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.     

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.      

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.     

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)      

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.     

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.     

Structure and evolution of the horse zeta globin locus

The equine zeta globin gene locus consists of an intact 5' gene and a truncated 3' pseudogene (psi zeta) that has only 5' control sequences and a first exon and intron. Nevertheless, the psi zeta gene has retained almost perfect homology with its neighbour, presumably by gene conversion. The first introns of both zeta and psi zeta genes contain a number of degenerate tandem repeats of a 14 base-pair sequence that has been found in the zeta genes of goats and humans and that is related to a family of human minisatellite sequences. Comparisons of sequences flanking the zeta and psi zeta genes reveal areas of considerable interspecies homology, which can be explained by a zeta gene duplication that pre-dated the mammalian radiation.     

A large deletion encompassing the entire alpha-like globin gene cluster in a family of northern European extraction.

We describe a new deletional form of alpha thalassemia segregating in three generations of a family of northern European origin. A full-term female girl had hypochromic, microcytic anemia since early infancy associated with delayed language development, slow growth and weight gain. Hematologic studies suggested the presence of alpha thalassemia. Gene-blotting studies showed no abnormal alpha-like globin gene fragments; however, studies of inheritance of informative polymorphic restriction fragments using zeta, alpha and 3'-alpha-hypervariable region (3'-HVR) probes showed evidence for an extensive deletion encompassing the entire alpha-like globin gene cluster. The 3' breakpoint of this deletion maps beyond the 3'-HVR, a region implicated as a hot spot for the generation of other large deletional events within the alpha-like cluster. The 5' breakpoint maps at least 10 kilobases (kb) 5' to the zeta-globin gene. The minimum size estimate for this deletion is greater than 47 kilobases.     

Analysis of a 70 kb segment of DNA containing the human zeta and alpha-globin genes linked to their regulatory element (HS-40) in transgenic mice.

We have ligated two cosmids through an oligonucleotide linker to produce a single fragment spanning 70 kb of the human alpha-globin cluster, in which the alpha-like globin genes (zeta 2, alpha 2 and alpha 1), their regulatory element (HS-40) and erythroid-specific DNase I hypersensitive sites accurately retain their normal genomic organization. The zeta (embryonic) and alpha (embryonic, fetal and adult) globin genes were expressed in all 17 transgenic embryos. Similarly, all fetal and adult mice from seven transgenic lines that contained one or more copies of the fragment, produced up to 66% of the level of endogenous mouse alpha-globin mRNA. However, as for smaller constructs containing these elements, human alpha-globin expression was not copy number dependent and decreased by 1.5-9.0 fold during development. These findings suggest that either it is not possible to obtain full regulation of human alpha-globin expression in transgenic mice or, more likely, that additional alpha-globin regulatory elements lie beyond the 70 kb segment of DNA analysed.     

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)     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences

The evolutionary history of the rabbit globin pseudogene psi beta 2 was studied by completing its nucleotide sequence and aligning the sequence with that of the rabbit adult globin gene beta 1 and the human minor adult globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were most similar to rabbit beta 1, but the large intervening sequence and the 3' untranslated region were most similar to human delta. Intron 1 and exon 2 were equally similar to both delta and beta 1. This pattern indicates that psi beta 2 was originally a delta-like gene that acquired the 5' portion of gene beta 1 by intrachromosomal gene conversion. The presence of a delta-globin gene sequence in both rabbits and humans shows that it is an ancient gene, predating the mammalian radiation that occurred over 85 Myr ago. Delta has shown a pronounced tendency to be altered in its 5' end during the course of mammalian evolution. Quantitative divergence analysis shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr ago, during which time the lagomorph beta-globin gene family apparently functioned without a pseudogene.      

Unusual features of CpG-rich (HTF) islands in the human alpha globin complex: association with non-functional pseudogenes and presence within the 3' portion of the zeta gene.

We have characterised a cluster of CpG rich (HTF) islands in the alpha-globin complex and report here two unusual features: The human embryonic zeta 2-globin gene is associated with an HTF island within its 3' portion rather than at the 5' end. Furthermore at least two non-functional pseudogenes within the cluster (psi zeta 1 and psi alpha 2) are associated with CpG rich islands.     

Genetic organization of the polymorphic equine alpha globin locus and sequence of the BII alpha 1 gene.

The equine alpha globin gene complex comprises two functional alpha genes and an alpha-like pseudogene arranged in the order 5'-alpha 2-(5kb)-alpha 1-(3kb)-psi alpha-3'. A single (embryonic) zeta-like sequence lies within a 12 kb region 5' to the alpha 2 gene. We have determined the sequence of the alpha 1 gene of the BII haplotype, one of two most common haplotypes (the other being BI) which encode alpha globins with either Tyr (BI) or Phe (BII) at codon 24 in both linked alpha genes. In BI and BII the non-allelic alpha 2 and alpha 1 genes respectively code for Gln or Lys at codon 60, thus accounting for the 4 alpha globin types seen in BI/BII heterozygotes. Genomic restriction enzyme maps of the BII alpha complex (24Phe/60Lys,Gln) and the allelic BI (24Tyr/60Lys,Gln) are identical to each other, and to those of a rarer normal haplotype, A, which encodes only alpha 24Tyr/60Gln globin, and a low expression mutant of BII which encodes only 24Phe/60Lys globin. These two latter haplotypes must therefore have a linked pair of alpha genes, as in BI and BII, but with identical coding properties, and it is suggested that this has arisen by gene conversion.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.