J Mol Evol (2002) 54:312–321
DOI: 10.1007/s00239-001-0046-5
Cloning and Molecular Characterization of the First Innexin of the Phylum
Annelida—Expression of the Gene During Development
Nicoletta Potenza, Rosanna del Gaudio, Loredana Rivieccio, Giuseppina Maria Rosaria Russo,
Giuseppe Geraci
Department of Genetics, General and Molecular Biology, University of Naples Federico II, Via Mezzocannone, 8, 80134 Naples, Italy
Received: 15 December 2000 / Accepted: 27 August 2001
Abstract. A novel member of the innexin family (cv-
inx) has been isolated from the annelid polychaete worm
Chaetopterus variopedatus using a PCR approach on ge-
nomic DNA and sequence analysis on genomic DNA
clones. The gene is present in a HindIII-HindIII segment
of 2250 bp containing an uninterrupted open reading
frame of 1196 bp encoding a protein of 399 amino acids.
The predicted protein shows the typical structural fea-
tures of innexins and consensus sites for phosphoryla-
tion. Analyses on genomic DNA demonstrate that cv-inx
is a single copy gene with no introns in the coding re-
gion, exactly corresponding to the cDNA sequence. The
gene expression is regulated during development as
shown by Northern blots analyses of the RNA and by
immunoreaction with antibodies against the protein at
several embryonic stages. The finding of an innexin in
the phylum Annelida, outside of the Ecdysozoa clade,
and its peculiar gene structure suggest the necessity to
reconsider the current hypothesis on the origin and evo-
lution of gap junctional proteins.
Key words: Innexin — Gap junctional protein — In-
vertebrate intercellular channel — Intronless gene —
Polycheate — Chaetopterus variopedatus
Introduction
Intercellular communication is a fundamental function of
multicellular organisms. A widely utilized form of com-
Correspondence to: G. Geraci; email: geraci@dgbm.unina.it
© Springer-Verlag New York Inc. 2002
munication is enabled by intercellular channels present
in the gap junctions, the gated connections that are clus-
tered where the plasma membranes of two cells are in
close apposition. The gap junctions allow ions, metabo-
lites and small molecules (<1 kDa) to flow between two
adjacent cells providing a mechanism for creating func-
tional compartments within cellular populations that can
result electrically and biochemically coupled. In verte-
brates, the channels of gap junctions are composed of
subunit proteins encoded by the multigene connexin
family (Simon and Goodenough, 1998).
Gap junctional communication is essential for cell
signalling during development and differentiation, as is
emerging from gene targeting in mice and from analyses
of genetic diseases in humans (Lo, 1999).
While electrical coupling of cells was discovered for
the first time in the crayfish and gap junctional commu-
nication has been well documented in many inverte-
brates, no connexins homologues have been found in
invertebrates. However, conventional genetic dissections
of Caenhorabditis elegans and Drosophila melanogaster
have identified a family of genes that, on the basis of the
phenotypes of mutant organisms, appear to code for pro-
teins that have a role in gap junction communication.
These bear no sequence similarity to the connexins, but
show the typical structural topology of those molecules
and are referred to as innexins (for invertebrate connex-
ins). Analysis of mutant organisms reveals neuronal de-
fects that affect cell-cell coupling via gap junctions that
appear to involve electrical synapses, suggesting that in-
nexins may represent the invertebrate functional coun-
terpart of the vertebrate connexin family. In fact, muta-

tions in l(1)ogre (lethal(1) optic ganglia reduced) gene
of D. melanogaster lead to death of post-embryonic neu-
roblasts, because that gene is essential for the genera-
tion and maintenance of both post-embryonic neuro-
blasts in the optic formation centers and the giant neu-
roblasts in the central nervous system (Lipshitz et al.
1985; Watanabe and Kankel 1990; Watanabe and Kankel
1992). Also mutants in shaking-B locus (encoding pasN
or shak-B(neural), and pasV or shak-B(lethal)) are lack-
ing specific synaptic connections. This is the case of
mutations in pasN, that cause inability to exhibit the
escape response, a jump followed by flight as a conse-
quence of a light-off stimulus, because the gap junctions
between giant fiber cells and their post-synaptic partners
are missing (Baird et al. 1990; Crompton et al. 1992;
Krishnan et al. 1995; Phelan et al. 1996; Sun and Wyman
1996).
Several mutations in genes of the innexins family
have been characterized also in the nematode C. elegans.
Mutations in unc-7 gene cause an uncoordinated pheno-
type, because they affect the neurons involved in regu-
lating locomotion (Starich et al. 1993). Mutations in
eat-5 impair the electric and dye coupling of particu-
lar pharyngeal muscles that are no longer synchro-
nized (Avery 1993; Starich et al. 1996). Mutations in the
unc-9 gene also result in abnormal forward locomo-
tion and egg-retention phenotype (Barnes and Hekimi
1997).
In addition to genetic evidence that innexins are in-
volved in mediating gap junctional communication that
is lost with mutations in the genes, direct evidence
that innexins are themselves structural components of
channels has been obtained by the recent demonstra-
tion that some members of that family, such as D. me-
lanogaster Shak-B(lethal) (Phelan et al. 1998), Dm-
Inx2, and Dm-Inx3 (Stebbings et al. 2000), and the C.
elegans Inx-3 (Landesman et al. 1999), are able to form
functional intercellular channels in paired X. laevis oo-
cytes.
So far, members of the innexin family have been re-
ported only in the nematode C. elegans and in the in-
sects, initially in D. melanogaster and, more recently, in
the grasshopper Schistocerca americana (Ganfornina et
al. 1999). Here we report the cloning and molecular char-
acterization of a new innexin from the polychaete anne-
lid worm Chaetopterus variopedatus and we show that
its expression is regulated during development, as it is
the case of many other innexins. This is a novel finding
that poses a question on the hypothesis that innexin pro-
teins are a prerogative and, consequently, a molecular
marker for the proposed new phyletic clade of the Ec-
dysozoa (Adoutte et al. 1999). Rather, our findings pro-
vide support to the hypothesis that this family of proteins
is commonly present in invertebrates and increase the
number of organisms where the functional properties of
intercellular communication can be studied.
313
Materials and Methods
Molecular cloning. A 200-bp fragment of cv-inx was amplified by PCR
using degenerate oligonucleotide primers on 700 ng of genomic DNA
extracted from sperm cells of a single male C. variopedatus (del Gau-
dio et al. 1998). The amplification products were cloned in pUC18 by
SureClone Ligation kit (Pharmacia Biotech) following the manufactur-
er’s protocol. A clone containing an insert coding for the first extra-
cellular loop was identified by sequence analysis and used as a probe
to hybridize a Southern blot of genomic DNA digested to completion
with different restriction enzymes. Digestion with HindIII generates a
DNA band corresponding to fragments averaging 2.2 kbp in length. In
order to clone that fragment, HindIII-restricted genomic DNA was
fractionated by electrophoresis on 0.8% (wt/vol) agarose gel in TBE
buffer (0.089 M Tris-HCl, 0.089 M sodium borate, 0.009 M EDTA, pH
8.3); the agarose gel slice containing the HindIII fragments of interest
was excised from the slab and the DNA was electroeluted. A genomic
library of these fragments was constructed in pBlueScript vector KS-
(Stratagene) using standard procedures (Sambrook et al. 1989). In brief,
the products of T4 DNA ligase (Roche) were used to transform by
electroporation competent E. coli Tg1 cells. Positive clones were de-
tected by colony hybridization using as a probe the 200 bp amplifica-
tion product labeled with [␣32P]-dCTP using the Multiprime labeling
system (Amersham). The hybridization procedure was as follows. Fil-
ters were prehybridized for 6 h at 65°C in 5× SSPE (150 mM NaCl, 10
mM Na-phosphate, 1 mM EDTA, pH 7.4), 0.1% SDS, 5× Denhardt’s
solution (0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvi-
nylpyrrolidone) and 100 ␮g/ml denatured herring sperm DNA. The
labeled probe was then added to this mixture and hybridization was
carried out for 18 h at 65°C in the same solution. Filters were then
sequentially washed in 2× SSPE, 0.1% SDS for 10 min at room tem-
perature, for 30 min at 65°C and finally in 1× SSPE, 0.1% SDS for 30
min at 65°C. Hybridization was revealed by exposing Fuji RX films to
the filters at −70°C, with intensifying screens. Clones showing the
strongest hybridization signal after washing to high stringency were
further analyzed by performing PCR analysis with specific primers by
resuspending each colony directly into the PCR reaction mix, as re-
ported (http://www.lifetech.com). The upstream primer was the oligo-
nucleotide from position 657 to 673 (5⬘-CAATATGTCGGCGACCC-
3⬘), the downstream primer was the oligonucleotide from position 833
to 815 (5⬘-GGGGACCCATTGGTAGTAG-3⬘). The PCR protocol was
as follows: 94°C for 3 min followed by 35 cycles at 95°C for 1 min,
50°C for 30 s; 72°C for 30 s, after which the samples were kept at 72°C
for 5 min. Only two clones gave amplification and contained the ex-
pected sequence. One positive clone (3(V)) was digested with HindII
and three fragments were produced that were subcloned in pBlueScript
KS- vector and sequenced on both strands using the T7 DNA poly-
merase kit (Sequenase version 2.0, Amersham).
Sequence analyses. BLAST search was used on the NCBI nonre-
dundant database to identify the coding sequence of cv-inx by homol-
ogy with other innexins. The identified cv-inx sequence was analyzed
with the PC-GENE software package. Transmembrane domain predic-
tion was performed with TMpred program (available on http://
www.ch.embnet.org) which uses a statistical analysis of a database of
naturally occurring transmembrane proteins. Multiple sequence align-
ments were performed utilizing the Clustal W (1.8) program (Thomp-
son et al. 1994) from the DDBJ Homology Search system (http://
www.ddbj.nig.ac.jp) with blosum matrix. The tree is calculated from
the matrix using the neighbor-joining method (Saitou and Nei 1987)
and plotted with Phylip package (Felsenstein 1988, 1996).
Analyses on genomic DNA. A series of PCR amplifications were
carried out on 500 ng of genomic DNA using the strategy shown in Fig.
4A: two sets of primers were used, indicated as 1,2 and 3,4. The 5⬘
primer of the first set was 5⬘-GCAGGTATAACTCAGCG-3⬘, corre-
sponding to positions 379 to 401 of the genomic clone and the 3⬘
314
primer was 5⬘-GGAACCCGTACAAATGATAGTCCG-3⬘, corre-
sponding to positions 1227 to 1204. The 5⬘ primer of the second set was
5⬘-GATCCTGTACCTTATCAACGTCG-3⬘, corresponding to posi-
tions 1145 to 1167 and the 3⬘ primer was 5⬘-GTGTAACCACCCATC-
TACGATTG-3⬘, corresponding to positions 1878 to 1856. The PCR
protocol was as follows: 94°C for 3 min for initial denaturation fol-
lowed by 35 cycles at 95°C for 1 min, 55°C for 40 s, 72°C for 1 min.
At the end of the cycles, samples were kept at 72°C for 5 min.
The copy number of the cv-inx gene was determined by digesting
both C. variopedatus genomic DNA and plasmid 3(V) with HindIII
restriction nuclease and comparing by Southern blot analysis the rela-
tive radioactive labeling of the gene bands in the two digests, using the
results of 3(V) DNA as reference values. In short, a set of increasing
amounts of the two hydrolyzed DNA samples were analyzed by elec-
trophoretic separation in adjacent lanes on 0.8% (wt/vol) agarose gel in
TBE buffer. The Southern blot was then hybridized with the cv-inx
segment spanning from position 657 to 833, labeled with [␣32P]-dCTP
using the Multiprime labeling system (Amersham). The different
amounts of DNA loaded in each lane of the two sets of digestions are
specified in the legend of Fig. 4. The amounts of radioactive probe
bound to the samples in the different lanes were determined by using
the PosphorImager Scanner (Molecular Dynamics).
cv-inx expression during the embryonic development. Total RNA
was isolated (Sambrook et al. 1989) from sperms, eggs, and embryos at
the indicated stages of development.
Thirty micrograms of each total RNA sample were run on a 1%
agarose gel containing 6.7% formaldehyde in a buffer of 20 mM 3-(N-
morpholino)propanesulfonic acid, pH 7.0, 8 mM sodium acetate, 1 mM
EDTA. The gel was stained with ethidium bromide and the RNA was
transferred to a nylon membrane (Amersham) by capillary diffusion in
20× SSC. Transcript sizes were determined by comparing with the
migration of 18S and 28S ribosomal RNA of S. cerevisiae and sea
urchin P. lividus. The blot was sequentially hybridized with three
probes corresponding to the N- and C-terminal regions containing also
part of the UTR sequences (from position 379 to position 588 and from
position 1431 to 1878 of the gene, respectively) and with the remaining
central part of cv-inx (from position 588–1431) that contains the more
conserved domains. The probes were labeled by PCR synthesis in the
presence of [␣32P]-dCTP. High stringency conditions were used: hy-
bridization solution contained 50% formamide, 5× SSPE, 5× Den-
hardt’s solution, 0.1% SDS and 100 ␮g/ml denatured herring sperm
DNA and the temperature was 42°C. The blot was washed at 65°C as
reported for Southern blot and exposed to a Fuji RX film with inten-
sifying screens.
Aliquots of total RNA (1 ␮g), after treatment with RNase-free
DNase (Promega), were reverse transcribed using AMV reverse tran-
scriptase (Genenco) according to the manufacturer’s protocol. The
cDNA was prepared in two partially overlapping fragments using as 3⬘
primers the two oligonucleotides used for the analyses on genomic
DNA and incubating the reactions for 1 h at 42°C. Aliquots of the
produced cDNAs were used to amplify the two gene segments em-
ploying the same PCR primers used on genomic DNA. The amplifi-
cation products were cloned in pUC18 and sequenced as previously
described.
Antibody preparation and immunodetection of Cv-Inx. Polyclonal
antibodies against the first extracellular loop of cv-inx were prepared
by inserting 123 bp of cv-inx (corresponding to amino acid sequence
from position 60 to position 100) in the expression vector pMAL-c2
(BioLabs, New England) as a fusion protein with maltose binding
protein. The fusion protein was expressed in E. coli Tg1, purified and
injected in three sequential aliquots in three rabbits to immunize them.
Antibodies were isolated from immune sera as reported (del Gaudio et
al. 1999). Embryos at stages L1, L2, and L3 (Irvine et al. 1999) were
suspended in the lysis buffer 5 mM Tris pH 8, 5 mM EDTA, 5 mM
EGTA in the presence of a mixture of protease inhibitors, chymostatin,
leupeptin and pepstain, and homogenized in a Dounce. Membranes
were prepared by differential centrifugation (Paul et al. 1995) and
resuspended in SDS buffer for SDS-PAGE followed by Western blot
analysis (del Gaudio et al. 1999). Protein concentration values were
determined with the BioRad reagent. In a typical Western blot analysis,
30 ␮g of proteins from embryos were separated by SDS gel electro-
phoresis on 10% acrylamide gel and transferred to a nitrocellulose
membrane. The immunoenzymatic reactions were performed using the
prepared rabbit polyclonal antibodies against Cv-Inx and secondary
goat anti-rabbit antibodies conjugated with horseradish peroxidase
(Sigma-Aldrich).
Results
Molecular Cloning and Sequence Analysis of C.
variopedatus Innexin
The search for innexin in C. variopedatus was initiated at
DNA level because there were no data concerning either
the gene in the phylum Annelida or its possible pattern of
expression. To identify the gene, oligos were constructed
that corresponded to the most conserved regions of in-
nexins, as resulted from the inspection of the sequences
of C. elegans and D. melanogaster genes in the data
banks. The successful oligos, in PCR amplification ex-
periments, were those corresponding to the sequence
QYVGXP, for the 5⬘ primer, and to YYQWVX for the 3⬘
primer because they produced among others, a fragment
of about 200 bp that, cloned and sequenced, showed high
homology to the first extracellular innexin loop. This 200
bp fragment was then used as a probe to extend the
analysis to the whole gene. To this aim, different 10 ␮g
aliquots of genomic DNA, isolated from the sperm cells
of a single male, were each digested with different re-
striction nucleases, fractionated by agarose gel electro-
phoresis, blotted and hybridized with the radioactively
labeled 200 bp DNA fragment previously identified (Fig.
1). HindIII enzyme restriction produced a DNA fragment
of about 2.2 kbp that gave a high intensity hybridization
signal. This fragment was considered to be convenient to
initiate the sequence analysis of the gene preparing a
restricted genomic DNA library by cloning the 2.2 kbp
HindIII DNA band positive to hybridization. To this aim,
the gel slice containing the 2.2 kbp DNA fragments was
excised from the agarose gel slab, the fragments were
electroeluted, and then cloned in pBlueScript KS- vector.
A first screening was performed by colony hybridization;
clones that resulted positive by the screening, were fur-
ther analyzed with a second screening performed by PCR
amplification using specific internal primers. Two
clones, 3(V) and 18(X), produced the expected amplifi-
cation of a DNA segment of about 200 bp in length.
Restriction analysis showed the same patterns for the two
clones indicating that they contained the same DNA in-
sert. The insert of clone 3(V) was cut into three frag-
ments by hydrolysis with HindII restriction nuclease,
each fragment was cloned in pBlueScript KS- and com-
pletely sequenced on the two strands.
The nucleotide sequence demonstrated the presence

Fig. 1. Hybridization analysis on genomic DNA hydrolyzed with the
indicated enzymes. 10 ␮g aliquots of digested DNA were run on aga-
rose gel, transferred to a nylon membrane and hybridized with the 200
bp PCR product (see Materials and Methods for details). ND: not
digested DNA.
of a start codon at 520 nucleotides downstream from the
HindIII site followed by an uninterrupted open reading
frame of 1196 nucleotides terminating with TAA. This
sequence encodes a protein of 399 amino acids, with a
predicted molecular mass of 46.817 kDa and an isoelec-
tric point of 9.19. Sequence analysis shows seven con-
sensus sequence sites for protein kinase C phosphoryla-
tion (position 44, 69, 121, 155, 194, 251, 384) and four
for casein kinase II phosphorylation (position 129, 135,
146, 258); there are, in addition, two potential sites for
N-glycosylation (positions 127, 272) and three for N-
myristoylation (positions 130, 268, 377). The sequence
of the putative encoded protein, analyzed by BLASTP
program, showed a high overall similarity with innexins
present in data banks, together with domain to domain
correspondence of the main properties (see also next
paragraph). For this reason this gene has been called
cv-inx. The Cv-Inx protein sequence shows, by hydro-
phobicity analysis, four putative membrane spanning
segments separating five protein domains, as typical of
the other innexins. In fact, the proposed folding for in-
nexins poses both N- and C-terminal domains inside the
cell cytoplasm and predicts the presence of one cytoplas-
mic and two extracellular loops.
Analysis of Sequence Similarity
The alignments of Cv-Inx sequence to those of members
of the innexin family isolated from D. melanogaster, S.
americana, and C. elegans are reported in Fig. 2. Cv-Inx
315
aligns well with the other proteins and the quality of the
alignment is good, as judged by the ability to correctly
align corresponding domains from protein sequences.
The various domains show different degrees of similarity
with a typical pattern of different percent of sequence
conservation. The sequence shows high conservation in
the predicted transmembrane regions and in the extracel-
lular domains; sequence gaps are present, instead, in the
cytoplasmic domains (the cytoplasmic loop, N- and C-
termini) denoting a lower conservativity for these re-
gions. Each predicted extracellular loop shows also the
two cysteines that are typical of the innexins. The longest
absolutely conserved stretch of amino acids is the pep-
tide YYQW(V), located near the end of the first extra-
cellular loop next to the second transmembrane segment.
This pentapeptide can be regarded as diagnostic for the
innexin family members, as it is not found in any other
open reading frame in the NCBI database (Barnes 1994).
A stretch of conserved residues is present in the same
region also in connexins (O’Brien et al. 1996) as well as
a conserved residue of prolin in the second transmem-
brane segment, reported to be important for voltage gat-
ing (Suchyna et al. 1993).
C. variopedatus represents the first invertebrate, dif-
ferent from insects and nematode, from which an innexin
gene has been reported. Hence, it is of interest to deter-
mine the relationship of this innexin to those in other
organisms. In the alignments of Fig. 2, Cv-Inx shows
global similarity values with the other innexins ranging
from 60% to 47%. The values are higher for C. elegans
innexins than for insect innexins. This result is confirmed
by the unrooted tree of Fig. 3 constructed on the basis of
multiple alignments of several other innexin sequences
identified from genome analysis. Interestingly, Cv-Inx
groups together with C. elegans innexins and is separated
from the group of insect innexins. The insect innexins
group together with a well supported node of internal
branching pattern (Ganfornina et al. 1999) and are sepa-
rated from the worm innexins (C. elegans and C. vari-
opedatus). At a variance, nodes organizing the clade of
worm innexins have low bootstrap values suggesting ei-
ther a non correlation or, more likely, a spreading and
divergent evolution of these molecules. Similar results
are obtained by creating the dendrogram using the
Clustal program of PC-GENE software package that also
places Cv-Inx in a group together with C. elegans innex-
ins, clearly separated from the group of insect innexins
(data not shown). An extensive search of innexins in
other phyla will be required to define evolutionary rela-
tionships and to identify the occurrence of possible sub-
groups within the innexin family, as it has been made for
the connexin family.
Genomic Structure and Copy Number
Sequencing of the genomic clone revealed no introns in
cv-inx. This is in contrast with the genomic structures of
316
Fig. 2. Multiple sequence alignments of innexins retrieved from data
bank. Unc-7, Unc-9, and Eat-5 are innexins from C. elegans; PasN,
PasV, and Ogre are innexins from D. melanogaster; G-Inx(1) and
G-Inx(2) are innexins from S. americana. The alignment was generated
by Clustal W 1.8. (*), identical residues; (:), conserved substitutions;
innexins so far identified in other organisms which all
contain several introns different in number and position.
In order to verify if this structure was a possible cloning
artifact, a series of PCR analyses were performed on
genomic DNA (Fig. 4A). When the different regions of
the cv-inx are amplified, only a single band of amplifi-
cation of the expected size is obtained for each experi-
ment (Fig. 4B), indicating the presence of a single copy
gene.
(.), semi-conserved substitutions. The black boxes indicate the approxi-
mate locations of the putative transmembrane domains and the grey
box the conserved peptide YYQW; 䊉, conserved cysteines in extra-
cellular loops. (Nucleotide Accession Number, EMBL AJ344621.)
These data are consistent with the results of the hy-
bridization pattern of genomic DNA hydrolyzed with
different enzymes (Fig. 1) that also suggest the presence
of only one gene for cv-inx with no variants. In fact, the
sequence from position 657 to 833, used as a probe, has
only one AatII recognition site at position 790. There-
fore, digestion with this enzyme is expected to produce
two hybridizing fragments and one has to show higher
hybridization signal because it contains the major part of

the probe sequence. The same consideration is valid also
for the hydrolysis with SnaBI enzyme that has a recog-
nition site at position 751. MspI has a recognition site
within the sequence used as a probe (position 690) and
another consensus site at position 1274 of the genomic
clone: in fact, two bands of hybridization can be ob-
served, one of about 600 bp with higher intensity of
hybridization, that contains the majority of the probe
sequence and another of about 3000 bp. BamHI, PstI,
SacI enzymes have no recognition sites within probe
sequence and within the sequence of the genomic clone:
we can observe, in fact, only bands of large size, greater
than the band produced by HindIII enzyme hydrolysis
used for the construction of the restricted genomic DNA
library. In some lanes (AatII and HindIII lanes) addi-
tional faint hybridizing bands are present: they might
represent related innexin sequences rather than products
of an incomplete digestion of the DNA because identical
results were obtained in independent experiments per-
formed with the same enzymes.
The question of whether cv-inx is present as a single
copy gene in the C. variopedatus genome was ap-
proached also by Southern blot analysis (Fig. 4C). Both
genomic DNA and the DNA of clone 3(V) were hydro-
lyzed with HindIII restriction nuclease to obtain the elec-
trophoretic band containing the gene. The number of
copies in the haploid genome of C. variopedatus was
then determined by comparing the hybridization signal
317
Fig. 3. Phylogenetic unrooted tree
based on Clustal W sequence
alignment. Bootstrap values are based
on 1000 replicates. Sequences were
retrieved from data bank and the
respective accession numbers are
shown. Worm innexins are on the
upper side and insect innexins on lower
side of the figure. Innexins
corresponding to those aligned in Fig. 3
are indicated by name.
of the genomic HindIII DNA band with that of the clone
used as a homologous reference value. The quantitative
results obtained by PhosphorImager measurements of the
radioactive probe bound to the genomic DNA bands
were consistent with each other and confirmed the indi-
cation obtained with the other approaches giving the
same average value corresponding to a single copy gene.
This value was calculated assuming that the haploid ge-
nome of C. variopedatus contains 1.45 pg of DNA (del
Gaudio et al. 1998).
cv-inx Expression During the Embryonic Development
Analyses on genomic DNA for cv-inx showed a peculiar
gene structure with no introns in the coding region. For
this reason it was considered necessary to analyze its
expression to exclude that it was pseudogene. To this
aim, total RNA was extracted from sperm and egg cells,
from embryos at different stages of development and
from the prostomium, as adult tissue, and examined by
Northern blot analysis (Fig. 5A). Hybridization with
three probes corresponding to different parts of the gene,
show that the expression of cv-inx is regulated. In fact,
the transcript is not detectable in RNA from sperms,
eggs, and from developmental stages until 46 h from
fertilization, stage L1 (Irvine et al., 1999), where a very
faint band is evident, while the hybridization signal is
318
Fig. 4. Analyses on genomic DNA. (A) Scheme of PCR amplifica-
tion performed on genomic DNA. The open reading frame is denoted
by the shaded box. (B) PCR amplification of DNA fragments per-
formed with primers 1 and 2 (lane 1) and with primers 3 and 4 (lane 2)
produced bands of about 850 and 730 bp, respectively; M, molecular
weight standard (1 kbp DNA ladder, Genenco). (C) Copy number of
cv-inx: clone 3(V) containing the gene and genomic DNA were hy-
drolyzed with HindIII, fractionated on agarose gel, transferred to a
nylon membrane and hybridized as described in the text. Lanes 1–6:
increasing amounts of the insert DNA of clone 3(V) (0.01 ng, 0.1 ng,
0.25 ng, 0.5 ng, 1.25 ng, 2 ng, respectively); lanes 7–10: increasing
amounts of genomic DNA (2.5 ␮g, 5 ␮g, 10 ␮g, 20 ␮g, respectively).
Only one DNA band, of about 2.2 kbp, hybridizes in all lanes with the
probe.
well evident at 97 h, stage L2–L3. Ethidium bromide
staining of the ribosomal RNA bands shows that similar
amounts of RNA were loaded in each lane (Fig. 5B). The
transcript is about 3 kbp, larger than the sequence con-
tained in the genomic clone, indicating that the analyzed
HindIII-HindIII DNA fragment contains the entire cod-
ing region but not the entire UTRs.
In order to properly identify the cv-inx mRNA,
cDNAs were prepared from embryos at 46 h and 97 h of
development and analyzed by PCR using oligos produc-
ing two overlapping fragments as described (see Mate-
rials and Methods). The analysis of the sequences of
these two fragments shows that the mRNA is identical to
the cloned gene not only for the coding region but also
for the 3⬘ and 5⬘ parts of the UTRs. This demonstrates
the presence of a mRNA exactly corresponding to the
identified gene.
In line with Northern blot results on RNAs, analyses
by Western blot of the proteins extracted from embryos,
Fig. 5. Analysis of cv-inx expression. Panel A: 30 ␮g of total RNA
were run on a formaldehyde agarose gel and transferred to a nylon
membrane. The blot was hybridized with probes reported in Materials
and Methods. S, E indicate sperm and egg cells respectively; A, indi-
cates the adult tissue examined (prostomium); 2–97 h indicate the
development times (hours) after fertilization based on laboratory cul-
ture at 18°C. Panel B: Ethidium bromide staining of 18S rRNA band on
the gel as indication of equal loading of RNA samples in the lanes.
using polyclonal antibodies against the first extracellular
loop of cv-inx, show the presence of an immunoreacting
protein at stages L2 and L3 not detectable at stage L1
(Fig. 6). These data indicate that the gene is transcribed
and the mRNA translated producing the expected pro-
tein. It is interesting to note that, at the stages where
immunoreaction is observed, the intensity of the band is
higher in samples corresponding to the membrane frac-
tion of the embryonic homogenates (compare lanes 3 and
4, and lanes 5 and 6, Fig. 6).
Discussion
Until recently, members of the innexin family have been
reported in worms only in the nematode C. elegans and
in insects, in the fruitfly D. melanogaster and grasshop-
per S. americana. We present here evidence that a novel
member of the innexin protein family, Cv-Inx, is present
in an annelid polychaete worm. In fact, the protein pre-
dicted by the genomic clone and by the sequence of the
cDNA has composition and molecular mass comparable
to those of the other innexins and, above all, it displays
the typical features of that protein type: four transmem-
brane segments with highly conserved two extracellular
loops, each with two cysteines, probably required for
gap-junctional channel formation (Laird 1996). These
structural elements are predicted to have the same topo-
logical organization for all known innexins. There is a
higher percent of amino acid sequence conservation for
the two extracellular loops and the four transmembrane
domains. Of the two extracellular loops, the second ap-
pears to have more conservativity. Perhaps it might have
a role for intercellular channel formation (junctional
compatibility), as it was shown for connexins (White et
al. 1994). There is very little conservativity in the se-
quence of the cytoplasmic regions because they probably

Fig. 6. Western blot analysis of Cv-inx. Samples from the stage L1
(lanes 1 and 2), stage L2 (lanes 3 and 4), and stage L3 (lanes 5 and 6)
immunoreacted with specific polyclonal antibodies. Lanes 1, 3, and 5:
proteins from embryo homogenates; lanes 2, 4, and 6: proteins from
fractions corresponding to cellular membranes.
mediate different properties of the junctions as has been
shown for the N-terminal domain of connexins (White et
al. 1995).
In addition, and in line with this observation, Cv-Inx
has the most part of phosphorylation sites in the intracy-
toplasmic loop where they may mediate interactions with
adapter proteins (Krishnan et al. 1993). Sequence analy-
sis with PC-GENE software locates similar potential
phosphorylation sites in almost all innexins. The possible
modulation gap junctional conductance by phosphoryla-
tion, as extensively documented for connexins, is still to
be investigated for innexins. The divergent portions of
the putative gap junction proteins, e.g. the differences in
amino acid sequence, mostly concerning the intracellular
loop, the length of the N- and C-termini, or the location
of phosphorylation sites, probably adapt channel activity
to specific time and tissue requirements, modulating the
function of proteins with an overall conserved sequence
and structure. Identification of Cv-Inx as an innexin finds
support in the results of the Western blot experiments.
They clearly show that the molecule is present on the
membrane fraction, as expected for this type of protein.
Although there is no amino acid sequence similarity be-
tween innexins and connexins, there are evident struc-
tural similarities: they share a common predicted mem-
brane topology of four transmembrane segments with
cytoplasmic amino and carboxy termini; they share the
same pattern of sequence conservation that is higher for
transmembrane domains and for the two extracellular
loops that share an invariant pattern of cysteine residues
(three for connexin and two for innexins). Therefore, it
appears that similar structural domains of proteins are
important for the particular functions for both families:
in the connexin family, for example, sequence conserva-
tion in the two extracellular loops is thought to reflect the
requirement for recognition and docking between gap
junction hemi-channels in apposed cells. Could the ul-
trastructural differences (noted in the gap junction
plaques examined by EM techniques) and the different
319
behavior under freeze-fracture (Bryant 1997) be in rela-
tion with sequence divergence of the two families? And
why should vertebrates and invertebrates use different
gene families for the same function? In fact, the innexins
that form functional intercellular channels in paired
Xenopus oocytes have striking degrees of conservation
with connexin channels in their functional properties
(White and Paul 1999). Classical phylogeny suggests
that functions common to both C. elegans and D. mela-
nogaster, if utilized, should be found also in vertebrates.
Two possible explanations have been proposed for the
similar predicted topology and functionality of proteins
without obvious sequence similarity: one is that these
families arose from a common ancestor but diverged in
their sequence while retaining their function, the other is
that they arose independently and attained a common
structural topology through convergent evolution, under
the selecting pressure of a common function as sug-
gested, for example, for the identity of the catalytic sites
of prokaryotic and eukaryotic serine proteases.
