Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.     

Complete amino acid sequences of the major early embryonic alpha-like globins of the chicken

Vertebrate embryos contain hemoglobins composed of globin polypeptides structurally distinct from those of adults. Together with fetal and adult globin chains, these early embryonic globins are encoded by two developmentally regulated multigene families. To facilitate analysis of the structure and evolution of early embryonic alpha-globin genes, we have determined the complete amino acid sequences of the pi and pi' alpha-like globins of the chick embryo. While differing from each other by an alanine/glutamic acid interchange at position 124, this pair of sequences differs from the major and minor adult alpha-globins by 43%. The early embryonic and adult alpha-like sequences appear to have diverged following an ancient gene duplication. We discuss specific amino acid substitutions in functional positions as possible mediators of the reduced Bohr effect and elevated oxygen affinity, which are characteristic of early embryonic hemoglobins.     

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.     

Carnivora: primary structure of the hemoglobins from ratel (Mellivora capensis)

The erythrocytes of adult ratel contain two hemoglobin components, with two alpha- and one beta-chains. In this paper, their complete amino acid sequences are presented. The two alpha-chains differ in one residue at position 34 (Ala----Val) only. The primary structure of the chains was determined by sequencing the N-terminal regions (45 steps) and the tryptic peptides after their isolation from the digests by reversed-phase high-performance liquid chromatography. The alignment of these peptides was deduced from homology with other carnivora globins. The alpha-chains show 21 and the beta-chains 11 exchanges compared with human globin chains. In the alpha-chains, one heme- and two alpha 1/beta 1 contacts are exchanged. In the beta-chains there are three exchanges which involve one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact. Between the ratel hemoglobin and those of carnivora a high degree of homology was found.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Molecular cloning and sequence analysis of two rat major globin cDNAs

Two cDNA clones for globins of the adult Wistar rat were isolated from a reticulocyte cDNA library and the nucleotide sequences of the inserts were determined. One clone contained a cDNA insert consisting of 556 bp and the other contained one of 577 bp, both covering the entire coding sequences for rat globins. Comparisons of their predicted amino acid sequences with known sequences of rat globins revealed that these cDNAs coded for a rat major alpha- and a major beta-globin, I alpha and II beta, respectively. The cause of diversity of rat globins was discussed in terms of the nucleotide sequences of cDNAs and known amino acid sequences of globins.     

The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA

We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

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.     

Amino acid sequence of a major globin from the sea cucumber Paracaudina chilensis

The sea cucumber Paracaudina chilensis (Echinodermata) contains three major globins I, II and III in coelomic cells. The complete amino acid sequence of globin I has been determined. It is composed of 157 amino acid residues, is acetylated at the N-terminus, and has a characteristic N-terminal extension of 9-10 residues when compared with vertebrate globins. The sequence of Paracaudina globin I showed slightly higher homology with human alpha globin (25%) rather than with the invertebrate Anadara alpha globin (22%). Paracaudina globin I also showed strong homology (59%) with globin D from another sea cucumber, Molpadia arenicola (Mauri, F.C. (1985) Ph.D. dissertation, University of Texas). The globin sequences from the phylum Echinodermata have an important position in the molecular evolution of the globins, because they are the invertebrate group most closely related to the vertebrates.     

