The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component

The erythrocytes of the marine polychaete Glycera dibranchiata contain a number of different, single-chain hemoglobins, some of which self-associate into a 'polymeric' fraction. An oligodeoxynucleotide probe was synthesized based on partial amino acid sequences determined by chemical methods, and used to screen a cDNA library constructed from the poly(A+)mRNA of Glycera erythrocytes (Simons, P.C. and Satterlee, J.D. (1989) Biochemistry 28, 8525-8530). The longest positive inserts found were sequenced using the dideoxy nucleotide chain termination method. One complete clone was obtained: clone 5A, 816 bases long, contained 59 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for 147 amino acids and a 3'-untranslated region of 316 bases. The derived amino acid sequence of Glycera globin P1 was in agreement with the partial amino acid sequences obtained by chemical methods. Three additional inserts obtained in the screening were also sequenced: the inferred amino acid sequences proved to be partial globin sequences which were different from each other and from the sequence of P1. Thus, the 'polymeric' fraction of the intracellular hemoglobin of Glycera probably consists of at least four different globin chains much like the 'monomeric' fraction. Comparison of the 'polymeric' sequence with the two known 'monomeric' sequences, M-II and M-IV, shows that they share 54 identical residues. At 74 positions, the identical residues in M-II and M-IV differ from the corresponding residue in P1, including at E-7, where P1 has a distal His, in contrast to Leu in M-II and M-IV. The alignment of Bashford et al. ((1987) J. Mol. Biol. 196, 199-216) and their templates were used to examine the principal differences between the two types of Glycera globin sequences. They appear to consist of uncommon surface amino acid residues at positions C6 (Phe vs. Ala), E10 (Val vs. Lys), E17 (Lys vs. Val), G1 (Arg vs. Lys), G10 (Met vs. Ala) and H5 (Arg vs. Lys). One or more of these residues could be responsible for the self-association exhibited by the 'polymeric' Glycera globins.     

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.     

cDNA cloning and predicted amino acid sequence of Glycera dibranchiata monomer hemoglobin IV

The three major monomer hemoglobins from Glycera dibranchiata erythrocytes isolated in this laboratory were sequenced from their N-termini. A stretch of amino acid sequence identity was used to determine the sequence of a mixed oligodeoxynucleotide that would be complementary to all 12 possible mRNA sequences coding for the amino acids. A cDNA library was constructed by using poly(A+) RNA from G. dibranchiata erythrocytes, the library was probed with the oligonucleotide, and the longest positive inserts found were subcloned into a sequencing plasmid and then sequenced. The first one was 745 bases long, containing 85 bases of 5'-untranslated RNA, an open reading frame of 444 bases coding for 148 amino acids, and a 3'-untranslated region of 216 bases. The predicted amino acid sequence matches the first 25 amino acids of G. dibranchiata monomer globin component IV. The sequence contains an N-terminal methionine plus 18 other mostly conservative sequence changes compared to the published sequence of Imamura et al. (1972), which appears from our partial sequencing to be monomer globin component II. We confirm the presence of leucine in the E7 position, which is histidine in most myoglobins and hemoglobins.     

Evolution of the sulfide-binding function within the globin multigenic family of the deep-sea hydrothermal vent tubeworm Riftia pachyptila

The giant extracellular hexagonal bilayer hemoglobin (HBL-Hb) of the deep-sea hydrothermal vent tube worm Riftia pachyptila is able to transport simultaneously O(2) and H(2)S in the blood from the gills to a specific organ: the trophosome that harbors sulfide-oxidizing endosymbionts. This vascular HBL-Hb is made of 144 globins from which four globin types (A1, A2, B1, and B2) coevolve. The H(2)S is bound at a specific location (not on the heme site) onto two of these globin types. In order to understand how such a function emerged and evolved in vestimentiferans and other related annelids, six partial cDNAs corresponding to the six globins known to compose the multigenic family of R. pachyptila have been identified and sequenced. These partial sequences (ca. 120 amino acids, i.e., 80% of the entire protein) were used to reconstruct molecular phylogenies in order to trace duplication events that have led to the family organization of these globins and to locate the position of the free cysteine residues known to bind H(2)S. From these sequences, only two free cysteine residues have been found to occur, at positions Cys + 1 (i.e., 1 a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., 11 a.a. from the same histidine) in globins B2 and A2, respectively. These two positions are well conserved in annelids, vestimentiferans, and pogonophorans, which live in sulfidic environments. The structural comparison of the hydrophobic environment that surrounds these cysteine residues (the sulfide-binding domain) using hydrophobic cluster analysis plots, together with the cysteine positions in paralogous strains, suggests that the sulfide-binding function might have emerged before the annelid radiation in order to detoxify this toxic compound. Moreover, globin evolutionary rates are highly different between paralogous strains. This suggests that either the two globin subfamilies involved in the sulfide-binding function (A2 and B2) have evolved under strong directional selective constraints (negative selection) and that the two other globins (A1 and B1) have accumulated more substitutions through positive selection or have evolved neutrally after a relaxation of selection pressures. A likely scenario on the evolution of this multigenic family is proposed and discussed from this data set.     

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.     

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26–33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human α and β globins strongly indicated that the leech globins first separated from globin lineage of annelids.     

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.     

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     

Amino acid sequence of the dimeric hemoglobin (Hb I) from the deep-sea cold-seep clam Calyptogena soyoae and the phylogenetic relationship with other molluscan globins

The deep-sea cold-seep clam Calyptogena soyoae has two homodimeric hemoglobins (Hbs I and II) in erythrocytes. The complete amino acid sequence of Hb I has been determined. It is composed of 144 amino acid residues, has a high content of hydrophobic residues, and a calculated molecular weight of 16,350 including a heme group. The sequence of Calyptogena Hb I showed high homology (42% identity) with that of Calyptogena Hb II (Suzuki, T., Takagi T. and Ohta, S. (1989) Biochem. J. 260, 177-182), although it has a long insertion of seven residues in the C-terminal region compared with Hb II. On the other hand, it showed low homology (12-20% identity) with other molluscan globins. As well as Hb II, Calyptogena Hb I lacked the N-terminal extension of 7-9 residues characteristic of molluscan intracellular hemoglobins, and the distal (E7) histidine was replaced by glutamine. A phylogenetic tree was constructed from 13 molluscan globins belonging to the five families Aplysiidae, Galeodidae, Potamididae, Arcidae and Vesicomyidae. The globin sequences of Calyptogena (Vesicomyidae) were found to be rather distant from other globin sequences, suggesting that they might conserve a primitive form of molluscan globins.     

Globin gene family evolution and functional diversification in annelids

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

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.     

Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume.

Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.     

Variation of folded polypeptide surface area with probe size

Three types of polypeptide surface area (contact, accessible, and molecular) have been studied as a function of the radius of a probe sphere used to map the surface. The surfaces are: (1) three alpha-helices, the H-helix of myoglobin, the E-helix of leghemoglobin, and an artificial polyalanine helix, each with 26 residues; (2) two globins, myoglobin and leghemoglobin, each with 153 residues; and (3) a two-center model system for which the three types of surface area have been calculated analytically. The two globin helices have almost identical surface areas as a function of probe size as do the two globins. The polyalanine helix surface area is smaller but similar in shape to the globin helix areas. All three helix contact areas tend to the same limit as the probe size increases, and the globin contact areas behave similarly. Fractal dimensions were calculated for the helix and globin contact and molecular surfaces. All fractal dimensions showed strong dependence on probe size. The contact fractal dimension peaks at larger values for both the helices and globins. Most residues do not make contact with large probes (15 A).     

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.     

Purification and Characterization of Recombinant Polymeric Hemoglobin P1 of Glycera dibranchiata

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the α-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).     

Structural Features of Glycera dibranchiata Monomer Hemoglobins. Primary Sequences of Monomer Hemoglobin Components II and III

Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785–2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151–164], it becomes clear that these three (GMG2–4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondences which unify disparate published isolations.     

Structural Features of Glycera dibranchiata Monomer Hemoglobins. Primary Sequences of Monomer Hemoglobin Components II and III

Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785–2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151–164], it becomes clear that these three (GMG2–4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondences which unify disparate published isolations.     

Characterization and expression of embryonic globin in the rainbow trout, Oncorhynchus mykiss: Intra-embryonic initiation of erythropoiesis

When fractionated by reverse-phase high performance liquid chromatography (HPLC), the embryonic hemoglobin of the rainbow trout, Oncorhynchus mykiss, consisted of eight globins different from adult globins in terms of retention time. Amino acid sequences of the N-terminal regions of some globins were determined. In addition, four cDNA clones for embryonic globins from 10-day embryos were isolated (at 15 degrees C), sequenced and the amino acid sequences predicted. In comparison with the sequences of previously characterized globins, they corresponded to two alpha-type and two beta-type globins and therefore were named em.alpha-1, em.alpha-2, em.beta-1 and em.beta-2. The N-terminal 36 amino acids of one (E2) of the embryonic globins isolated by HPLC were identical to those of the sequence deduced from a cDNA, em.beta-2. The phylogenetic relationship between the embryonic globins and other globins previously reported was discussed. The present study is the first demonstration of amino acid sequences of embryonic globins in a teleost. To understand the initiation of erythropoiesis in the early development of the rainbow trout, histochemistry using o-dianisidine/hydrogen peroxide, immunohistochemistry using an antibody against embryonic hemoglobin, and northern blotting and whole embryo in situ hybridization using antisense RNA probe for em.beta-2 were performed. Embryonic globin mRNA, globin and hemoglobin appeared first in the anterior part of the intermediate cell mass (ICM) located in the median line beneath the notochord of embryos 6-7 days after fertilization at 15 degrees C (Vernier's stages 16-20). Shortly after that, the expression signal extended to the posterior part of the ICM and spread out laterally to blood islands on the posterior yolk sac. Thus, the initiation of erythropoiesis in the early embryo of rainbow trout is intraembryonic.