From the Dipartimento di Biologia and Centro
di Ricerca Interdipartimentale per le Biotecnologie Innovative,
Università di Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy,
the ¶ Dipartimento di Scienze e Tecnologie Avanzate,
Università del Piemonte Orientale Amedeo Avogadro, Corso
Borsalino 54 I-15100 Alessandria, Italy, and the
Dipartimento di Biologia and Consiglio
Nazionale delle Ricerche Center for the Study of Metalloproteins,
Università di Padova, via Ugo Bassi 58/B, 35131 Padova, Italy,
and the
Department of Biochemistry, University of Antwerp UIA,
B-2610 Wilrijk, Belgium
Received for publication, January 19, 2001, and in revised form, April 2, 2001
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ABSTRACT |
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The evolution of extracellular hemoglobins
of annelids, vestimentiferans, and pogonophorans was
investigated by applying cladistic and distance-based approaches to
reconstruct the phylogenetic relationships of this group of respiratory
pigments. We performed this study using the aligned sequences of globin
and linker chains that are the constituents of these complex molecules.
Three novel globin and two novel linker chains of Sabella
spallanzanii described in an accompanying paper (Pallavicini, A.,
Negrisolo, E., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens,
L., and Lanfranchi, G. (2001) J. Biol. Chem. 276, 26384-26390) were also included. Our results allowed us to test
previous hypotheses on the evolutionary pathways of these proteins
and to formulate a new most parsimonious model of molecular
evolution. According to this novel model, the genes coding for the
polypeptides forming these composite molecules were already present in
the common ancestor of annelids, vestimentiferans, and pogonophorans.
The extracellular hemoglobins of Annelida, Vestimentifera, and
Pogonophora together with the chlorocruorin, a variant extracellular hemoglobin restricted to four polychaete families, have been recognized to form a unique group according to their physicochemical properties (1). These molecules are complexes of a large number of polypeptide components that assemble into hierarchical ordered quaternary structures.
The extracellular Hbs and
Chls1 of Annelida are formed
by two kinds of polypeptide chains, the globins and the linkers (2). Vestimentifera have two types of extracellular Hbs (2) that are
indicated here as heavy hemoglobin (HbH) and light
hemoglobin (HbL). HbH is very similar in shape
and molecular mass (~ 3,000 kDa) to the Hbs and Chls of
Annelida and is also composed of globin and linker chains. In contrast,
the HbL has a molecular mass of ~ 400 kDa and
contains globin chains only. Pogonophora contain an Hb very similar to
the HbL of Vestimentifera; therefore, it is also indicated
here as HbL (2).
In their pioneering work on the molecular evolution of
extracellular Hbs, Gotoh et al. (3) divided the globin
polypeptides into two groups with a common origin. Successively, Suzuki
and Riggs (4) demonstrated that linker chains share similarities with
the low density lipoprotein receptor and that they cannot be related to
the globins because of the high divergence of their primary structures.
More recently, Yuasa et al. (5) have proposed a general
evolutionary model for the Hbs, HbH, and
HbL. According to these authors the common ancestor
of all these molecules was a protein formed by globin chains only.
During the successive evolution the linker chains would have been added
to the final structure of Hbs and HbH. Chls were not
included in their analysis, because no sequences were available at that
time. Moreover, their model implies an independent evolution of Hbs and
HbH. As a consequence two points still remain unsettled:
(i) the placement of Chl in this scenario and (ii) the
evolutionary pathway followed by Hbs, Chls, and HbH with
respect to HbL.
To investigate these points we have sequenced the globin and linker
chains of Chl of the polychaete Sabella spallanzanii (6). They have been aligned with other available sequences of Annelida, Vestimentifera, and Pogonophora to perform phylogenetic analyses applying both cladistic and distance-based methods. Using this approach
we have been able to retrace the molecular evolution of Hbs, Chls,
HbHs, and HbLs. The comparison of our new
phylogenetic reconstruction with the previously advanced hypotheses (3, 5) lead us to propose an alternative model for the evolution of annelid hemoglobins.
