Annelid noncirculating intracellular globins are gen-erally encountered as monomers [9,10], and only the Keywords annelid; dehaloperoxidase; extracellular globin; intracellular globin; m
Trang 1in annelids
Xavier Bailly1,*,, Christine Chabasse1,*, Ste´phane Hourdez1, Sylvia Dewilde2, Sophie Martial1, Luc Moens2and Franck Zal1
1 Equipe Ecophysiologie: Adaptation et Evolution Mole´culaires, UPMC – CNRS UMR 7144, Station Biologique, BP 74, Roscoff, France
2 Biochemistry Department, University of Antwerp, Belgium
Globins are heme-containing proteins that reversibly
bind oxygen and other gaseous ligands, and are
wide-spread in the three major kingdoms of life [1,2]
Despite the great diversity of their amino-acid
sequences, the basic functional unit is assumed to be a
monomeric globin with a specific and highly conserved
fold referred to as the ‘globin-fold’ On the basis of
this conserved basic structure and its prevalence in
living organisms, it has been suggested that globin
genes evolved from a common ancestral gene which,
after successive duplications and speciation events,
led to the genes that encode the widespread globin
superfamily [1–5]
Three types of globin have been described in anne-lids: (a) noncirculating intracellular globin [e.g myo-globin (Mb) found in the cytoplasm of muscle cells] [5,6]; (b) circulating intracellular globin [e.g hemo-globin (Hb) found in erythrocytes] [7]; (c) extracellu-lar globin dissolved in circulating fluids [7,8] These three types of globin display diversity in sequence, quaternary structure and functions such as binding and transport of oxygen and hydrogen sulfide, and activity of superoxide dismutase and mono-oxygenase [8]
Annelid noncirculating intracellular globins are gen-erally encountered as monomers [9,10], and only the
Keywords
annelid; dehaloperoxidase; extracellular
globin; intracellular globin; myoglobin
Correspondence
F Zal, Equipe Ecophysiologie: Adaptation et
Evolution Mole´culaires, UPMC – CNRS
UMR 7144, Station Biologique, BP 74,
29682 Roscoff cedex, France
Fax: +33 (0) 2 98 29 23 24
Tel: +33 (0) 2 98 29 23 09
E-mail: zal@sb-roscoff.fr
Present address
Department of Cell Biology and Comparative
Zoology, Institute of Biology, University of
Copenhagen, Denmark
*These authors contributed equally to this
work
(Received 8 December 2006, revised 12
March 2007, accepted 20 March 2007)
doi:10.1111/j.1742-4658.2007.05799.x
Globins are the most common type of oxygen-binding protein in annelids
In this paper, we show that circulating intracellular globin (Alvinella pom-pejanaand Glycera dibranchiata), noncirculating intracellular globin (Areni-cola 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 intracellu-lar and extracelluintracellu-lar 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 intra-cellular 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 com-mon ancestry with globins In A ornata, DHP may be a derived globin with an unusual enzymatic function
Abbreviations
DHP, dehaloperoxidase; Hb, hemoglobin; HBL-Hb, hexagonal bilayer hemoglobin; Mb, myoglobin; nMb, nerve myoglobin.
