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.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.     

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.     

Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA

Genomic DNA from a fetal sheep homozygous for the beta A gene was used to construct a library of one million cloned DNA fragments using the bacteriophage vector, Charon 4A. Screening of 150,000 plaques from this library using radioactive beta-globin gene sequences resulted in the isolation of two recombinant bacteriophage containing globin genes. One of these, S beta AG-21, contains the complete adult beta A-globin gene as demonstrated by hybridization and restriction endonuclease analysis. In common with adult globin genes from other species, the beta A gene contains small (105 base pairs) and large (900 base pairs) intervening sequences. The second recombinant bacteriophage, SG-4, contains a complete embryonic beta-like globin gene which is expressed in the sheep embryo as demonstrated by hybridization analysis with cDNA made from sheep embryonic globin mRNA. Although differing in its restriction endonuclease map from the adult beta-globin genes, SG-4 appears to contain a large intervening sequence of at least 750 base pairs in length. Finally, preliminary evidence is discussed which indicates that a Pvu II site just 5' to the Cap site may be a common feature of sheep globin genes.     

Cloning of a new mouse foetal beta-globin mRNA sequence.

A novel globin cDNA recombinant (pFG5) has been isolated from a 14-15 day Porton mouse foetal liver cDNA library. It codes for a beta-like globin mRNA expressed in foetal liver-derived erythroblasts and erythrocytes but not in adult reticulocytes nor in yolk sac derived nucleated erythrocytes. It is also found in Friend cells induced to differentiate by DMSO. The nucleotide sequence of pFG5 confirms that it does not code for the beta major or beta minor globin chains nor the embryonic epsilon Y2 globin chain; but it is identical to the published partial sequence of the epsilon Y3 globin gene over the region of overlap (78 nucleotides).     

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.     

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.      

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.     

Molecular cloning of DNA sequences coding for mouse embryonic globins

Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two are alpha-like and two are beta-like. Pure globin messenger RNAs from these cells were used as templates for two successive polymerizing reactions and a mixture of double stranded cDNAs coding for the four globins was obtained. These molecules were blunt-end ligated to an ECoR1 digested pBR322 plasmid and the recombinant plasmids were used to transform E. coli Hb101. Bacterial clones which proved positive upon hybridization with 32P-labelled embryonic globin cDNA were amplified and their plasmid DNA was isolated. Three different plasmids were studied, namely no. 2, 16 and 54. The restriction map of these plasmids showed that: 1) plasmid no. 2 and 54 had lost extensive DNA sequences comprising the genes responsible for tetracycline resistance; 2) the size of inserted sequences ranges from 427 base pairs of plasmid no. 16 to about 280 base pairs of plasmid no. 54; 3) plasmid no. 2 does not share any of the studied restriction sites with the other plasmids, while no. 2 and 54 have at least one site in common. The coding properties of inserted DNA were determined by positive hybrid translation showing that no. 2 codes for the alpha-like embryonic chain x, while no. 16 and 54 code for a beta-like embryonic chain, either y or z.     

The detection of a cluster of beta-globin genes in the long arm of chromosome 2 in the domestic hen by using DNA-DNA in situ hybridization]

Using isotopic and nonisotopic methods of in situ hybridization, the localization the beta-like globin gene cluster was established. Molecular hybridization of DNA probes containing embryonic epsilon-globin and adult beta-globin genes revealed the localization of the cluster of the long arm on chromosome 2 (2q).     

A study of the chromatin structure of human beta-like hemoglobin genes in K562 cell line with a modified assay of nick-translation of nuclei

A modified assay of nick-translation of nuclei has been developed to study the chromatin structure of human beta-like globin genes in nuclei of K 562 cell line. Nuclei were gently digested with DNase I and nick-translated with E. coli DNA polymerase I in the presence of 32P-triphosphate nucleotides. The total DNA from the labelled nuclei was used as probes to hybridize restricted fragments of beta-like globin genes which have been immobilized on Diazobenzyloxymethyl (DBM) paper. Using this approach we have observed that in K 562 nuclei all beta-like globin genes, including epsilon, gamma, delta, and beta-globin genes and human 18 S ribosomal genes are preferentially labelled in comparison to alpha-lactalbumin and c-sis genes which do not express in K 562 cells, but the total DNA from nick-translated nuclei of a nonerythroid cell line hybridized none of those genes except for 18 S ribosomal gene. This assay is a simple and fast method for surveying chromatin structure of any individual DNA sequence in nuclei once the corresponding clone is available.