Sequence Alignment
We aligned all the sequences of extracellular Hbs, Chls,
HbHs, and HbLs of Annelida, Pogonophora, and
Vestimentifera available in the literature including the newly
presented globin and linker sequences of S. spallanzanii
(6). Globin sequences were aligned manually according to the
nonvertebrate globin template (7), and linker sequences were aligned
using the ClustalW program (8).
Pairwise Comparisons
Pairwise comparisons were performed on the coding and 3'-UTR
portions of the S. spallanzanii globin cDNAs and some
selected globins of Annelida using the program ALIGN. Similar
comparisons were carried out on the linker sequences using the ClustalW
program (8).
Phylogenetic Analyses
Sequence alignments were used to build phylogenetic trees using
the maximum parsimony2 (9)
and neighbor-joining (NJ) methods (10).
Outgroup Choice--
Globin sequences with known x-ray
structures were chosen as outgroups. Based on the phylogeny of Metazoa
(11), the sequences of the closest relatives of Annelida,
Vestimentifera, and Pogonophora were selected as outgroups. These
groups are (i) the globins of the mollusk Scapharca
inaequivalvis, (ii) the globin of the echiuran Urechis
caupo, and (iii) the intracellular globins of the annelid Glycera dibranchiata. No outgroup for the linker sequences
could be selected because their evolutionary position is unknown
(4).
Cladistic Analysis--
The cladistic analyses were done using
the program PAUP, version 3.1.1. (12). Globin evolution was studied
using a heuristic approach because the number of sequences involved
excludes the exhaustive approach (12). All characters were weighted
equally, and those uninformative were excluded from the analysis. After a series of trials, we set the PAUP options to perform more efficiently in finding the shortest trees. For the optimization of characters, the
ACCTRAN option was set. For the heuristic searches, the
options set were: (i) keep minimal trees only, (ii)
collapse zero-length branches, (iii) starting tree sources,
get by stepwise addition, (iv) swap on, minimal trees
only, (v) addition sequence, random, (vi) replications,
50, (vii) swapping algorithm, TBR tree
bisection-reconnection, (viii) MULPARS on, and (ix)
steepest descent on.
For the phylogenetic analysis of linker chains, we applied an
exhaustive approach because of the smaller size of the data set (12).
Uninformative characters were also excluded, and the remaining
characters were weighted equally. The PAUP optimization of characters
(ACCTRAN on) and exhaustive search (keep minimal trees
only and collapse zero-length branches on) options were set.
Tree Indexes in the Cladistic Analyses--
Two indexes are
traditionally used to test the robustness of the most parsimonious tree
obtained by cladistic analyses, the consistency and retention indexes
(13, 14). These indexes were calculated as implemented in PAUP (12).
Values of the consistency index and retention index > 0.5 indicate that convergent/parallel evolution does not affect strongly
the phylogenetic reconstruction and that the obtained topologies of
trees are reliable. Molecular synapomorphies in the cladistic analysis
were detected using the program MacClade (Version 3), which allows the
tracking of the evolution of each character (15).
Neighbor-joining Analysis--
The distance-based phylogenetic
reconstructions were performed on globin and linker alignments
according to the NJ method (10) as implemented in the TREECON program
(Version 1.3b) (16). The NJ trees were created by applying the
following settings: (i) distance calculation, Kimura 83;
(ii) alignment positions, all; and (iii) insertions and
deletions, not taken into account.
Bootstrap Test--
The bootstrap resampling (17) was performed
to test the robustness of the trees obtained by cladistic and
neighbor-joining phylogenetic reconstructions. In both cases, 500 replicates were run.
Sequence Alignments
Globin Alignment--
In Fig. 1 we
present the results of a global alignment of the Sabella
globin sequences with the globins of other annelids, vestimentiferans,
pogonophorans, and the canonical globin fold (7). Eight sequences
chosen according to the criteria described under "Materials and
Methods" were added as outgroups. An inspection of the alignment
clearly indicates that the key residues Trp (A12), Phe (CD1), His (E7),
and His (F8), consistent with the canonical globin fold, are absolutely
conserved. Conversely, the presence of Pro (C2), which usually
determines the bend of the corner between helices B and C, is not
universal to all the globins. In two Sabella globins (Glb1
and Glb2), the small residues Ala and Cys are found at positions B6 and
E8. In these sites the majority of annelid globins have a Gly residue,
with the exception of globin I of Tylorrhynchus, in which a
Phe is found at position B6. These substitutions in the
Sabella globins would result in a closer crossing of the B
and E helices. All the extracellular annelid globins shown in the
alignment have in common the Cys residues at positions NA2 and H11.
These amino acids are important for the formation of the first
supramolecular aggregate in the assembly of the whole Hb (18), which is
confirmed by the fact that they are replaced in the intracellular
globins of Glycera, Urechis, and
Scapharca by other residues. Four other residues, namely Arg
(E10), Phe (E4), His (F3), and Gln (F7), are highly conserved in the
extracellular globins, but the structural and functional significance
of this conservation remains to be clarified.
Linker Alignment--
A similar alignment was carried out for the
linker sequences of Sabella with the five other linkers
available in the data bases (Fig. 2). The
main feature that seems to be conserved is a cysteine-rich segment
((Cys-X6)3-Cys-X5-Cys-X10-Cys)
that is typical for all the linker chains sequenced thus far and has
been related to the low density lipoprotein-receptor motif (4). Considering the current global alignment (Fig. 2), the cysteine-rich segment can be written more precisely as
((Cys-X5-6)2-Cys-X6-Cys-Asp-X3-Asp-Cys-X4-Asp-Glu-X4-Cys).
Pairwise Comparison--
The analysis of the pairwise comparison
of the globin cDNAs (Fig. 3) shows a
higher percentage of identity in the open reading frame portion
(57.59% mean value) than in the 3'-UTR portion (47.82% mean value).
This is consistent with a higher degree of variability of the globin 3'
UTRs that are less subject to structural constrains. However, it should
be noted that Lumbricus d1 globin and d2 are more similar in
3' regions than in the coding portions.
The pairwise comparisons performed on the linker chains (Fig.
4) give the following main results. (i)
Tylorrhynchus L2 and Neanthes L2 markedly differ
from the general structure of other linkers. In fact they share 74.58%
of identical amino acids, whereas the mean for the whole data set is
25.79%. The latter value decreases to 23.35%, if the comparison of
Tylorrhynchus L2 versus Neanthes L2 is
excluded from the computation. (ii) Roughly 40% of the amino acids are
specific for each chain (mean of dissimilar amino acids = 39.45%). (iii) Identical amino acids represent one fourth of the whole
data when sequences are pairwise-compared. However, the residues common
to all the linker chains in the global alignment are only 15 (5.7%)
(Fig. 2), and they are mainly restricted to the cysteine-rich
segment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of globin chains. The
sequences have been aligned in the coding portions using as
reference the tertiary structure template of invertebrate globins (7).
