Use of long sequence alignments to study the evolution and regulation of mammalian globin gene clusters

The determination of long segments of DNA sequences encompassing the beta- and alpha-globin gene clusters has provided an unprecedented data base for analysis of genome evolution and regulation of gene clusters. A newly developed computer tool kit generates local alignments between such long sequences in a space-efficient manner, helps the user analyze the alignments effectively, and finds consistently aligning blocks of sequences in multiple pairwise comparisons. Such sequence analyses among the beta-like globin gene clusters of human, galago, rabbit, and mouse have revealed the general patterns of evolution of this gene cluster. Alignments in the flanking regions are very useful in assigning orthologous relationships. Investigation of such matches between the mouse and human beta-like globin gene clusters has led to a reassessment of some orthologous assignments in mouse and to a revision of the proposed pathway for evolution of this gene cluster. In general, the interspersed repetitive elements have inserted independently, presumably via a retrotransposition mechanism, in the different mammalian lineages. However, some examples of ancient L1 repeats are found, including one between the epsilon- and gamma-globin genes that appears to have been in the ancestral eutherian gene cluster. Prominent matching sequences are found in a long region 5' to the epsilon-globin gene, the locus control region (LCR) that is a positive regulator of the entire gene cluster. Three-way alignments among the human, goat, and rabbit sequences can extend for > or = 3 kb in part of the LCR (DNase hypersensitive site 3), indicating that the cis-acting components of this complex regulatory region cover a long segment of DNA. In contrast to the beta-like globin gene clusters, the alpha-like globin gene clusters of many mammals occur in very G+C-rich isochores and contain prominent CpG islands. The regions between the alpha-like globin genes are evolving faster than the intergenic regions of the beta-like globin gene clusters. The contrasts between the two gene clusters can be attributed to differences in DNA metabolism in the isochore. The proximal control elements of the rabbit alpha-globin gene are located both 5' to and within the gene. All of this region is part of a prominent CpG island that may be acting as an extended, enhancer- independent promoter. One can hypothesize that the analogue to the LCR in the alpha-globin gene cluster may interface with the distinctive alpha-globin promoter in ways different from the interaction between the beta LCR and the promoters of beta-like globin genes.(ABSTRACT TRUNCATED AT 400 WORDS)      

Sequence and comparative analysis of the rabbit alpha-like globin gene cluster reveals a rapid mode of evolution in a G + C-rich region of mammalian genomes

A sequence of 10,621 base-pairs from the alpha-like globin gene cluster of rabbit has been determined. It includes the sequence of gene zeta 1 (a pseudogene for the rabbit embryonic zeta-globin), the functional rabbit alpha-globin gene, and the theta 1 pseudogene, along with the sequences of eight C repeats (short interspersed repeats in rabbit) and a J sequence implicated in recombination. The region is quite G + C-rich (62%) and contains two CpG islands. As expected for a very G + C-rich region, it has an abundance of open reading frames, but few of the long open reading frames are associated with the coding regions of genes. Alignments between the sequences of the rabbit and human alpha-like globin gene clusters reveal matches primarily in the immediate vicinity of genes and CpG islands, while the intergenic regions of these gene clusters have many fewer matches than are seen between the beta-like globin gene clusters of these two species. Furthermore, the non-coding sequences in this portion of the rabbit alpha-like globin gene cluster are shorter than in human, indicating a strong tendency either for sequence contraction in the rabbit gene cluster or for expansion in the human gene cluster. Thus, the intergenic regions of the alpha-like globin gene clusters have evolved in a relatively fast mode since the mammalian radiation, but not exclusively by nucleotide substitution. Despite this rapid mode of evolution, some strong matches are found 5' to the start sites of the human and rabbit alpha genes, perhaps indicating conservation of a regulatory element. The rabbit J sequence is over 1000 base-pairs long; it contains a C repeat at its 5' end and an internal region of homology to the 3'-untranslated region of the alpha-globin gene. Part of the rabbit J sequence matches with sequences within the X homology block in human. Both of these regions have been implicated as hot-spots for recombination, hence the matching sequences are good candidates for such a function. All the interspersed repeats within both gene clusters are retroposon SINEs that appear to have inserted independently in the rabbit and human lineages.     

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.     

Comparison of the beta-like globin gene families of rabbits and humans indicates that the gene cluster 5'-epsilon-gamma-delta-beta-3' predates the mammalian radiation

The members of the rabbit and human beta-like globin gene families have been compared both by a computer-generated dot matrix graphical analysis of each entire gene and by calculating divergences in the coding regions. The rabbit-human gene pairs beta 4-epsilon, beta 3- gamma, psi beta 2-delta, and beta 1-beta were identified as orthologous on the basis of sequence similarities found in flanking and intervening sequences as well as by quantitative divergence calculations. The orthologous genes are in the same order on the chromosome in each species, which suggests that an ancestral family with the arrangement 5'-epsilon-gamma-delta-beta-3' preceded the mammalian radiation. Descendants of ancestral epsilon have diverged more slowly than other beta-like genes and are expressed only in embryonic life. Descendants of ancestral gamma and beta diverged at a higher rate and are expressed at wider range of developmental times. Descendants of delta have undergone nonreciprocal recombination at a high frequency and are often pseudogenes. Paralogous comparisons among the rabbit beta-like globin genes show that the beta 4-beta 3 and psi beta 2-beta 1 pairs are most similar and that beta 4 and beta 3 are more closely related to beta 1 than to psi beta 2. This fits with a branching pattern where the primordial beta split into ancestral epsilon/gamma and delta/beta genes, which later split into epsilon and gamma or delta and beta, respectively. Rabbit genes beta 4 and beta 1 acquired similar 3' untranslated regions after the epsilon/gamma split but prior to the mammalian radiation, presumably via a gene conversion event. The 5' end of beta 2 apparently converted with beta 1 after the radiation, and afterward it became a pseudogene.      

Block duplications of a zeta-zeta-alpha-theta gene set in the rabbit alpha-like globin gene cluster

In order to understand the coordinate regulation between the alpha-like and beta-like globins during the developmental switches in hemoglobin synthesis, we have studied the rabbit alpha-like globin gene family. A cluster of six linked genes arranged 5'-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3' has been isolated as a set of overlapping clones from a library of rabbit genomic DNA. Blot-hybridization analysis of genomic DNA not only confirms this linkage arrangement but also reveals the presence of additional zeta and theta genes. We propose that this gene cluster was generated by a block duplication of a set of alpha-like genes; the proposed duplication unit is zeta-zeta-alpha-theta. Further duplications of a zeta-zeta-theta set are also proposed to have occurred. As expected for a duplicated locus, the rabbit alpha-like gene cluster contains long blocks of internal homology. The Z homology block is about 7.2 kilobase pairs long and contains the zeta genes; the T homology block is about 4.7 kilobase pairs long and contains a theta gene. Surprisingly, both Z and T homology blocks are flanked by a common junction sequence (J) which contains a region very similar to the 3'-untranslated sequence of an alpha-globin gene. Analysis of the J sequences suggests a recombination mechanism by which the alpha gene could have been deleted from the second set of genes in the cluster (zeta 2-zeta 3-theta 2). The relationships among the genes in characterized alpha-like gene clusters in mammals are summarized. The rabbit gene cluster differs from those of other mammals principally in the loss of a gene orthologous to the human psi alpha 1 and in the block duplication of the zeta-zeta-alpha-theta gene set.     

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.     

Localization and characterization of members of a family of repetitive sequences in the goat beta globin locus.

We have characterized a family of repetitive DNA elements in the beta-globin locus of the goat. These sequences are structurally analogous to the Alu families of repeats of other mammals. Repetitive elements are located both in the intervening sequences and in the intergenic regions of the goat beta-globin locus. Nucleotide sequence analysis of five repetitive elements located within the large intervening sequence of the beta-like globin genes, and four repeats located 5' to the major developmentally regulated beta-globin genes has resulted in the definition of a consensus sequence for this family of repeats.     

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.      

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.     