The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata

The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 199-216) showed that all five globins have identical residues at 39 positions. At 44 positions, the three polymeric globins share identical residues that differ from the identical residues at the corresponding locations in the monomeric sequences M-II and M-IV including position E7, where the latter have leucine instead of the distal histidine. At 15 positions, there occurs an alteration from polar to nonpolar or from a small nonpolar to a larger nonpolar residue in going from the monomeric to the polymeric globins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.Abbreviations used GMHX the holo-protein (including b-type heme, Glycera dibranchiata monomer hemoglobin Component X (X=2, 3, or 4) - GMGX the apo-protein, or globin, Glycera dibranchiata monomer globin derived from Component X (X=2, 3, or 4) - rec-gmg the globin derived from a recombinant holoprotein of a Glycera dibranchiata monomer hemoglobin, rec-gmh, whose sequence has been inferred from an isolated cDNA insert - CB label refers to peptides generated from cyanogen bromide cleavage of GMG4 - HPLC high-performance liquid chromatography - T label refers to peptides generated from trypsin digests of GMG4 - Mb myoglobin - MCS monomer hemoglobin crystal structure from Glycera dibranchiata. H, N-terminal sequence of GMG4 - SWMb sperm whale myoglobin     

Transcriptome Profiling of Embryonic Development Rate in Rainbow Trout Advanced Backcross Introgression Lines

In rainbow trout (Oncorhynchus mykiss) and other fishes, embryonic development rate is an ecologically and evolutionarily important trait that is closely associated with survival and physiological performance later in life. To identify genes differentially regulated in fast and slow-developing embryos of rainbow trout, we examined gene expression across developmental time points in rainbow trout embryos possessing alleles linked to a major quantitative trait loci (QTL) for fast versus slow embryonic development rate. Whole genome expression microarray analyses were conducted using embryos from a fourth generation backcross family, whereby each backcross generation involved the introgression of the fast-developing alleles for a major development rate QTL into a slow-developing clonal line of rainbow trout. Embryos were collected at 15, 19, and 28 days post-fertilization; sex and QTL genotype were determined using molecular markers, and cDNA from 48 embryos were used for microarray analysis. A total of 183 features were identified with significant differences between embryonic development rate genotypes. Genes associated with cell cycle growth, muscle contraction and protein synthesis were expressed significantly higher in embryos with the fast-developing allele (Clearwater) than those with the slow-developing allele (Oregon State University), which may associate with fast growth and early body mass construction in embryo development. Across time points, individuals with the fast-developing QTL allele appeared to have earlier onset of these developmental processes when compared to individuals with the slow development alleles, even as early as 15 days post-fertilization. Differentially expressed candidate genes chosen for linkage mapping were localized primarily to regions outside of the major embryonic development rate QTL, with the exception of a single gene (very low-density lipoprotein receptor precursor).     

The multigenic family of the extracellular hemoglobin from the annelid polychaete Arenicola marina

The extracellular hemoglobin of the lugworm Arenicola marina which inhabits on the intertidal area, a sulfide-rich environment, comprises eight globin chains previously determined by mass spectrometry. We have cloned and sequenced five of the globin components. The deduced amino-acid sequences exhibit an extracellular signal peptide and two cysteine residues involved in an internal disulfide bond. The molecular weights calculated from the globin primary structures obtained from complete cDNA sequences are in good agreement with the mass spectrometry values obtained with the native hemoglobin. Phylogenetic analysis has allowed assigning the five A. marina sequences to the different globin sub-families. Two of the globins were found to be A2 globin chains lacking the cysteine residues proposed to be involved in the binding of hydrogen sulfide by such hemoglobin. We discuss the unusual absence of these cysteines in the light of their invariant occurrence in the A2 subfamily of hemoglobins from annelids inhabiting sulfide-rich environments.     

Differential hemoglobin synthesis in murine erythroleukemic cells: hemin effects

The addition of butyric acid to murine erythroleukemic cells (clone T3Cl2) induced the cells to differentiate, producing adult hemoglobin (A, alpha 2,beta 2) and an embryonic hemoglobin (E2, alpha 2Y2). The subsequent addition of hemin to the differentiating cells increased the synthesis of adult hemoglobin four-fold and the synthesis of embryonic hemoglobin two-fold; the relative synthesis of the alpha and beta globins increased more than the y globin. The embryonic hemoglobin was expressed prior to the adult hemoglobin in differentiating cells.     

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.     

Characterization of embryonic globin genes of the zebrafish

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching.