The globin gene repertoire of lampreys: convergent evolution of hemoglobin and myoglobin in jawed and jawless vertebrates.

Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in agnathans and gnathostomes involved the convergent co-option of different precursor proteins in the ancestral globin repertoire of vertebrates.

Conservation of globin genes in the "living fossil" Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family.

The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the "living fossil" Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a "globin fossil". Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O2 supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O2 supply. Latimeria possesses two α- and two ß-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic ß-chains - with the exception of a more complex pattern in the coelacanth and amphibians - display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of globins in certain taxa may be associated with changes in the oxygen-dependent metabolism. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Negative information for building phylogenies.

An absent word (also called a forbidden word or an unword in other contexts) in a sequence is a segment that does not appear in the given sequence. It is a minimal absent word if all its proper factors occur in the given sequence. In this article, we review the concept of minimal absent words, which includes the notion of shortest absent words but is much stronger. We present an efficient method for computing the minimal absent words of bounded length for DNA sequence using a Trie of bounded depth, representing bounded length factors. This method outputs the whole set of minimal absent words and furthermore our technique provides a linear-time algorithm with less memory usage than previous solutions. We also present an approach to distinguish sequences of different organisms using their minimal absent words. Our solution applies a length-weighted index to discriminate sequences and the results show that we can build phylogenetic tree based on the patent collected information.

Phylogenetic analysis reveals wide distribution of globin X.

The vertebrate globin gene repertoire consists of seven members that differ in terms of structure, function and phyletic distribution. While hemoglobin, myoglobin, cytoglobin, and neuroglobin are present in almost all gnathostomes examined so far, other globin genes, like globin X, are much more restricted in their phyletic distribution. Till today, globin X has only been found in teleost fish and Xenopus. Here, we report that globin X is also present in the genomes of the sea lamprey, ghost shark and reptiles. Moreover, the identification of orthologs of globin X in crustacean, insects, platyhelminthes, and hemichordates confirms its ancient origin.

Phylogenetic diversification of the globin gene superfamily in chordates.

Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this article is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this, we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges.

Structure and reactivity of hexacoordinate hemoglobins.

The heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called "pentacoordinate" hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called "hexacoordinate hemoglobins", which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal.

Tracing globin phylogeny using PSIBLAST searches based on groups of sequences.

PSIBLAST search of a protein database with a query sequence is a widely used tool for the detection of related but evolutionarily distant sequences. Iterations carried out until convergence (i.e., until the majority of the sequences most similar to the query sequence are extracted from the database) also produces an ordered list of more distantly related (i.e., less similar sequences) and false positives belonging to other protein families. Thus, a PSIBLAST search based on one group of globin sequences will provide sequences left in the database that are more distantly related (i.e., belong to other groups of globins) ordered according to the E value or bit score. The relative order of the scores should yield a clue to the relative similarity of the query group to the other groups of globins. Histograms of E values or bit scores from PSIBLAST searches using vertebrate myoglobins, cytoglobins, alpha- and beta-globins, and neuroglobins as query groups show distributions that are congruent with the accepted phylogenetic tree. An illustration of more distant relationships is demonstrated by the results using neuroglobins as the query group, which show a striking similarity to bacterial single-domain globins and flavohemoglobins from bacteria and eukaryotes. Furthermore, it is observed that sequences belonging to the three undoubtedly ancient globin lineages form very broad distributions, while recently evolved groups such as cytoglobins have narrow distributions. Thus, the breadth of a distribution of E values or bit scores for a query group may be related to its evolutionary age.

Genomic evidence for independent origins of beta-like globin genes in monotremes and therian mammals.

Phylogenetic reconstructions of the beta-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult beta-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic beta-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the "epsilon-globin" gene in birds is not orthologous to the epsilon-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto beta-globin gene. Here, we report evidence that the early and late expressed beta-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto beta-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the epsilon- and beta-globin genes of therian mammals arose via duplication of a proto beta-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of beta-like globin genes that originated via duplication of a proto beta-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto beta-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development.

Complex signatures of selection and gene conversion in the duplicated globin genes of house mice.

Results of electrophoretic surveys have suggested that hemoglobin polymorphism may be maintained by balancing selection in natural populations of house mice, Mus musculus. Here we report a survey of nucleotide variation in the adult globin genes of house mice from South America. We surveyed nucleotide polymorphism in two closely linked alpha-globin paralogs and two closely linked beta-globin paralogs to test whether patterns of variation are consistent with a model of long-term balancing selection. Surprisingly high levels of nucleotide polymorphism at the two beta-globin paralogs were attributable to the segregation of two highly divergent haplotypes, Hbbs (which carries two identical beta-globin paralogs) and Hbbd (which carries two functionally divergent beta-globin paralogs). Interparalog gene conversion on the Hbbs haplotype has produced a highly unusual situation in which the two paralogs are more similar to one another than either one is to its allelic counterpart on the Hbbd haplotype. Levels of nucleotide polymorphism and linkage disequilibrium at the two beta-globin paralogs suggest a complex history of diversity-enhancing selection that may be responsible for long-term maintenance of alternative protein alleles. The alternative two-locus beta-globin haplotypes are associated with pronounced differences in intraerythrocyte glutathione and nitric oxide metabolism, suggesting a possible mechanism for selection on hemoglobin function.

A phylogenomic profile of globins.

BACKGROUND: Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and "truncated" hemoglobins with a 2-over-2 instead of the canonical 3-over-3 alpha-helical fold. RESULTS: A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only approximately 25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in approximately 65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, approximately 33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. CONCLUSION: Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.

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.

Finding biologically relevant protein domain interactions: conserved binding mode analysis.

Proteins evolved through the shuffling of functional domains, and therefore, the same domain can be found in different proteins and species. Interactions between such conserved domains often involve specific, well-determined binding surfaces reflecting their important biological role in a cell. To find biologically relevant interactions we developed a method of systematically comparing and classifying protein domain interactions from the structural data. As a result, a set of conserved binding modes (CBMs) was created using the atomic detail of structure alignment data and the protein domain classification of the Conserved Domain Database. A conserved binding mode is inferred when different members of interacting domain families dock in the same way, such that their structural complexes superimpose well. Such domain interactions with recurring structural themes have greater significance to be biologically relevant, unlike spurious crystal packing interactions. Consequently, this study gives lower and upper bounds on the number of different types of interacting domain pairs in the structure database on the order of 1000-2000. We use CBMs to create domain interaction networks, which highlight functionally significant connections by avoiding many infrequent links between highly connected nodes. The CBMs also constitute a library of docking templates that may be used in molecular modeling to infer the characteristics of an unknown binding surface, just as conserved domains may be used to infer the structure of an unknown protein. The method's ability to sort through and classify large numbers of putative interacting domain pairs is demonstrated on the oligomeric interactions of globins.

Characterizing conserved structural contacts by pair-wise relative contacts and relative packing groups.

To adequately deal with the inherent complexity of interactions between protein side-chains, we develop and describe here a novel method for characterizing protein packing within a fold family. Instead of approaching side-chain interactions absolutely from one residue to another, we instead consider the relative interactions of contacting residue pairs. The basic element, the pair-wise relative contact, is constructed from a sequence alignment and contact analysis of a set of structures and consists of a cluster of similarly oriented, interacting, side-chain pairs. To demonstrate this construct's usefulness in analyzing protein structure, we used the pair-wise relative contacts to analyze two sets of protein structures as defined by SCOP: the diverse globin-like superfamily (126 structures) and the more uniform heme binding globin family (a 94 structure subset of the globin-like superfamily). The superfamily structure set produced 1266 unique pair-wise relative contacts, whereas the family structure subset gave 1001 unique pair-wise relative contacts. For both sets, we show that these constructs can be used to accurately and automatically differentiate between fold classes. Furthermore, these pair-wise relative contacts correlate well with sequence identity and thus provide a direct relationship between changes in sequence and changes in structure. To capture the complexity of protein packing, these pair-wise relative contacts can be superimposed around a single residue to create a multi-body construct called a relative packing group. Construction of convex hulls around the individual packing groups provides a measure of the variation in packing around a residue and defines an approximate volume of space occupied by the groups interacting with a residue. We find that these relative packing groups are useful in understanding the structural quality of sequence or structure alignments. Moreover, they provide context to calculate a value for structural randomness, which is important in properly assessing the quality of a structural alignment. The results of this study provide the framework for future analysis for correlating sequence changes to specific structure changes.

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.

[Quantitative analysis of human globin gene expression in beta-thalassemia using real-time RT-PCR].

Whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with beta-thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and 39 with beta-thalassemia major (beta(T)/beta(T),alphaalpha/alphaalpha). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for beta-thalassemia genotype analysis. Different types of beta globin gene mutations were analyzed using reverse dot blotting (RDB) method. Total RNA were extracted and subjected to real-time RT-PCR for quantitative measurement of alpha, beta and gamma globin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of alpha, beta and gamma human globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of alpha, beta and gamma globin mRNA were calculated and the ratios of alpha/beta, alpha/(beta + gamma ) and gamma /(beta + gamma ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, beta-thalassemia minor and beta-thalassemia major patients. The resultant data were analyzed using SPSS 10.0 software to determine statistical significance of human globin gene expression among normal controls and beta-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of alpha/beta + 1, alpha/(beta + gamma ) + 1 and gamma /(beta + gamma ) +1 was used for statistical analyses and intergroup comparison. The alpha/beta globin gene mRNA ratios were determined to be 4.62+/-1.20, 7.81+/-2.89, 13.51+/-5.12, and 188.24+/-374.04 for normal healthy adult (beta(A)/beta(A),alphaalpha/alphaalpha), infant (beta(A)/beta(A),alphaalpha/alphaalpha), beta- thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and beta-thalassemia major(beta(T)/beta(T),alphaalpha/alphaalpha) respectively. The alpha/(beta+ gamma ) ratios were 4.43+/-1.17, 0.56+/-0.49, 9.62+/-4.37, and 2.14+/-1.58 for normal healthy adult (beta(A)/beta(A),alphaalpha/alphaalpha), infant (beta(A)/beta(A),alphaalpha/alphaalpha), beta- thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and beta- thalassemia major(beta(T)/beta(T),alphaalpha/alphaalpha) respectively. The gamma /(beta+ gamma ) ratios were 0.04+/-0.03, 0.92+/-0.06, 0.28+/-0.18, and 0.95+/-0.04 for normal healthy adult (beta(A)/beta(A),alphaalpha/alphaalpha), infant (beta(A)/beta(A),alphaalpha/alphaalpha), beta- thalassemia minor (beta(T)/beta(A),alphaalpha/alphaalpha) and beta- thalassemia major (beta(T)/beta(T),alphaalpha/alphaalpha) respectively. Following statistical analyses, the alpha/beta and alpha/(beta+ gamma ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, beta- thalassemia minor and beta- thalassemia major). The gamma /(beta + gamma ) globin gene mRNA ratio was significantly different among all groups except for between infant and beta- thalassemia major patients. Human beta globin gene mRNA levels decrease progressively and dramatically from normal adults to beta-thalassemia patients with beta-thalassemia major having the lowest levels. On the other hand, the gamma globin gene mRNA levels increase progressively from normal adult to beta-thalassemia patients with beta-thalassemia major having the highest levels. Infants have relatively lower levels of beta but higher levels of gamma globin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of alpha, beta or gamma globin genes varied but inter-related among different ages of normal individuals and different beta-thalassemia genotypes.

Neuroglobin and cytoglobin in search of their role in the vertebrate globin family.

Neuroglobin and cytoglobin are two recent additions to the family of heme-containing respiratory proteins of man and other vertebrates. Here, we review the present state of knowledge of the structures, ligand binding kinetics, evolution and expression patterns of these two proteins. These data provide a first glimpse into the possible physiological roles of these globins in the animal's metabolism. Both, neuroglobin and cytoglobin are structurally similar to myoglobin, although they contain distinct cavities that may be instrumental in ligand binding. Kinetic and structural studies show that neuroglobin and cytoglobin belong to the class of hexa-coordinated globins with a biphasic ligand-binding kinetics. Nevertheless, their oxygen affinities resemble that of myoglobin. While neuroglobin is evolutionarily related to the invertebrate nerve-globins, cytoglobin shares a more recent common ancestry with myoglobin. Neuroglobin expression is confined mainly to brain and a few other tissues, with the highest expression observed in the retina. Present evidence points to an important role of neuroglobin in neuronal oxygen homeostasis and hypoxia protection, though other functions are still conceivable. Cytoglobin is predominantly expressed in fibroblasts and related cell types, but also in distinct nerve cell populations. Much less is known about its function, although in fibroblasts it might be involved in collagen synthesis.

Automatic generation and evaluation of sparse protein signatures for families of protein structural domains.

We identified key residues from the structural alignment of families of protein domains from SCOP which we represented in the form of sparse protein signatures. A signature-generating algorithm (SigGen) was developed and used to automatically identify key residues based on several structural and sequence-based criteria. The capacity of the signatures to detect related sequences from the SWISSPROT database was assessed by receiver operator characteristic (ROC) analysis and jack-knife testing. Test signatures for families from each of the main SCOP classes are described in relation to the quality of the structural alignments, the SigGen parameters used, and their diagnostic performance. We show that automatically generated signatures are potently diagnostic for their family (ROC50 scores typically >0.8), consistently outperform random signatures, and can identify sequence relationships in the "twilight zone" of protein sequence similarity (<40%). Signatures based on 15%-30% of alignment positions occurred most frequently among the best-performing signatures. When alignment quality is poor, sparser signatures perform better, whereas signatures generated from higher-quality alignments of fewer structures require more positions to be diagnostic. Our validation of signatures from the Globin family shows that when sequences from the structural alignment are removed and new signatures generated, the omitted sequences are still detected. The positions highlighted by the signature often correspond (alignment specificity >0.7) to the key positions in the original (non-jack-knifed) alignment. We discuss potential applications of sparse signatures in sequence annotation and homology modeling.

Neuroglobin and other hexacoordinated hemoglobins show a weak temperature dependence of oxygen binding.

Mouse and human neuroglobins, as well as the hemoglobins from Drosophila melanogaster and Arabidopsis thaliana, were recombinantly expressed in Escherichia coli, and their ligand-binding properties were studied versus temperature. These globins have a common feature of being hexacoordinated (via the distal histidine) under deoxy conditions, as evidenced by a large amplitude for the alpha absorption band at 560 nm and the Soret band at 426 nm. The transition from the hexacoordinated form to the CO bound species is slow, as expected for a replacement reaction Fe-His --> Fe --> FeCO. The intrinsic binding rates would indicate a high oxygen affinity for the pentacoordinated form, due to rapid association and slow (100 ms-1 s) dissociation. However, the competing protein ligand results in a much lower affinity, on the order of magnitude of 1 torr. In addition to decreasing the affinity for external ligand, the competitive internal ligand leads to a weaker observed temperature dependence of the ligand affinity, since the difference in equilibrium energy for the two ligands is much lower than that of ligand binding to pentacoordinated hemoglobin. This effect could be of biological relevance for certain organisms, since it could provide a globin with an oxygen affinity that is nearly independent of temperature.

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 alpha and beta globins strongly indicated that the leech globins first separated from globin lineage of annelids.

Globin genes are present in Ciona intestinalis.

The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that-like some molluscs and arthropods-the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enzymatic instead of respiratory functions.