A possible clue to decide on the origin and evolution
of gap junctional proteins is provided by the absolutely
peculiar gene structure of cv-inx: it has an uninterrupted
ORF. This gene structure is very different from those of
innexins of other organisms that have many introns dif-
ferent in number, position and length for the different
genes even in the same organism. Data presented here
suggest that the various innexins may have resulted from
a very early duplication, rather than by independent ap-
pearance in different groups of organisms. Once a gene
had duplicated, each copy may have come under the
influence of different promoters, allowing distinct alter-
ations of gene structure and distinct regulation of their
expression. In this case, the proteins themselves may
have evolved to tailor their physiological activities to the
requirements of the particular cell type in which they are
expressed. In favor of this hypothesis it can be observed
that innexin genes from C. elegans that are physically
closer to each other on the chromosome (for example,
innexins with the accession number Q23593 and Q23594
that have a spacer of only 926 bp) are more similar to
each other in the gene structure: the introns lie in the
same regions of the protein and the amino acid sequences
are very similar. Similar considerations hold also in D.
melanogaster. The more related proteins, Pas and Prp6
on one hand and Prp7, Ogre and Prp33 on the other hand,
have their genes in clusters along the X chromosome
(Curtin et al. 1999). This clustering is consistent with the
idea that the different genes originated by repeated and
independent gene duplications. The clearest example of
possible gene duplication is represented by pasV and
pasN. These genes have distinct N-terminal regions, but
have common exons for the C-terminal regions. The
level of identity between the N-termini of the two pro-
teins is high (69%) suggesting that they arose by partial
320
gene duplication from a common ancestral sequence and
subsequently evolved to perform distinct roles (Cromp-
ton et al. 1995).
In contrast to innexins, with only few exceptions, ver-
tebrate connexins have no introns in their ORFs: the
entire coding region is present within a single exon
(O’Brien et al. 1996; O’Brien et al. 1998; Sohl et al.
1998). So, cv-inx has a gene structure reminiscent of
connexin genes. In line with the hypothesis of a common
origin for both innexins and connexins, cv-inx could rep-
resent the situation of an ancestral gene, especially if the
evolutionary relationship and the antiquity of the phylum
Annelida are considered (Adoutte et al. 1999; Aguinaldo
et al. 1997). Under this hypothesis, and according to the
introns-late theory (Rzhetsky and Ayala, 1999), the gene
structure, initially simple, accumulated intron sequences
in independent ways during the evolution in the different
organisms. Indeed, in each evolutionary lineage, it ap-
pears that some independent expansion of the gene has
occurred, because, as it can be seen in the phylogenetic
tree, the known sequences of the worm and of the fly are
much more similar within species than between species.
The evolutionary value of having different innexin or
connexin genes might be the possibility that they might
form heteromeric junctions extending the range of struc-
tural and thus of physiological properties of the junctions
as concerns different pore-size, gating properties, or pos-
sibility of biochemical modifications (for example, phos-
phorylation) than otherwise possible with homomeric
junctions. Distinct assortments of innexins could produce
the formation of specific gap junctions between specific
groups of cells. This mechanism might be operative in
particular between neurons, whose function appears af-
fected in organisms in which innexins have been mutated
as detailed in the Introduction. Distinct assortments of
innexins would constitute a biological version of an elec-
tronic multiplexer by permitting the transmission of dif-
ferent molecular or electrical messages from a neuron,
through its axon, in a specific manner to each one of
several other neurons connected to it through their den-
drites. The existence of a complex set of innexins with
different, though sometimes overlapping, expression pat-
terns supports a combinatorial character of gap junction
formation in invertebrates as well as in vertebrates, in
line with the above proposed mechanism.
As a final comment, the existence of innexin in the
phylum of Annelida contributes to the hypothesis that
innexin family is commonly present in invertebrates
rather than being a prerogative of the new proposed phy-
letic clade of Ecdysozoa, formed by nematodes, arthro-
pods and other moulting animals (Phelan et al. 1998;
Todman et al. 1999). The finding of innexin outside of
this clade supports the possibility that innexin family
may represent indeed the functional counterpart of ver-
tebrate connexins. Given the multigenic character (re-
dundancy and compensatory expression) of this family in
the fruitfly and in the nematode, it is expected that other
innexins, though different in composition, should exist
also in the annelid C. variopedatus, probably differen-
tially expressed during development and in adult tissues.
In favor of this hypothesis is the finding of weakly hy-
bridizing bands observed when genomic DNA is ana-
lyzed by restriction and high stringency hybridization
using cv-inx as probe (Fig. 1). These bands might indeed
be due to other innexin genes closely related to cv-inx. A
search for these variants is necessary, also to approach,
through a comparative study in the different phyla, the
mechanism of gap junction communication in regulating
events during development and differentiation.
Acknowledgments. This work has been supported in part by funds of
the Italian MURST (40%) and in part by CNR contract number
96.3292.
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