They are shown with the accession numbers for the Swiss-Prot, NCBI, and
EMBL data banks in parentheses: Scapharca I (P02213),
S. inaequivalvis; Scapharca IIA (P14821),
S. inaequivalvis; Urechis F1 (P06148), U. caupo; Glycera MH4 (P15447), G. dibranchiata; Glycera mmc (P02216), G. dibranchiata; Glycera I (P23216), G. dibranchiata; Glycera II (P21659), G. dibranchiata; Glycera III (P21660), G. dibranchiata; Oligobrachia b (5), Oligobrachia
mashikoi; Tylorrhynchus I (P02219), Tylorrhynchus
heterochaetus; Tylorrhynchus IIA (P09966),
Tylorrhynchus heterochaetus; Lamellibrachia BI
(23), Lamellibrachia sp.; Oligobrachia a5 (5),
Oligobrachia mashikoi; Lamellibrachia AIII
(P15469), Lamellibrachia sp.; Riftia b (P80592),
Riftia pachyptila; Pontodrilus I (24),
Pontodrilus matsushimensis; Pheretima sie. I
(P11740), Pheretima sieboldi; Pheretima com. I
(24), Pheretima comunissima; Pheretima hil. I
(25), Pheretima hilgendorfi; Lumbricus d1
(U55073), Lumbricus terrestris; Lumbricus d2
(U55074), Lumbricus terrestris; Tubifex 1 (P18202), Tubifex tubifex; Sabella Glb3
(AJ131285), S. spallanzanii; Lumbricus II
(P02218), Lumbricus terrestris; Lamellibrachia
BIV (23), Lamellibrachia sp.; Lumbricus III
(P11069), Lumbricus terrestris; Tylorrhynchus IIB
(P13578), Tylorrhynchus heterochaetus;
Tylorrhynchus IIC (P02220), Tylorrhynchus
heterochaetus; Lumbricus IV (P13579), Lumbricus
terrestris; Oligobrachia c (5), Oligobrachia
mashikoi; Lamellibrachia BII (23),
Lamellibrachia sp.; Sabellastarte E (D58418),
Sabellastarte indica; Sabella Glb1 (AJ131283),
S. spallanzanii; and Sabella Glb2 (AJ131284),
S. spallanzanii. The amino acids common among all the
globins of Annelida, Pogonophora, and Vestimentifera are
indicated by asterisks. Red, invariant
amino acids; blue, amino acids common to the majority of
sequences; green, synapomorphic amino acids;
black, autoapomorphic amino acids.
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Fig. 2.
Alignment of linker chains. Amino acid
sequences of the following species were used (with the accession
numbers from Swiss-Prot, NCBI, and EMBL data banks in parenthesis):
Tylorrhynchus L2 (P18208), Tylorrhynchus
heterochaetus; Neanthes L2 (D58413), Neanthes
diversicolor; Sabella L1 (AJ131900), S. spallanzanii; Lumbricus L1 (A46587), Lumbricus
terrestris; Tylorrhynchus L1 (P18207),
Tylorrhynchus heterochaetus; Lamellibrachia LAV1
(P16222), Lamellibrachia sp.; and Sabella L3
(AJ131286), S. spallanzanii. Red, invariant amino
acids; blue, amino acids common to the majority of
sequences.
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Fig. 3.
Pairwise comparison of open reading
frames and 3' UTRs of globin cDNAs. Black bars,
globin open reading frame; white bars, globin 3' UTRs;
SAS.g1, Sabella Glb1 globin; SAS.g2,
Sabella Glb2 globin; SAS.g3, Sabella
Glb3 globin; SAI.gl, Sabellastarte E globin;
LUT.d1, Lumbricus d1 globin; LUT.d2,
Lumbricus d2 globin; LUT.III,
Lumbricus III globin.
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Fig. 4.
Pairwise comparison of amino acid sequences
of linker chains. Black bars, identical amino acids;
striped bars, strongly similar amino acids; dotted
bars, weakly similar amino acids; white bars, different
amino acids; TY2, Tylorrhynchus L2 linker;
NEA, Neanthes L2 linker; SA1,
Sabella L1 linker; LUM, Lumbricus L1
linker; TY1, Tylorrhynchus L1 linker;
LAM, Lamellibrachia LAV1 linker; SA3,
Sabella L3 linker.
Phylogenetic Analyses
Globin Phylogenetic Analysis--
We have applied both cladistic
and distance-based methods to study the molecular evolution of annelid
globins. The cladistic analysis results in 18 equally parsimonious
cladograms. The resulting strict consensus (SC) tree is presented in
Fig. 5A together with the
distance-based tree (Fig. 5B). Both phylogenetic
reconstructions recognize a monophyletic origin of Hbs, Chls,
HbHs, and HbLs. A more detailed analysis of the
ingroup shows that the extracellular Hbs, Chls, HbHs, and
HbLs can be divided into two distinct groups. However, the
actual position of the Lumbricus III globin cannot be
resolved in the SC tree. The topologies of the two trees show some
discrepancies. In particular the placement of the
Lamellibrachia BIV globin seems controversial. In fact, in
the cladogram it is placed into the A group according to the
classification of Gotoh et al. (3), whereas in the NJ tree
it is included in the B group. Both hypotheses are poorly supported by
bootstrap values, revealing the weakness of the more basal nodes. In
Table I we list the clades that belong to
the ingroup and are supported by one or more molecular synapomorphies.
