that the LIF gene is highly conserved between marsupials and monotremes in terms of sequence and genomic organisation. Critical functional residues within the LIF sequence were also conserved including residues implicated in intracellular LIF activity, and in interaction with the receptor subunits LIFR and gp130. These findings suggest that the structure and biochemical function of the protein is likely to be conserved. Consistent with this, purified recombinant S. crassicaudata LIF interacted functionally with mouse receptor components and was sufficient for maintenance of mouse embryonic stem (ES) cells in the undifferentiated state. Conservation of LIF outside eutherians is intriguing given the markedly divergent reproductive strategies which include, for some marsupial species, embryonic diapause, and in monotremes, the absence of implantation. The availability of marsupial LIF probes provides an opportunity to investigate conservation of expression and function in these mammals.     

Marsupials and Eutherians reunited: genetic evidence for the Theria hypothesis of mammalian evolution

The three living monophyletic divisions of Class Mammalia are the Prototheria (monotremes), Metatheria (marsupials), and Eutheria (`placental' mammals). Determining the sister relationships among these three groups is the most fundamental question in mammalian evolution. Phylogenetic comparison of these mammals by either anatomy or mitochondrial DNA has resulted in two conflicting hypotheses, Theria and Marsupionta, and has fueled a ``genes versus morphology' controversy. We have cloned and analyzed a large nuclear gene, the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R), from representatives of all three mammalian groups, including platypus, echidna, opossum, wallaby, hedgehog, mouse, rat, rabbit, cow, pig, bat, tree shrew, colugo, ringtail lemur, and human. Statistical analysis of this nuclear gene unambiguously supports the morphology-based Theria hypothesis that excludes monotremes from a clade of marsupials and eutherians. The M6P/IGF2R was also able to resolve the finer structure of the eutherian mammalian family tree. In particular, our analyses support sister group relationships between lagomorphs and rodents, and between the primates and Dermoptera. Statistical support for the grouping of the hedgehog with Feruungulata and Chiroptera was also strong. Received: 8 December 2000 / Accepted: 01 February 2001     

The evolution of the thyroid hormone distributor protein transthyretin in the order insectivora, class mammalia

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.     

Duplication of a four-gene set during the evolution of the goat beta-globin locus produced genes now expressed differentially in development

Genomic clones which link the goat preadult (beta C) and adult (beta A) beta-globin genes have been isolated. These overlapping clones contain a previously unidentified embryonic like globin gene (epsilon III) and establish the following linkage map of eight genes in the goat beta-globin locus: epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A. This linkage map and the nucleotide sequence of the eight genes document a relatively recent duplication of a four-gene set: epsilon-epsilon-psi beta-beta. This duplication produced two genes (beta C and beta A) which are now expressed differentially during development. An embryonic like globin gene located downstream from beta A has also been isolated. The embryonic nature of this gene as well as the adult beta-like sequence of the goat fetal globin gene (gamma) suggest that a duplication of the four-gene set also produced the globin gene now expressed during fetal development.     

Molecular evolution of monotreme and marsupial whey acidic protein genes

Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.     

M6P/IGF2R imprinting evolution in mammals

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.     

Immunoglobulin genetics of Ornithorhynchus anatinus (platypus) and Tachyglossus aculeatus (short-beaked echidna)

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.     

The karyotype of Alligator mississippiensis,and chromosomal mapping of the ZFY/X homologue,Zfc

Comparative mapping studies of X-linked genes in mammals have provided insights into the evolution of the X chromosome. Many reptiles including the American alligator, Alligator mississippiensis, do not appear to possess heteromorphic sex chromosomes, and sex is determined by the incubation temperature of the egg during embryonic development. Mapping of homologues of mammalian X-linked genes in reptiles could lead to a greater understanding of the evolution of vertebrate sex chromosomes. One of the genes used in the mammalian mapping studies was ZFX, an X-linked copy of the human ZFY gene which was originally isolated as a candidate for the mammalian testis-determining factor (TDF). ZFX is X-linked in eutherians, but maps to two autosomal locations in marsupials and monotremes, close to other genes associated with the eutherian X. The alligator homologue of the ZFY/ZFX genes, Zfc, has been isolated and described previously. A detailed karyotype of A. mississippiensis is presented, together with chromosomal in situ hybridisation data localising the Zfc gene to chromosome 3. Further chromosomal mapping studies using eutherian X-linked genes may reveal conserved chromosomal regions in the alligator that have become part of the eutherian X chromosome during evolution.     

Concerted evolution of the cow epsilon 2 and epsilon 4 beta-globin genes

The nucleotide sequences of the cow epsilon 2 and epsilon 4 globin genes were determined. The sequences were 95% identical. These genes arose via a four-gene block duplication that also gave rise to the bovine fetal (gamma) and adult (beta) genes. Their deduced amino acid sequences are unlike any previously reported fetal or adult globins; rather, comparison to other mammalian globin genes indicates that they are embryonic in nature. The sequence data indicate that these two genes have converted each other during evolution. Pairwise comparison to the corresponding goat genes shows greater similarity between paralogues than between more directly related orthologues. This is in direct contrast to the situation between the cow and goat fetal and adult genes. These observations suggest that the frequency of DNA conversion or the fixation of conversion events may vary in different locations of the cow beta-globin cluster.      

Nucleotide sequence of the goat embryonic alpha globin gene (zeta) and linkage and evolutionary analysis of the complete alpha globin cluster

In previous studies we identified and sequenced clones containing two adult alpha globin genes of the goat. Additional studies have revealed the presence of an embryonic alpha globin gene termed zeta. Sequence analysis of the gene shows that it is the largest mammalian or avian globin gene cloned to date. Its unusual size is mainly due to a 14 base-pair tandem repeat sequence in its first intron. A similar sequence is also found in the first intron of the human zeta gene. The goat zeta coding sequence differs greatly from that of the adult alpha, particularly at amino acid position 38, where it codes for the amino acid replacement of Gln for Thr. This change may confer a higher intrinsic O2 affinity on the zeta globin protein, ensuring a sufficient O2 supply for the developing goat embryo. The cloning and sequencing of this gene completes the alpha globin locus of the goat, composed of three genes in the following order 5'-zeta-I alpha-II alpha-3'. Evolutionary comparisons of the goat alpha locus with other amphibian, avian and mammalian loci reveal several interesting features. Statistical analysis confirms the hypothesis that the embryonic alpha gene is much older (400 million years) than the embryonic beta gene (200 million years), and that it is descended from a primordial gene, whose present-day counterpart is the Xenopus larval alpha globin gene. Our results also suggest that after the divergence of the avian line, the alpha A gene converted the alpha D gene during the evolution of the pre-mammalian line. The alpha D globin gene remains unconverted in the avian line, potentially because of insertion/deletion sequences that may prevent any gene conversion event. The divergence rates of specific globin genes have been analyzed and found to form an essentially straight line, in agreement with the neutralist view of evolution.