Identification by nucleotide sequence analysis of a goat pseudoglobin gene

We have recently prepared a recombinant library of goat genomic DNA and have isolated clones containing th known goat globin genes. These include the alpha, gamma, beta C and beta A genes. In addition to these, another beta-like sequence has been observed. In this communication we report the complete nucleotide sequence of this gene, excluding a portion of the large intervening sequence. Several features suggest that this is a non-functional or pseudoglobin gene. The alterations include a frameshift mutation, substitution of the heme-binding histidines, a mutated termination codon, a change in the GT/AG excision sequence of the 5' end of the first intervening sequence, an AT rich sequence in the 3' untranslated region, and a mutated Hogness-Goldberg box. We conclude that this gene cannot function in the synthesis of globin.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.     

Complete nucleotide sequence of the rabbit beta-like globin gene cluster. Analysis of intergenic sequences and comparison with the human beta-like globin gene cluster

The nucleotide sequence of the entire beta-like globin gene cluster of rabbits has been determined. This sequence of a continuous stretch of 44.5 x 10(3) base-pairs (bp) starts about 6 x 10(3) bp upstream from epsilon (the 5'-most gene) and ends about 12 x 10(3) bp downstream from beta (the 3'-most gene). Analysis of the sequence reveals that: (1) the sequence is relatively A + T rich (about 60%); (2) regions with high G + C content are associated with OcC repeats, a short interspersed repeated DNA in rabbits; (3) the distribution of polypurines, polypyrimidines and alternating purine/pyrimidine tracts is not random within the cluster; (4) most open reading frames are associated with known globin coding regions, OcC repeats or long interspersed repeats (L1 repeats); (5) the most prominent open reading frames are found in the L1 repeats; (6) different strand asymmetries in base composition are associated with embyronic and adult genes as well as the tandem L1 repeats at the 3' end of the cluster; and (7) essentially all the repeats appear to have been inserted by a transposon mechanism. A comparison of the sequence with itself by a dot-plot analysis has revealed nine new members of the OcC family of repeats in addition to the six previously reported. The OcC repeats tend to be clustered, particularly in the epsilon-gamma and gamma-psi delta intergenic regions. Dot-plot comparisons between the rabbit and the human clusters have revealed extensive sequence matches. Homology starts about 6 x 10(3) bp 5' to epsilon or as far upstream as the rabbit sequence is available. It continues throughout the entire cluster and stops about 0.7 x 10(3) bp 3' to beta, at which point several repeats have inserted in both rabbits and humans. Throughout the gene cluster, the homology is interrupted mainly by insertions or deletions in either the rabbit or the human genome. Almost all of the insertions are of known short or long repeated DNAs. The positions of the insertions are different in the two gene clusters, which indicates that both short and long repeats have been transposing throughout the genome for the time since the mammalian radiation. An alignment of rabbit and human sequences allows the calculation of the substitution rate around epsilon. Sequences far removed from the gene are evolving at a rate equivalent to the pseudogene rate, although some short regions show an apparently higher rate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and functional analysis of the goat epsilon-globin genes

Since none of the vertebrate beta-globin loci studied to date has more than two functional embryonic beta-like globin genes, it would be unique if all six goat embryonic beta-globin genes were required for its survival. In this study we have asked whether all six embryonic genes in the goat are functional. This question has been addressed by examining the transient expression of these genes in HeLa cells and correlating these results with the sequence information obtained to date. Our studies show that only epsilon I and epsilon II are functional while the remaining four epsilon-globin genes are nonfunctional, i.e., pseudogenes. Interestingly, the two active epsilon-globin genes are located at the 5' end of the locus. While this unusual inactivation pattern may be the result of chance, it could also have resulted because the two duplication events, of the ancestral gene set epsilon-epsilon-psi beta-beta, did not include distally located regulatory region(s) essential for epsilon-globin gene expression. Once separated from the 5'-regulatory sequences the remaining four embryonic genes (epsilon III, epsilon IV, epsilon V and epsilon VI) accumulated mutations and became pseudogenes.     

Primate evolution of the alpha-globin gene cluster and its Alu-like repeats

The arrangement of alpha-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate alpha-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of alpha-globin genes. In contrast, the arrangement and correction of galago alpha-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5' and 3'-flanking regions of the human alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.