Several of them are also sustained by high bootstrap values.
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Linker Phylogenetic Analysis--
The cladistic analysis of linker
sequences produced two equally parsimonious cladograms. The SC tree is
showed in Fig. 6, where the NJ tree is
also presented. Cladistic and distance-based analyses were performed
only on the available sequences of linker chains because no convincing
putative outgroups are known. In fact, with the exception of the
cysteine-rich segment that relates the linkers with the low density
lipoprotein receptor (4), no other significant similarity is known
between linker chains and other proteins. Nevertheless,
Tylorrhynchus L2 and Neanthes L1 were used to
root the trees, because they are much more similar to each other than
to other linkers.
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The SC and NJ trees show some discrepancies in their topology. The
cladogram structure favors a strict relationship between Tylorrhynchus L1, Lamellibrachia LAV1, and
Sabella L3. This clade is also supported by a very high
bootstrap value. Conversely, the NJ tree supports the two groups
Tylorrhynchus L1 + Lumbricus L1 and
Sabella L1 + Sabella L3. The first is also
corroborated by the bootstrap value (72%), whereas the second does not
receive strong support by the bootstrap test (48%). Both cladistic and distance-based analyses strongly favor the grouping of
Tylorrhynchus L2 and Neanthes L2 with respect to
other linker chains.
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DISCUSSION |
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The cladistic and distance-based analyses that we performed on the extracellular Hbs of annelids, pogonophorans, and vestimentiferans confirm the previous results of Gotoh et al. (3). These authors proposed the division of the globin chains into two main groups, A and B, each divided further into two subgroups, A1/A2 and B1/B2. This classification was successively applied to HbHs and HbLs (19, 20). Recently, a correction of this nomenclature has been proposed that recommends an inversion of the names of the two main strains (21).
However the identification of homologous chains, i.e. globins that are the products of orthologous genes, does not seem to be a trivial task. In both our analyses Tylorrhynchus IIA and Tylorrhynchus I are grouped together more closely than the respective "homologous" sequences. Therefore it seems problematic to name them Tylorrhynchus A2 and Tylorrhynchus A1 as previously suggested (22). In this light Lumbricus d1 and Lumbricus d2 can be defined either as two allelic forms of the same gene or as the products of a very recent gene duplication. The same reasoning can be applied to Sabella Glb1 and Sabella Glb2.
The subdivision into four homologous groups of globins could be an oversimplification of the real situation. In fact we have found a higher number of globin chains in the Chl, purified from a single specimen of Sabella (6). A study of the entire portion of the genome coding for these proteins should be the best way to understand these discrepancies.
The globins and linkers, forming the Chls, are tightly associated in the phylogenetic reconstructions with those included in the Hbs. This clearly identifies the Chl as a variant of Hb. On this assumption the name chlorocruorin must be considered only as a descriptive term.
The phylogenetic reconstruction presented in this paper reveals that the genes coding for the polypeptides that form Chls, Hbs, HbHs, and HbLs appeared before the separation of Vestimentifera and Pogonophora phyla from Annelida (Sensu stricto) (11). The evolutionary pathway does not change even if we consider pogonophorans and vestimentiferans as members of the class Opisthochaeta within the phylum Annelida (20). The critical question is: what was the scenario in which Chls, Hbs, HbHs, and HbLs evolved?