Trang 2amino-acid sequences from the polychete, Arenicola
marina, [11] and the nucleotide sequence from the
polychete, Aphrodite aculeata, [24] have been obtained
previously To date, only cDNA and amino-acid
sequences of circulating intracellular Hb belonging to
the marine polychete, Glycera dibranchiata, are known
[12,13] Annelid extracellular hexagonal bilayer
hemo-globins (HBL-Hbs) are assembled into a large
multi-subunit structure with molecular mass of 3–4 MDa
Some nucleotide and amino-acid sequences are already
known Extracellular globin chains are encoded by
genes belonging to a multigenic family, the molecular
phylogeny of which has previously been studied
[15,16] Only three annelid families are currently
known to express simultaneously the three types of
globin: the Terebellidae, the Alvinellidae and the
Opheliidae [17,18] The sporadic co-occurrence of the
three globin types may be more common in the annelid
phylum, as they were probably already present in a
common ancestor
Despite studies on the evolution of noncirculating
intracellular globins (Mbs) [5] and extracellular globins
[16,19,20], the phylogenetic relationships between these
different globins in annelids remain unclear because of
the lack of available sequences
To understand the emergence and evolution of these
globins, we have sequenced new annelid extracellular
and intracellular globin polypeptides, cDNAs and
genes such as (a) the nucleotide sequences of two
extra-cellular globins from the polychete, Ar marina, (b) the
nucleotide sequence of an Ar marina Mb, (c) the
amino-acid and nucleotide sequence of the intracellular
circulating globin of the polychete, Alvinella pompejana,
(d) the nucleotide sequence of the intracellular
circula-ting globin of the polychete, G dibranchiata, and (e)
the nucleotide sequence of dehaloperoxidase A
(DHPA) of the marine annelid polychete, Amphitrite
ornata In the light of a molecular phylogeny including
intracellular and extracellular globins of annelids,
mol-luscs and arthropods, we address here a likely
evolu-tionary scenario for the origin of extracellular and
intracellular globins in annelids
To complete and strengthen the phylogenetic
analy-sis, we also carried out a study on globin gene
struc-ture (intron positions), which provides an obvious
opportunity to explore gene evolution because genes
sharing the same intron positions are thought to be
homologous and closely related The typical pattern of
two introns⁄ three exons (with intron positions in B12.2
and G7.0) already found in numerous eukaryotic
glo-bin genes [1] has previously been reported in four
annelid extracellular globin genes from Lumbricus
terrestris [21], Eudystilia vancouverii [22] and Riftia
pachyptila [23] Apart from the nerve myoglobin (nMb) of Aph aculeata in which the first intron is missing [24], neither intracellular circulating nor non-circulating globin gene structures are known For this survey, we have identified (a) gene structures of the two new extracellular globins from Ar marina, (b) the gene structures of four extracellular globins from the vestimentiferan, R pachyptila, (c) the gene structure of
Ar marinaMb, (d) the gene structure of the new intra-cellular circulating globin from Al pompejana, (e) the gene structure of intracellular circulating globin from
G dibranchiata, and (f) the gene structure of DHPA from A ornata
Interestingly, blast searches revealed a strong amino-acid similarity between intracellular Hbs from
Al pompejana and DHP from A ornata involved in halometabolite detoxication (converts halogenated phenols into quinones in the presence of hydrogen per-oxide) [25] These heme-containing enzymes exhibit conserved distal and proximal histidines found in most globin sequences [26], and the crystal structure of native DHP exhibits a typical globin fold [27] These data suggest that DHP activity may have arisen by duplication of a globin gene [27], but do not rule out a possible evolutionary convergence In this paper,
we also show that this annelid DHP protein illustrates
an original case of functional diversification from a globin
Results
Identification of the A2 and B2 extracellular globin chains of Ar marina
Two extracellular globin chains of Ar marina (acces-sion numbers AJ880690 and AJ880691 for cDNAs, Q53I65 and Q53I64 for amino-acid sequences) were aligned and compared with other globins in the mul-tiple sequence alignment (Fig 1 and Table 1) The sequence of the extracellular globin from Lumbricus terrestris [28] was used as reference for the helix assignment (Fig 1) These two new globin chains pos-sess the well-conserved globin amino acids, Pro-C2, Phe-CD1, His-F8 and Trp-H4, as well as the two cysteines NA2 and H7 known to be involved in the formation of an intrachain disulfide bridge [29,30] Strong molecular signatures and phylogenetic analyses allowed the unambiguous assignment of these two new globin chains to A2 and B2 extracellular Hb clusters according to the classification proposed by Suzuki et al [31] in which the A and B families are, respectively, subdivided into A1, A2 and B1, B2 sub-families
Trang 3A2 chain
This sequence contains an ORF of 157 codons
(inclu-ding the initiation codon) As in other annelid
extracel-lular globins, residues 1–16 correspond to a signal
peptide This signal peptide was removed in the
align-ment presented in Fig 1
This sequence clearly belongs to the A family, as
evidenced by typical molecular features: Lys-A9,
Trp-B10 and also a deletion of three residues
between the A and B helices, and a deletion of one
residue between the F and G helices Moreover,
the two residues Gly-Pro (at position A1-A2)
indi-cate that this sequence belongs to the A2 group
(Fig 1)
B2 chain This sequence contains an ORF of 165 codons (inclu-ding the initiation codon) Residues 1–16 correspond
to a signal peptide This signal peptide is not shown in the alignment (Fig 1)
This sequence exhibits amino acids that are typical
of the B family: Asp-A4, Trp-A16, Phe-B10 and Leu-B13 Furthermore, it shows an insertion of three resi-dues between the A and B helices, an insertion of one residue between the F and G helices, and a three-resi-due motif Pro-Gln-Val at position G17-19 Moreover, the three-residue motif Thr-Gly-Arg between the A and B helices indicates that this sequence belongs to the B2 group (Fig 1)
Fig 1 Multiple alignment of annelid DHP,
extracellular and intracellular globins
(circula-ting and noncircula(circula-ting) amino-acid
sequences Intracellular globin sequences
are shaded Positions of intron 1 (B12.2) and
2 (G7.0) are indicated by dashed lines The
conserved amino-acid residues are indicated
in black Letters above the sequence
indi-cate the helical designation, based on the
Lumbricus terrestris helical structure [28].
Signal peptides, when present, have been
removed See Table 2 for abbreviations.
Trang 4Intracellular circulating globin of Al pompejana
The partial cDNA sequence (accession number
AJ880693) was obtained using degenerate primers
designed from the amino-acid sequence obtained by Edman degradation This sequence displays the key residues as Pro-C2, Phe-CD1, His-F8 and Trp-H4 (Fig 1) However, as in Ar marina myoglobin MbIIa and A ornata DHP, the conserved Trp-A12 is replaced
by an Ile residue
Gene structure Introns were sequenced and characterized for Ar mar-inaA2 and B2 extracellular globins (accession numbers AJ880690 and AJ880691, respectively) and myoglobin MbIIa (accession number AJ880692), the extracellular A1, B1a, B1b and B1c globin chains from R
pachypti-la, intracellular Hb from Al pompejana, Hb mIV from
G dibranchiata, and DHPA from A ornata Bailly reported the intron position of R pachyptila A2 and B2 [23] The position and length of each intron are summarized in Table 2 For all the sequences, the insertion positions of the two introns correspond to the usual B12.2 and G7.0 positions previously reported for many other globin sequences including L terrestris [32] and E vancouverii globins [1,22] (Fig 1)
The splicing sites have also been analyzed: it was shown that the 5¢ splice donor is marked by an eight-nucleotide conserved sequence, the 3¢ acceptor site cor-responds to a pyrimidine-rich region of 11 nucleotides followed by (C⁄ T)AG, and the typical branch point signal corresponds to a five-nucleotide sequence that functions as a signal for the spliceosome [33,34] In all introns, splicing donor and acceptor sequences con-form to the consensus sequences (Table 2)
Phylogenetic relationships The Bayesian tree based on annelid globin sequences only is shown in Fig 2 The NJ tree shows a similar topology (data not shown) Two well-supported main clusters can be identified: one comprises all intracellu-lar globins and the other all the extracelluintracellu-lar globins The intracellular cluster includes the nerve myoglobin (nMb) and all Mbs and intracellular Hbs The extra-cellular cluster is divided into two groups: the A and B families [15], as expected
Al pompejana intracellular Hb and A ornata DHP are most closely related to each other and obvi-ously belong to a well-supported intracellular cluster, distinct from a second cluster of intracellular globins which includes G dibranchiata Hb and Aph aculeata nMb
In Fig 3, which includes annelid, mollusc and arth-ropod globins, annelid extracellular and intra-cellular globins do not cluster together, but annelid
Table 1 Globin amino-acid sequences shown in the multiple
align-ment and molecular phylogeny.
Accession number
MbIa-Are Kleinschmidt [11]
Tylorrhynchus heterochaetus A1-Tyl P02219
Biomphalaria glabrata HBD2Biom and
HBD3Biom
Q683R3
HB9Art
Q7M454
Chironomus thummi thummi B1CHITH P02221
a
New sequences presented in this work.
Trang 5intracellular globins are at the base of a cluster
com-posed of extracellular globins from the insect,
Chirono-mus thummi thummi.The extracellular globin family of
the other arthropod, Artemia salina, surprisingly
clus-ters independently of C thummi thummi This topology
illustrates the high level of divergence among
extracel-lular globin families between crustacean and insects in
the Arthropoda phylum, reflecting specific adaptations
This is particularly obvious when the exon⁄ intron
structures of arthropod globin genes (summarized in
[35]) are compared, showing the presence of the
canon-ical B12.2 and G7.0 introns position in the Artemia
crustacean and their absence in the Chironomus insects
Discussion
Annelid globin gene structure
All introns described in this article exhibit the splicing
donor GT and acceptor AG sites (Table 2), whereas in
the L terrestris B1 globin gene, the donor GT is
replaced by GC [32] The typical branch point
sequence, involved in spliceosome binding, sometimes
diverges from the standard consensus sequence
(CTRAY), but studies have shown that this consensus sequence may not reflect the majority of branch point signals [34]
We used the intron insertion position pattern as ref-erence in order to follow gene evolution relationships
We assume that identical intron position between genes is a strong argument to reject evolutionary convergence between proteins exhibiting structural similarity
We have shown that R pachyptila and Ar marina extracellular globins (A2 and B2), Ar marina Mb and
Al pompejana and G dibranchiata intracellular Hb all share the same typical two intron⁄ three exon pattern (Table 2) These results allow us to rule out the possi-bility of structural and functional convergence between circulating and noncirculating intracellular globin and extracellular globin genes in annelids: these genes prob-ably evolved from a common globin-like gene ancestor and did not emerge independently of unrelated genes All the annelid intracellular globin gene structures reported here show the same gene structure, whereas Aph aculeatanMb gene lacks the first intron [24] This might be explained by the loss of the first intron in the Aph aculeatanMb evolutionary lineage [24]
Table 2 Position, length of introns, exon ⁄ intron splice junctions and possible branch points in Ar marina, Al pompejana, R pachyptila, G di-branchiata globins and A ornata globin and DHP genes Sequences in italic correspond to branch points where the well-conserved C or T or
A is not found BP, Branch point The consensus sequences presented are from Mount et al [33,52].
Splicing donor
A ⁄ C
AG gt a ⁄ g
Splicing acceptor yyn c ⁄ t
ag G G ⁄ T
Trang 6Distinct evolutionary history of extracellular
globins with respect to intracellular globins
in annelids
Working in the field of comparative biochemistry and
especially on intracellular hemerythrins and
hemo-globins, Manwell & Baker [36] drew attention to a
neglected problem in annelid globin evolution: ‘The
transition between intracellular and extracellular
res-piratory proteins represents a profound evolutionary
accomplishment It is much more than placing an
appropriate signal polypeptide portion, labeling a
pro-tein for extracellular export.’
Molecular signatures (Fig 1) clearly indicate that
circulating and noncirculating intracellular globins
share more common features among them than with
extracellular globins Phylogenetic analyses (Fig 2)
show that these intracellular globins cluster
independ-ently of extracellular globins, with high bootstrap
values (NJ method, data not shown) This strongly suggests that extracellular globin lineages have a dis-tinct evolutionary history with respect to circulating and noncirculating intracellular globins Experiments performed on the polychete, Travisia japonica, by Fushitani et al [37] showed that antibodies against extracellular globins did not cross-react with intracellu-lar globins This supports early or rapid divergence between intracellular and extracellular globin lineages Globins are found in many unicellular organisms such as archae, eubacteria and lower eukaryota [2,38– 40] The acquisition of a signal peptide for the secre-tion of extracellular globins is probably an apomorphic characteristic in multicellular organisms with respect to unicellular ones It has been proposed that the secre-tory peptide may have been acquired by insertion of some other secreted protein gene by genetic recombi-nation [41,42] Therefore, the secretory peptide found
in all annelid extracellular globins must have been
Fig 2 Bayesian phylogenetic tree based on annelid extracellular and intracellular globins (circulating and noncirculating) and DHP amino-acid sequences Posterior probability values are indicated above the branches.
Trang 7acquired by the original ancestral globin gene, which
has duplicated to give rise to the extracellular globin
multigenic family in this phylum (it is not
parsimoni-ous to envisage a repeated peptide signal acquisition in
the extracellular globin multigenic family)
Even though acquisition of a signal peptide must have
been a determinant step, other features are specific to
annelid extracellular globins Extracellular state seems
to correlate with the formation of a
high-molecular-mass complex, limiting the protein’s contribution to the
total colloid osmotic pressure of body fluids (vascular
blood and coelomic fluids) [43] and to minimizing rapid
Hb loss by excretion It is clear that the evolutionary
elaboration of the HBL-Hb structure simultaneously
involved the possibility for globins and linkers (proteins
involved in the structure of HBL-Hb) to interact and to
bind to dimers or other aggregation states while
conser-ving functionality Indeed, linkers are thought to result
from duplication of globins and rearrangement of exons
[41] It has been suggested that these
‘nonoxygen-bind-ing subunits’ may also intrinsically possess superoxide
dismutase and⁄ or methemoglobin reductase activities
necessary to keep the Hb functional [36] Evolution of
HBL-Hbs implies the coevolution of extracellular
glo-bins and linkers, which also represents a unique feature
of annelids with respect to other living organisms
Mol-luscs and arthropods, which also sometimes express
extracellular Hbs, do not exhibit a HBL-Hb quaternary structure and do not possess linkers Arthropod extra-cellular globin sequences also exhibit a signal peptide, but possess neither internal disulfide bridge nor
HBL-Hb quaternary structure, which suggests a different evo-lutionary history from extracellular globins in annelids
In addition, annelid extracellular globins do not cluster together with arthropod and mollusc extracellular glo-bins as shown in Fig 3 To date, it is not possible to state whether annelid, arthropod and mollusc extracel-lular globins come from an extracelextracel-lular globin already present in a common ancestor before their radiation Showing a clear homology between these extracellular globins would require additional globin sequences However, in the Annelida phylum, the HBL-Hb extracellular globins represent a phylum-specific inno-vation, and the common gene structure shared between all annelid globin types attest that extracellular globins are homologous with the intracellular ones This rules out functional and structural convergence occurring by
a gene co-option process
DHP is a derived globin
A ornata DHP, Al pompejana intracellular Hb and Ar marina Mb exhibit obvious molecular sig-natures between each other and with the intracellular
Fig 3 Bayesian phylogenetic tree based on
annelid, mollusc and arthropod extracellular
and intracellular globins and DHP amino-acid
sequences Posterior probability values are
indicated above the branches.
Trang 8monomeric globin N-terminal sequence of the
poly-chete, Enoplobranchus sanguineus [44] These results,
suggesting a close relationship between DHP and
intracellular globin, are supported by molecular
phylo-genic analyses showing that A ornata DHP, Al
pom-pejana intracellular Hb, Ar marina Mb and Ophelia
bicornis Mb cluster together with high bootstrap
values, with respect to other intracellular globins
(G dibranchiata and Aph aculeata nMb) and
extra-cellular globins Interestingly, we have found that, in
A ornata, the DHP-encoding gene exhibits the same
gene structure as extracellular globins, Ar marina Mb
and intracellular globin from Al pompejana and
G dibranchiata
The conserved intron positions between the three
annelid globin types and DHP, the DHP globin fold
and the amino-acid similarities between protein
sequences including globin and DHP allow us to rule
out structurally convergent evolution and indicate the
homology (i.e common ancestry) of these genes In
addition, like other typical globins, A ornata DHP is
able to reversibly bind oxygen It is found in the
oxy-ferrous (Fe2+) state when natively purified [26] and
also exhibits a globin fold, a heme group and distal
histidine [26,27]
We have confirmed by a molecular genetic approach
that DHP is a globin with a derived function, using
its heme to bind the peroxide ligand in order to
cata-lyze the oxidative dehalogenation of polyhalogenated
phenols
The DHP function, derived from a canonical globin
structure encoded by a globin-like gene, may have
been an innovation selected after annelid radiation
from an oxygen carrying Hb as an adaptation driven
by selection based on territorial war between annelids
excreting halogenous compounds [25]
A ornata possesses a monomeric circulating Hb in
its coelomic circulating cells [45], but the amino-acid
sequence is still unknown It is not possible to state
whether DHP and intracellular Hb of A ornata are
the same protein Further molecular studies are needed
to confirm whether they are the same protein with
several functions or the result of gene duplication with
subsequent acquisition of a new function
Experimental procedures
Collection of biological material
Juvenile specimens of the lugworm, Ar marina, were
collec-ted at low tide from a sandy shore near Roscoff (Penpoull
Beach), Nord Finiste`re, France, and kept in local running
sea water for 24 h
Specimens of Al pompejana were collected at 2500 m depth on the East Pacific Rise (950¢N at the M-vent site)
by the manned submersible Nautile during the HOPE¢99 cruise Once on board, the animals were kept in chilled sea water (10C) until used for tissue collection (usually less than 5 h) Tissues were then frozen in liquid nitrogen until they were used
Specimens of the hydrothermal vent tube worm,
R pachyptila, were collected on the EPR (950¢N at the Riftia Field site) at a depth of about 2500 m, during the French oceanographic cruise HOT 96 and the American cruise LARVE’99 The worms were sampled using the tele-manipulated arms of the submersibles Nautile and Alvin, brought back alive to the surface inside a temperature-insu-lated basket, and immediately frozen and stored in liquid nitrogen after their recovery on board
Specimens of A ornata were collected at Debidue flats,
in the North Inlet estuary (Georgetown, SC, 3320¢N, 7910¢W) and immediately placed in 70% alcohol until used for DNA extraction
Specimens of G dibranchiata were collected in Maine, USA and immediately preserved in 70% alcohol until used for DNA extraction
Preparation of Al pompejana intracellular Hb
Coelomic fluid was collected by carefully opening the dorsal body wall in the middle part of the body The coelomic fluid was centrifuged at 1000 g for 3 min at 4C, and the cells were washed twice with filtered sea water After the last cen-trifugation, three volumes of distilled water were added to the pellet of cells obtained, inducing cell lysis The suspension obtained was then centrifuged at 10 000 g for 5 min at 4C The supernatant, containing the cell extract, was then separ-ated and frozen in liquid nitrogen To prevent hydrolysis by proteases, phenylmethanesulfonyl fluoride was added to a final concentration of 1 lmolÆL)1before freezing Intracellu-lar Hb was prepared as previously described [18]
Protein sequencing of Al pompejana intracellular Hb
Heme was extracted by acid acetone precipitation
‘De-hemed’ Hb was pyridylethylated as described by Allen [46] and subsequently dialysed against 0.1% trifluoroacetic acid The protein was modified with maleic anhydride and cleaved with trypsin and CNBr An Asp-Pro cleavage was performed as described by Allen [46] The tryptic peptides were separated by HPLC on a reversed-phase Vydac C4 column developed with 0.1% trifluoroacetic acid⁄ acetonitrile The CNBr and Asp-Pro peptides were separated by SDS⁄ PAGE [47], and subjected to electroblot-ting The peptides were sequenced in an ABI 471-B sequencer (Applied Biosystems, Foster City, CA, USA)
Trang 9operated as recommended by the manufacturer The
N-terminal sequence was obtained by subjecting intact Hb
to Edman degradation
Total RNA extraction and cDNA synthesis
Entire juvenile specimens of Ar marina and Al pompejana
were crushed in liquid nitrogen Total RNA was extracted
using RNAble buffer (Eurobio, Courtaboeuf, France),
and poly(A) RNA was then isolated using an mRNA
Puri-fication Kit (Amersham, Little Chalfont,
Buckingham-shire, UK) RT-PCR was carried out using an anchor
5¢-CTCCTCTCCTCTCCTCTTCC(T)17primer
Isolation of genomic DNA
Whole specimens of Ar marina, Al pompejana, R
pachypti-la, G dibranchiata and A ornata were washed in deionized
water, and then incubated in 700 lL PK Buffer (50 mm
Tris⁄ HCl, 100 mm NaCl, 25 mm EDTA and 1% SDS, pH 8)
with 15 lL Proteinase K (10 lgÆlL)1) at 65C for 1 h The
supernatant was separated by centrifugation at 12 000 g for
5 min at 4C, and added to 700 lL phenol The DNA was
separated by a standard phenol⁄ chloroform extraction The
resulting DNA was precipitated with propan-2-ol, kept at
)20 C overnight, and centrifuged at 12 000 g for 15 min
The pellet was then washed once with 75% ethanol Finally,
the DNA pellet was resuspended in 100 lL TE buffer
(10 mm Tris⁄ HCl, 0.1 mm EDTA, pH 8) and stored at 4 C until used
Amplification of cDNA and genomic DNA
PCR was carried out in a total volume of 25 lL containing 10–50 ng template cDNA⁄ gDNA, 100 ng each degenerate primer, 200 lm dNTPs, 2.5 mm MgCl2 and 1 U DNA polymerase (Uptima, Interchim, Montluc¸on, France) PCR conditions were as follows: an initial denaturation step at
95C for 5 min, 35 cycles consisting of denaturation at
95C for 30 s, annealing for 30 s, extension at 72 C for
40 s, and a final elongation step at 72C for 10 min Prim-ers are given in Table 3
Cloning and sequencing
The PCR products were cloned using a TOPO-TA cloning Kit (Invitrogen, Cergy Pontoise, France) The pos-itive recombinant clones were isolated, and plasmid DNA was prepared with the FlexiPrep Kit (Amersham) Purified plasmids containing the putative globin insert were used in
a dye–primer cycle sequencing reaction, using the primer T7 and the Big Dye Terminator V3.1 Cycle Sequencing kit (Applied Biosystems) PCR products were subsequently run on a 3100 Genetic Analyser (Applied Biosystems) at Roscoff Sequencing Core Facility Ouest Genopole Plateform
Table 3 Primer sequences used for the PCR amplification CDS, Coding sequence.
Trang 10Rapid amplification of cDNA ends
cDNA ends were obtained by PCR using the 5¢-RACE and
3¢-RACE kit (Roche, Grenzacherstrasse, Switzerland)
according to the manufacturer’s instructions Buffer,
rea-gents and other conditions for the nested PCR were as
described by the manufacturer The RACE products
were purified, cloned with the TOPO-TA cloning kit
(Amersham), and sequenced as described above
Sequence analyses
Signal peptide
The peptide signal cleavage site was predicted by the
SignalP 3.0 Server [48] (http://www.cbs.dtu.dk/services/
SignalP)
Database analysis
The tblastn and tblastx search algorithms [49] were
employed to search data on the Uniprot database (http://
www.ebi.ac.uk)
Globin multiple alignment
We performed a global multiple alignment including
anne-lid, mollusc and arthropod intracellular and extracellular
globins We present here only the annelid globin multiple
alignment; the global one including molluscs and
arth-ropods is available on request All the globin sequences
used in the multiple alignment are listed in Table 1
Amino-acid sequences were aligned with the program
muscle [50] (http://phylogenomics.berkeley.edu/cgi-bin/
muscle/input_muscle.py) and adjusted manually
Molecular phylogeny
For the two sets of aligned globins (annelids on one hand
and annelids, molluscs and arthropods on the other),
Baye-sian analysis was carried out using mrbayes 3.1.1 (http://
mrbayes.csit.fsu.edu/index.php) and the JTT transition
mat-rix [51] Four chains were run simultaneously for 106
gener-ations, and trees were sampled every 100 genergener-ations,
producing a total of 104trees
Acknowledgements
We thank Dr Joa˜o Gil for collecting specimens of
G dibranchiata, and Dr David Lincoln for collecting
specimens of A ornata We gratefully acknowledge the
captain and crew of the NO L’Atalante, the pilots and
groups of the French and US submersibles Nautile
and Alvin, respectively We are also very grateful to
the chief scientists of the HOT’96, LARVE’99 and
HOPE’99 oceanographic cruises This work was sup-ported by CNRS, European grant (FEDER no pres-age 3814) and the Conseil Re´gional de Bretagne (contract no 809) (FZ) SD is a postdoctoral fellow of the FWO (Fund for Scientific Research Flanders)
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