Yuasa et al. (5) previously suggested that Hb, Chl, and
HbH evolved from HbL, which is made only of
globin chains, adding linker chains to form the final hexagonal
bilayered structure. If we accept this hypothesis we must make four
assumptions (Fig. 7A): (i)
HbL was present in the common ancestor of Annelida,
Vestimentifera, and Pogonophora, (ii) in the Annelida phylum
HbL disappeared, originating Hb and Chl, (iii) in
Vestimentifera HbH evolved from HbL but the
latter did not disappear, and (iv) HbH evolved separately from Hb and Chl. The last assumption is supported by the fact that
vestimentiferans and pogonophorans are sister groups, independent from
their systematic position. As a consequence, HbH could not be present in their common ancestor (an annelid) because otherwise we
should admit its subsequent loss in Pogonophora, which contrasts with
the starting hypothesis.
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Our analyses performed on linkers of Hb, Chl, and HbH show that the genes coding for these polypeptides were already present in the common ancestor of Annelida, Vestimentifera, and Pogonophora. We therefore suggest an alternative scenario for the evolution of this group of respiratory pigments. Our most parsimonious hypothesis is based only on the following three assumptions (Fig. 7B). (i) The common ancestor of Annelida, Vestimentifera, and Pogonophora had in its genome the whole set of genes coding for globin and linker chains. As a consequence Hb, Chl, and HbH did not evolve independently. (ii) The common ancestor of Vestimentifera and Pogonophora evolved the HbL from HbH, starting from the set of genes coding for the latter. (iii) Pogonophora lost HbH during evolution.
The data deduced from our phylogenetic analyses fit nicely in this new
hypothesis, which requires fewer ad hoc assumptions to
explain the origin of this group of proteins. The new evolutionary model that we propose can be applied also to the Hbs of leeches, which
were not considered in our study because the available sequences are
not sufficiently complete. However, the data based on the amino termini
of some leech globins clearly show that they are homologous to other
extracellular Hbs (21).
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ACKNOWLEDGEMENTS |
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We thank Dr. Lucio Cariello, Director of the Stazione Zoologica "A. Dhorn" (Napoli, Italy), for providing the specimens of S. spallanzanii.
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FOOTNOTES |
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* This work was supported in part by the Italian Ministry of University and of Scientific and Technological Research MURST Grant-Cofinanziamento Protocol 9805192993-002 (to A. G.-M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
** Post-doctoral fellow of the Fund for Scientific Research-Flanders (FWO).
§§ To whom correspondence should be addressed. Tel.: 39-04982-76221; Fax: 39-04982-76280; E-mail: lanfra@cribi.unipd.it.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M100557200
2 Glossary of phylogenetic terms: apomorphy, a derived state of a character that represents an evolutionary novelty with respect to the ancestral state; autoapomorphy, a derived character state that is unique for a particular sequence; bootstrap test, a statistical test used to verify the robustness of the topology of an evolutionary tree; bootstrap value, the result of the bootstrap test (in this paper it is expressed as a percentage); cladistic analysis, a phylogenetic reconstruction based on the principle of maximum parsimony; cladogram, a tree depicting the phylogenetic relationships that results from a cladistic approach; clade, a monophyletic group in a cladistic context; ingroup, a set of sequences that are considered the focus of interest; monophyletic group, a group that includes all the sequences that originate from a common ancestor; most parsimonious tree, a tree produced in a cladistic analysis that has the shortest length; orthologous sequences, two sequences derived from a speciation event (i.e. the same sequence in different species); outgroups, a set of sequences that are brought into the analysis to determine the root of the ingroup and ancestral states; strict consensus tree, a tree derived from a set of trees in which all conflicting branching patterns are collapsed into multifurcations; synapomorphy, a derived state of a character that is shared by all the taxa belonging to a clade; tree length in the cladistic analysis, the value obtained by computing the sum of the minimum numbers of substitutions in all the positions of the alignment.
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ABBREVIATIONS |
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The abbreviations used are: Chl, chlorocruorin; HbH, heavy hemoglobin; HbL, light hemoglobin; UTR, untranslated region; NJ, neighbor-joining; SC, strict consensus.
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